Smelling in the dark: Phylogenomic insights into the chemosensory system of a subterranean beetle.

The chemosensory system has experienced relevant changes in subterranean animals, facilitating the perception of specific chemical signals critical to survival in their particular environment. However, the genomic basis of chemoreception in cave-dwelling fauna has been largely unexplored. We generated de novo transcriptomes for antennae and body samples of the troglobitic beetle Speonomus longicornis (whose characters suggest an extreme adaptation to a deep subterranean environment) in order to investigate the evolutionary origin and diversification of the chemosensory gene repertoire across coleopterans through a phylogenomic approach. Our results suggested a diminished diversity of odourant and gustatory gene repertoires compared to polyphagous beetles that inhabit surface habitats. Moreover, S. longicornis showed a large diversity of odourant-binding proteins, suggesting an important role of these proteins in capturing airborne chemical cues. We identified a gene duplication of the ionotropic coreceptor IR25a, a highly conserved single-copy gene in protostomes involved in thermal and humidity sensing. In addition, no homologous genes to sugar receptors or the ionotropic receptor IR41a were detected. Our findings suggest that the chemosensory gene repertoire of this cave beetle may result from adaptation to the highly specific ecological niche it occupies, and that gene duplication and loss may have played an important role in the evolution of gene families involved in chemoreception. Altogether, our results shed light on the genomic basis of chemoreception in a cave-dwelling invertebrate and pave the road towards understanding the genomic underpinnings of adaptation to the subterranean lifestyle at a deeper level.

Phylogeny and taxonomic revision of Deronectina Galewski, 1994 (Coleoptera: Dytiscidae: Hydroporinae: Hydroporini).

The subtribe Deronectina Galewski, 1994 (Dytiscidae, Hydroporinae, Hydroporini) is distributed in the Nearctic, in the north of the Neotropical region, and in the Palaearctic and Afrotropical regions. It is currently composed of 194 species and 13 subspecies in eight genera: Amurodytes Fery Petrov, 2013, Boreonectes Angus, 2010, Deronectes Sharp, 1882, Nebrioporus Régimbart, 1906, Oreodytes Seidlitz, 1887, Scarodytes Gozis, 1914, Stictotarsus Zimmermann, 1919, and Trichonectes Guignot, 1941. We present a morphological and a molecular phylogeny of the species of the subtribe, and a revision of their taxonomy to accommodate our phylogenetic results. The morphological phylogeny is based on the study of 54 characters of the adults of 189 species and 2 subspecies, of which 114 species and the 2 subspecies were coded in the morphological matrix. For the molecular phylogeny we investigated 115 species and 11 subspecies, using a combination of fragments of four mitochondrial (COI, 16S rRNA, tRNA-Leu and NAD1) and two nuclear genes (18S rRNA and H3), analysed with maximum likelihood and Bayesian methods. For both datasets we included the type species of all genus-group taxa. The morphological, molecular and combined phylogenies mostly agree with the current classification of the group, but in some cases our results are in contradiction with established genera. Most remarkable are the polyphyly of Stictotarsus and Nebrioporus, the low support for the monophyly and internal phylogeny of Oreodytes, and the low support for the monophyly of Deronectina with molecular data. Thus, we introduce some taxonomic changes in the current classification to accommodate the generic concepts to our phylogenetic results. Nine new genera are established: Clarkhydrus n. gen. (type species Hydroporus roffii Clark, 1862), Hornectes n. gen. (type species Hydroporus quadrimaculatus Horn, 1883), Iberonectes n. gen. (type species Deronectes bertrandi Legros, 1956), Larsonectes n. gen. (type species Potamonectes minipi Larson, 1991), Leconectes n. gen. (type species Hydroporus striatellus LeConte, 1852), Mystonectes n. gen. (type species Deronectes neomexicanus Zimmerman Smith, 1975), Nectoboreus n. gen. (type species Hydroporus aequinoctialis Clark, 1862), Nectomimus n. gen. (type species Oreodytes okulovi Lafer, 1988), and Zaitzevhydrus n. gen. (type species Hydroporus formaster Zaitzev, 1908). Three genera are reinstated as valid: Deuteronectes Guignot, 1945 (stat. rest.) (type species Hydroporus picturatus Horn, 1883), Nectoporus Guignot, 1950 (stat. rest.) (type species Hydroporus abbreviatus Fall, 1923), and Neonectes J. Balfour-Browne, 1940 (stat. rest.) (type species Hydroporus natrix Sharp, 1884). Thirty-six new combinations for species and subspecies thus far treated in the genera Boreonectes, Nebrioporus, Oreodytes and Stictotarsus result from the new classification: Clarkhydrus corvinus (Sharp, 1887) n. comb., C. decemsignatus (Clark, 1862) n. comb., C. deceptus (Fall, 1932) n. comb., C. eximius (Motschulsky, 1859) n. comb., C. falli (Nilsson, 2001) n. comb., C. interjectus (Sharp, 1882) n. comb., C. minax (Zimmerman, 1982) n. comb., C. opaculus (Sharp, 1882) n. comb., C. roffii (Clark, 1862) n. comb., C. spectabilis (Zimmerman, 1982) n. comb., Deuteronectes angustior (Hatch, 1928) n. comb., Hornectes quadrimaculatus (Horn, 1883) n. comb., Iberonectes bertrandi (Legros, 1956) n. comb., Larsonectes minipi (Larson, 1991) n. comb., Leconectes striatellus (LeConte, 1852) n. comb., Mystonectes coelamboides (Fall, 1923) n. comb., M. grammicus (Sharp, 1887) n. comb., M. neomexicanus (Zimmerman Smith, 1975) n. comb., M. panaminti (Fall, 1923) n. comb., M. titulus (Leech, 1945) n. comb., Nectoboreus aequinoctialis (Clark, 1862) n. comb., N. dolerosus (Leech, 1945) n. comb., N. funereus (Crotch, 1873) n. comb., Nectomimus okulovi (Lafer, 1988) n. comb., Nectoporus angelinii (Fery, 2015) n. comb., N. congruus (LeConte, 1878) n. comb., N. crassulus (Fall, 1923) n. comb., N. obesus obesus (LeConte, 1866) n. comb., N. obesus cordillerensis (Larson, 1990) n. comb., N. rhyacophilus (Zimmerman, 1985) n. comb., N. sanmarkii sanmarkii (C.R. Sahlberg, 1826) n. comb., N. sanmarkii alienus (Sharp, 1873) n. comb., N. sierrae (Zimmerman, 1985) n. comb., N. subrotundus (Fall, 1923) n. comb., Zaitzevhydrus formaster formaster (Zaitzev, 1908) n. comb., and Z. formaster ulanulana (C.-K. Yang, 1996) n. comb.

Speciation below ground: Tempo and mode of diversification in a radiation of endogean ground beetles.

Dispersal is a critical factor determining the spatial scale of speciation, which is constrained by the ecological characteristics and distribution of a species' habitat and the intrinsic traits of species. Endogean taxa are strongly affected by the unique qualities of the below-ground environment and its effect on dispersal, and contrasting reports indicate either high dispersal capabilities favoured by small body size and mediated by passive mechanisms, or low dispersal due to restricted movement and confinement inside the soil. We studied a species-rich endogean ground beetle lineage, Typhlocharina, including three genera and more than 60 species, as a model for the evolutionary biology of dispersal and speciation in the deep soil. A time-calibrated molecular phylogeny generated from >400 individuals was used to delimit candidate species, to study the accumulation of lineages through space and time by species-area-age relationships and to determine the geographical structure of the diversification using the relationship between phylogenetic and geographic distances across the phylogeny. Our results indicated a small spatial scale of speciation in Typhlocharina and low dispersal capacity combined with sporadic long distance, presumably passive dispersal events that fuelled the speciation process. Analysis of lineage growth within Typhlocharina revealed a richness plateau correlated with the range of distribution of lineages, suggesting a long-term species richness equilibrium mediated by density dependence through limits of habitat availability. The interplay of area- and age-dependent processes ruling the lineage diversification in Typhlocharina may serve as a general model for the evolution of high species diversity in endogean mesofauna.

Gondwanian relicts and oceanic dispersal in a cosmopolitan radiation of euedaphic ground beetles.

Anillini are a tribe of minute, euedaphic ground beetles (Carabidae) characterized by the loss of eyes, loss of wings and high levels of local endemism. Despite their presumed low dispersal, they have a nearly cosmopolitan distribution, including isolated islands such as New Zealand and New Caledonia. We used a time calibrated molecular phylogeny to test, first, if the tribe as currently understood is monophyletic and, second, whether the time of divergence is compatible with an early vicariant diversification after the breakup of Gondwana. We sequenced portions of 6 mitochondrial and 3 nuclear genes for 66 specimens in 17 genera of Anillini plus 39 outgroups. The resulting phylogenetic tree was used to estimate the time of diversification using two independent calibration schemes, by applying molecular rates for the related genus Carabus or by dating the tree with fossil and geological information. Rates of molecular evolution and lineage ages were mostly concordant between both calibration schemes. The monophyly of Anillini was well-supported, and its age was consistent with a Gondwanian origin of the main lineages and an initial diversification at ca. 100Ma representing the split between the eyed Nesamblyops (New Zealand) and the remaining Anillini. The subsequent diversification, including the split of the Nearctic Anillinus and the subsequent splits of Palaearctic lineages, was dated to between 80 and 100Ma and thus was also compatible with a tectonic vicariant origin. On the contrary, the estimated age of the New Caledonian blind Orthotyphlus at ca. 30±20Ma was incompatible with a vicariant origin, suggesting the possibility of trans-oceanic dispersal in these endogean beetles.

The evolutionary conserved oil body associated protein OBAP1 participates in the regulation of oil body size.

A transcriptomic approach has been used to identify genes predominantly expressed in maize (Zea mays) scutellum during maturation. One of the identified genes is oil body associated protein1 (obap1), which is transcribed during seed maturation predominantly in the scutellum, and its expression decreases rapidly after germination. Proteins similar to OBAP1 are present in all plants, including primitive plants and mosses, and in some fungi and bacteria. In plants, obap genes are divided in two subfamilies. Arabidopsis (Arabidopsis thaliana) genome contains five genes coding for OBAP proteins. Arabidopsis OBAP1a protein is accumulated during seed maturation and disappears after germination. Agroinfiltration of tobacco (Nicotiana benthamiana) epidermal leaf cells with fusions of OBAP1 to yellow fluorescent protein and immunogold labeling of embryo transmission electron microscopy sections showed that OBAP1 protein is mainly localized in the surface of the oil bodies. OBAP1 protein was detected in the oil body cellular fraction of Arabidopsis embryos. Deletion analyses demonstrate that the most hydrophilic part of the protein is responsible for the oil body localization, which suggests an indirect interaction of OBAP1 with other proteins in the oil body surface. An Arabidopsis mutant with a transfer DNA inserted in the second exon of the obap1a gene and an RNA interference line against the same gene showed a decrease in the germination rate, a decrease in seed oil content, and changes in fatty acid composition, and their embryos have few, big, and irregular oil bodies compared with the wild type. Taken together, our findings suggest that OBAP1 protein is involved in the stability of oil bodies.

Nucleotide substitution rates for the full set of mitochondrial protein-coding genes in Coleoptera.

The ages of cladogenetic events in Coleoptera are frequently estimated with mitochondrial protein-coding genes (MPCGs) and the "standard" mitochondrial nucleotide substitution rate for arthropods. This rate has been used for different mitochondrial gene combinations and time scales despite it was estimated on short mitochondrial sequences from few comparisons of close related species. These shortcomings may cause greater impact at deep phylogenetic levels as errors in rates and ages increase with branch lengths. We use the full set of MPCGs of 15 species of beetles (two of them newly sequenced here) to estimate the nucleotide evolutionary rates in a reconstructed phylogeny among suborders, paying special attention to the effect of data partitioning and model choices on these estimations. The optimal strategy for nucleotide data, as measured with Bayes factors, was partitioning by codon position. This retrieved Adephaga as a sister group to Myxophaga with strong support (expected-likelihood weights test 0.94-1) and both sisters to Polyphaga, in contradiction with the most currently accepted views. The hypothesis of Archostemata being sister to the remaining Coleoptera, which is in agreement with morphology, was increasingly supported when third codon sites were recoded or completely removed, sequences were analyzed as AA, and heterogeneous models were implemented but the support levels remained low. Nucleotide substitution rates were strongly affected by the choice of data partitioning (codon position versus individual genes), with up to sixfold levels of variation, whereas differences in the molecular clock algorithm produced changes of only about 20%. The global mitochondrial protein coding rate using codon partitioning and an estimated age of 250 million years (MY) for the origin of the Coleoptera was 1.34% per branch per MY, which closely matches the 'standard' clock of 1.15% per MY. The estimation of the rates on alternative topologies gave similar results. Using local molecular clocks, the evolutionary rate in the Polyphaga and Archostemata was estimated to be nearly twice as fast as in the Adephaga and Myxophaga (1.03% versus 0.53% per MY). Rates across individual genes varied from 0.55% to 8.61% per MY. Our results suggest that cox1 might not be an optimal gene for implementing molecular clocks in deep phylogenies for beetles because it shows relatively slow rates at first and second codon positions but very fast rates at third ones. In contrast, nad5, nad4 and nad2 perform better, as they exhibit more homogeneous rates among codon positions.

Ancient origin of a Western Mediterranean radiation of subterranean beetles.

BACKGROUND: Cave organisms have been used as models for evolution and biogeography, as their reduced above-ground dispersal produces phylogenetic patterns of area distribution that largely match the geological history of mountain ranges and cave habitats. Most current hypotheses assume that subterranean lineages arose recently from surface dwelling, dispersive close relatives, but for terrestrial organisms there is scant phylogenetic evidence to support this view. We study here with molecular methods the evolutionary history of a highly diverse assemblage of subterranean beetles in the tribe Leptodirini (Coleoptera, Leiodidae, Cholevinae) in the mountain systems of the Western Mediterranean. RESULTS: Ca. 3.5 KB of sequence information from five mitochondrial and two nuclear gene fragments was obtained for 57 species of Leptodirini and eight outgroups. Phylogenetic analysis was robust to changes in alignment and reconstruction method and revealed strongly supported clades, each of them restricted to a major mountain system in the Iberian peninsula. A molecular clock calibration of the tree using the separation of the Sardinian microplate (at 33 MY) established a rate of 2.0% divergence per MY for five mitochondrial genes (4% for cox1 alone) and dated the nodes separating the main subterranean lineages before the Early Oligocene. The colonisation of the Pyrenean chain, by a lineage not closely related to those found elsewhere in the Iberian peninsula, began soon after the subterranean habitat became available in the Early Oligocene, and progressed from the periphery to the centre. CONCLUSIONS: Our results suggest that by the Early-Mid Oligocene the main lineages of Western Mediterranean Leptodirini had developed all modifications to the subterranean life and were already present in the main geographical areas in which they are found today. The origin of the currently recognised genera can be dated to the Late Oligocene-Miocene, and their diversification can thus be traced to Miocene ancestors fully adapted to subterranean life, with no evidence of extinct epigean, less modified lineages. The close correspondence of organismal evolution and geological record confirms them as an important study system for historical biogeography and molecular evolution.

Phylogeny and diversification of diving beetles (Coleoptera: Dytiscidae).

Dytiscidae is the most diverse family of beetles in which both adults and larvae are aquatic, with examples of extreme morphological and ecological adaptations. Despite continuous attention from systematists and ecologists, existing phylogenetic hypotheses remain unsatisfactory because of limited taxon sampling or low node support. Here we provide a phylogenetic tree inferred from four gene fragments (cox1, rrnL, H3 and SSU, ≈ 4000 aligned base pairs), including 222 species in 116 of 174 known genera and 25 of 26 tribes. We aligned ribosomal genes prior to tree building with parsimony and Bayesian methods using three approaches: progressive pair-wise alignment with refinement, progressive alignment modeling the evolution of indels, and deletion of hypervariable sites. Results were generally congruent across alignment and tree inference methods. Basal relationships were not well defined, although we identified 28 well supported lineages corresponding to recognized tribes or groups of genera, among which the most prominent novel results were the polyphyly of Dytiscinae; the grouping of Pachydrini with Bidessini, Peschetius with Methlini and Coptotomus within Copelatinae; the monophyly of all Australian Hydroporini (Necterosoma group), and their relationship with the Graptodytes and Deronectes groups plus Hygrotini. We found support for a clade formed by Hydroporinae plus Laccophilini, and their sister relationship with Cybistrini and Copelatinae. The tree provided a framework for the analysis of species diversification in Dytiscidae. We found a positive correlation between the number of species in a lineage and the age of the crown group as estimated through a molecular clock approach, but the correlation with the stem age was non-significant. Imbalances between sister clades were significant for several nodes, but the residuals of the regression of species numbers with the crown age of the group identified only Bidessini and the Coptotomus + Agaporomorphus clade as lineages with, respectively, above and below expected levels of species diversity. © The Willi Hennig Society 2008.

A comprehensive phylogeny of beetles reveals the evolutionary origins of a superradiation.

Beetles represent almost one-fourth of all described species, and knowledge about their relationships and evolution adds to our understanding of biodiversity. We performed a comprehensive phylogenetic analysis of Coleoptera inferred from three genes and nearly 1900 species, representing more than 80% of the world's recognized beetle families. We defined basal relationships in the Polyphaga supergroup, which contains over 300,000 species, and established five families as the earliest branching lineages. By dating the phylogeny, we found that the success of beetles is explained neither by exceptional net diversification rates nor by a predominant role of herbivory and the Cretaceous rise of angiosperms. Instead, the pre-Cretaceous origin of more than 100 present-day lineages suggests that beetle species richness is due to high survival of lineages and sustained diversification in a variety of niches.