Cave Beetles Research Articles

Rediscovery and conservation status of six short‐range endemic Pseudanophthalmus cave beetles (Carabidae: Trechini)

Abstract The North American endemic cave beetle genus Pseudanophthalmus is exceptionally diverse, with >150 described taxa in karst regions of the eastern United States. Eighty‐seven per cent of taxa, however, are at risk of extinction due to small, restricted distributions, low abundance, and several potential anthropogenic threats to their habitats. Six species in Tennessee are exceedingly rare and are candidates for listing under the U.S. Endangered Species Act: Coleman Cave Beetle (P. colemanensis), Fowler's Cave Beetle (P. fowlerae), Inquirer Cave Beetle (P. inquisitor), Bakers Station Cave Beetle (P. insularis), Noblett's Cave Beetle (P. paulus), and Soothsayer Cave Beetle (P. tiresias). Each species is an extreme short‐range endemic. Four species have not been observed in several decades, and two species (P. insularis and P. paulus) are considered possibly extinct. We searched 57 caves in 15 counties in Tennessee, including eight of the nine historical localities of the six Pseudanophthalmus species between July 2013 and March 2016 to determine if populations were still extant, to search for new populations, and to estimate relative abundance. We confirmed the continued existence of all six species, including P. fowlerae, P. insularis, P. paulus, and P. tiresias, which had not been observed in 52, 60, 50, and 42 years, respectively. We also discovered five new populations in total, one for each species except for P. paulus. Although U.S. Fish and Wildlife Service ruled that all six species do not warrant federal listing, all species continue to have restricted ranges and remain at an elevated risk of extinction.

Molecular phylogeny of the cave beetle genus Hadesia (Coleoptera: Leiodidae: Cholevinae: Leptodirini), with a description of a new species from Montenegro

Hadesia is a charismatic and scientifically important genus of cave beetles. It is a classical representative of the subterranean fauna of the Dinaric Karst and the model upon which the ultra-specialized ecomorphological form of semi-aquatic, hygropetricolous filter feeders became known. We describe a new species, Hadesia zetae sp.n., from southwestern Montenegro, and employ new morphological characters to build an identification key to all five species of the genus. Based on DNA sequence data from two nuclear and two mitochondrial genes and using multispecies coalescent methods, we corroborated their reciprocal monophyly and inferred a well-supported phylogenetic hypothesis. Molecular dating suggests that the most recent common ancestor of the extant Hadesia species lived about three million years ago, during the warm and wet mid-Pliocene, and diversified allopatrically through range fragmentation caused by habitat reduction during the Pleistocene. Because of their extreme rarity and observed negative impact of collection pressure, we discourage collecting of Hadesia at known sites.

The Iberian genus Paraphaenops Jeannel, 1916 (Coleoptera: Carabidae: Trechini): Morphology, phylogeny and geographical distribution

The species Paraphaenops breuilianus (Jeannel, 1916) is an Iberian iconic cave beetle with “aphaenopsian” facies, monospecific until now. An extensive field work over the last years allowed the revision of this peculiar genus using the combinations of morphological, molecular and ecological approaches and led to the description of new taxa Paraphaenops fadriquei Ortuño and Faille sp. nov., Paraphaenops breuilianus espanoli Ortuño and Faille ssp. nov. and the full larval diagnosis of the third larval instar of the type species. The sequencing of two mitochondrial (cox1, rrnl+tRNA-Leu+nad1) and one nuclear (LSU) gene fragments evidenced a strong divergence for all markers considered between P. breuilianus and the new species P. fadriquei sp. nov. The strong genetic differences between the two taxa contrast singularly with the extreme morphological homogeneity of the genus. The ecological data recorded for Paraphaenops Jeannel, 1916 species show that they are stenotherms and stenohygrobionts, perfectly adapted to the subterranean environment, living at temperatures between 4.4 and 10°C with high relative humidity. The new localities and taxa enlarge the former distribution area from 72 to 5091 square kilometres sprinkled between twelve caves: six in the Mola de Catí (P. breuilianus breuilianus); three in the aforementioned karst region towards the south, crossing the geographical barrier “Barranc de Regatxol” (P. breuilianus espanoli ssp. nov.); and three, 60km towards the west, in a different karst region: the “Sierra de la Dehesas” (P. fadriquei sp. nov.) This geographical range overlaps a geostructural area quoted as “junction area” between the Iberian System and the Catalan coastal Mountain range, an area up to 1000 meters above sea level.

The cave beetle genus Anthroherpon is polyphyletic; molecular phylogenetics and description of Graciliella n. gen. (Leiodidae, Leptodirini)

The subtribe Anthroherponina form an iconic group of obligate cave beetles, typical representatives of the Dinaric subterranean fauna, which is considered to be the richest in the world. Phylogenetic studies within this subtribe are scarce and based only on morphological characters, which, due to troglomorphic convergence, are frequently unreliable. Moreover, morphological stasis and morphological polymorphism make classification of taxa difficult. To test if characters that have traditionally been accepted as informative for Anthroherponina classification are indeed reliable, we evaluated the monophyly of the most speciesrich genus of this subtribe - Anthroherpon Reitter, 1889. Our study, based on a molecular phylogenetic analysis of fragments of the 18S, 28S, and COI (both 5’ and 3’ end) loci revealed that the genus Anthroherpon as conventionally defined is polyphyletic. To resolve this polyphyly, we defined one new additional genus, Graciliella n. gen., for which we then examined the intrageneric diversity using molecular and morphometric approaches. Molecular phylogenetic analysis of two COI mitochondrial gene fragments revealed the presence of four species inside Graciliella n. gen., including two new species, which we here describe as G. kosovaci n. sp. and G. ozimeci n. sp. To analyze interspecific morphological differences within Graciliella we performed a discriminant analysis based on 40 linear morphometric measurements. The results showed that differences between species and subspecies inside Graciliella, however subtle they may seem, are measurable and reproducible. All species of the genus are briefly diagnosed, an identification key is proposed and a distribution map of all taxa of Graciliella is provided.

A Minimal Invasive Method to Forecast the Effects of Anthropogenic Disturbance on Tropical Cave Beetle Communities.

Many tropical landscapes are changing rapidly, with uncertain outcomes for biodiversity, landscape function, and the corresponding landscape services. Therefore, monitoring and adaptively managing the drivers and consequences of landscape change while sustaining the production of essential resources have become research and policy priorities. In this perspective, we have applied a recent framework, the stochastic dynamic methodology (StDM), with the purpose of understanding the effects of natural and anthropogenic disturbances on caves' integrity using cave beetle communities (Coleoptera) as ecological indicators. The proposed method was preceeded by a generalized linear model for discriminating significant relationships between the selected indicators, the structural changes in the caves, and the epigean habitats associated. The obtained results showed different ecological trends in response to the environmental changes. Overall, the simulation results seem to demonstrate the StDM reliability in determining the effects of habitat dynamics, that is, the expansion of agricultural activities, in areas near the caves in the structure of cave beetle communities. The applied method, based on universal information-theoretic principles, can be easily implemented and interpreted by environmental managers and decision makers, enabling anticipating impacts and supporting the development of measures aimed at minimizing the identified problems.

On the Way to Speciation: Shedding Light on the Karstic Phylogeography of the Microendemic Cave Beetle Aphaenops cerberus in the Pyrenees.

The highly modified morphology and ecological features of cave-dwelling organisms are a strong obstacle to dispersion. Hence, they represent ideal models for the study of historical biogeography at both large and fine timescales. Here, we study the phylogeography of Aphaenops cerberus, an endemic hypogean ground beetle with a fragmented distribution in the French Northern Pyrenees. We extracted 75 exemplars of 17 populations of A. cerberus and sequenced one mitochondrial and one nuclear marker to assess the geographic structuration as well as the recent biogeographic history of this species. We used Bayesian Inference and Maximum Likelihood to reconstruct the relationships among most of the extant populations of this species across its distributional range. We inferred divergence time estimates using carabid substitution rates and reconstructed haplotype networks to investigate the recent biogeographic history of this lineage. We recover a strong geographic structuration of the populations across the mountain range. The strong impact of geology on the structure of the populations is evidenced although geological continuity does not systematically lead to continual gene flow. The origin of the species is dated from the Early Pleistocene and the dispersal predates the main Last Glacial Maximum. Our results indicate broad similitudes between islands and karsts, which make cave organisms an excellent model for the study of evolution mechanisms.

Lack of evolutionary adjustment to ambient temperature in highly specialized cave beetles.

BackgroundA key question in evolutionary biology is the relationship between species traits and their habitats. Caves offer an ideal model to test the adjustment of species to their surrounding temperature, as they provide homogeneous and simple environments. We compared two species living under different thermal conditions within a lineage of Pyrenean beetles highly modified for the subterranean life since the Miocene. One, Troglocharinus fonti, is found in caves at 4-11°C in the ancestral Pyrenean range. The second, T. ferreri, inhabits the coastal area of Catalonia since the early Pliocene, and lives at 14-16°C.ResultsWe found no differences in their short term upper thermal limit (ca. 50°C), similar to that of most organisms, or their lower thermal limit (ca. -2.5°C), higher than for most temperate insects and suggesting the absence of cryoprotectants. In longer term tests (7 days) survival between 6-20°C was almost 100% for both species plus two outgroups of the same lineage, but all four died between 23-25°C, without significant differences between them.ConclusionsOur results suggest that species in this lineage have lost some of the thermoregulatory mechanisms common in temperate insects, as their inferred default tolerance range is larger than the thermal variation experienced through their whole evolutionary history.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-015-0288-2) contains supplementary material, which is available to authorized users.

Developmental constraints in cave beetles.

In insects, whilst variations in life cycles are common, the basic patterns typical for particular groups remain generally conserved. One of the more extreme modifications is found in some subterranean beetles of the tribe Leptodirini, in which the number of larval instars is reduced from the ancestral three to two and ultimately one, which is not active and does not feed. We analysed all available data on the duration and size of the different developmental stages and compared them in a phylogenetic context. The total duration of development was found to be strongly conserved, irrespective of geographical location, habitat type, number of instars and feeding behaviour of the larvae, with a single alteration of the developmental pattern in a clade of cave species in southeast France. We also found a strong correlation of the size of the first instar larva with adult size, again regardless of geographical location, ecology and type of life cycle. Both results suggest the presence of deeply conserved constraints in the timing and energy requirements of larval development. Past focus on more apparent changes, such as the number of larval instars, may mask more deeply conserved ontogenetic patterns in developmental timing.

High variability in the expression of circadian rhythms in a cave beetle population

Cave-dwelling organisms belong to widely different evolutionary lineages but they show parallel or convergent evolution in several morphological and physiological traits such as elongated appendages, lower metabolism, specialized sensory systems, loss of eye and pigmentation. Circadian rhythms in locomotor activity have already been documented in hypogean insects but ultradian rhythmicity in cave-dwelling organisms has been reported only by recent authors. We selected Laemostenus latialis as a good candidate to investigate the biological clock functioning in a species thought to be at its beginning of the process of cave evolution in the darkness. We based our analysis on inferential methods used here to obtain the quantitative resolution of the rhythm features. An automatic electronic device was developed to this purpose so to continuously monitoring the general locomotor activity of individual cave-dwelling beetles. We found significant locomotor rhythms to be present and that Laemostenus maintained a 24-h circadian rhythm but with a different amplitude. We observed two different and opposite behavioural patterns: the “Rhythmic group” showed a typical circadian rhythmicity of surface-dwelling animals with a surprisingly and extremely stable circadian period and with a restricted variance and a high amplitude. Differently, within the “arrhythmic group” we have observed an evanescent circadian period with high variability in tau, a larger variance and a lower amplitude. DFT spectral analysis showed the presence of ultradian rhythms with several significant peaks in the range 1–10 h and a main peak at 12 h, similar to those found in mysid crustaceans, cockroaches and mice. Ultradian rhythm looked independent from the circadian one and we did not observe any regularity. Further experimental studies are still needed to track the variation of this trait along the process of adaptation to cave life, particularly in closely related populations and species of troglophilic animals.

Life-history specialization was not an evolutionary dead-end in Pyrenean cave beetles

Research on subterranean organisms has focused on the colonization process and some of the associated phenotypic changes, but little is known on the long-term evolutionary dynamics of subterranean lineages and the origin of some highly specialized complex characters. One of the most extreme modifications is the reduction of the number of larval instars in some Leptodirini beetles from the ancestral 3 to 2 and ultimately a single instar. This reduction is usually assumed to have occurred independently multiple times within the same lineage and geographical area, but its evolution has never been studied in a phylogenetic framework. Using a comprehensive molecular phylogeny, we found a low number of independent origins of the reduction in the number of instars, with a single transition, dated to the Oligocene-Miocene, from 3 to 2 and then 1 instar in the Pyrenees, the best-studied area. In the Pyrenees, the 1-instar lineage had a diversification rate (0.22 diversification events per lineage per million years) significantly higher than that of 3- or 2-instar lineages (0.10), and similar to that seen in other Coleopteran radiations. Far from being evolutionary dead-ends, ancient lineages fully adapted to subterranean life seem able to persist and diversify over long evolutionary periods.

Ultrastructural analysis and polymorphisms in Coarazuphium caatinga (Coleoptera: Carabidae: Zuphiini), a new Brazilian troglobitic beetle.

Coarazuphium caatinga sp. n. occurs in limestone caves located in Campo Formoso municipality, in the Brazilian Caatinga (Bahia, Brazil). The new species is close to C. formoso although they are morphologically distinct by the elytra sinuosity, which is more pronounced in C. caatinga; the aedeagus is more tapered at the tip in this last species. Important traits found in C. caatinga are the variable size presented by the eyes, and the remarkable variability of body pigmentation among specimens; both traits do not seem to be correlated. Coarazuphium Gnaspini, P., Vanin, S.A. & Godoy, N.M., 1998, species exhibit advanced troglomorphic characters in comparison to other Brazilian cave beetles, as are increased extra-optic sensory structures, presence of particular sensilla, and sensory and gustatory receptors. These characters are not detected under routine microscopy and thus require ultrastructural methods for their study.

Application of molecular genetics and geometric morphometrics to taxonomy and conservation of cave beetles in central Italy

Integration of molecular genetic techniques and geometric morphometrics represent a valuable tool in the resolution of taxonomic uncertainty and the identification of significant units for conservation. We combined mitochondrial DNA cytochrome c oxidase subunit II gene sequence data and geometric morphometric analysis to examine taxonomic status and identify units for conservation in four species of the hypogean beetle Duvalius (Coleoptera, Trechinae) using mainly museum specimens collected in central Italy. Previous taxonomic studies based on morphological traits described several subspecies often inhabiting geographically distinct caves. Phylogenetic analysis identified two well supported monophyletic lineages and a number of different clades with relatively small genetic differences, suggesting a short divergence time in line with known geological history of the study area. Geometric morphometrics, on the other hand, recovered a high level of distinctiveness among specimens. Both genetic and morphometric analyses did not entirely corroborate former taxonomic nomenclature, suggesting possible rearrangements and the definition of evolutionary significant units. Beetles of the genus Duvalius are protected by regional laws and the majority of taxa considered in this study inhabit caves located outside protected areas. Our study advocates the importance of devoting protection efforts to networks of cave ecosystems rather than single locations or species.

A unique midgut-associated bacterial community hosted by the cave beetle Cansiliella servadeii(Coleoptera: Leptodirini) reveals parallel phylogenetic divergences from universal gut-specific ancestors

BackgroundCansiliella servadeii (Coleoptera) is an endemic troglobite living in deep carbonate caves in North-Eastern Italy. The beetle constantly moves and browses in its preferred habitat (consisting in flowing water and moonmilk, a soft speleothem colonized by microorganisms) self-preens to convey material from elytra, legs, and antennae towards the mouth. We investigated its inner and outer microbiota using microscopy and DNA-based approaches.ResultsAbundant microbial cell masses were observed on the external appendages. Cansiliella’s midgut is fully colonized by live microbes and culture-independent analyses yielded nearly 30 different 16S phylotypes that have no overlap with the community composition of the moonmilk. Many of the lineages, dominated by Gram positive groups, share very low similarity to database sequences. However for most cases, notwithstanding their very limited relatedness with existing records, phylotypes could be assigned to bacterial clades that had been retrieved from insect or other animals’ digestive traits.ConclusionsResults suggest a history of remote separation from a common ancestor that harboured a set of gut-specific bacteria whose functions are supposedly critical for host physiology. The phylogenetic and coevolutionary implications of the parallel occurrences of these prokaryotic guilds appear to apply throughout a broad spectrum of animal diversity. Their persistence and conservation underlies a possibly critical role of precise bacterial assemblages in animal-bacteria interactions.

Biological Clocks and Visual Systems in Cave-Adapted Animals at the Dawn of Speleogenomics

Cave-adapted animals are characterized by extreme reduction or complete absence of eyes, reflecting their lack of exposure to daylight. Given the overall constancy of abiotic variables in the cave environment, one would also expect the relaxation and eventual loss of circadian rhythms, and with it, the regressive evolution of the underlying genetic biological clock. Remarkably, however, recent behavioral and molecular studies converge with earlier evidence that the biological clock remains conserved in cave-adapted species. Reviewing the structural and behavioral data on the relationship between the visual system and the biological clock in cave species, I assess the potential of deep sequencing for elucidating their evolutionary conservation and adaptive significance in the subterranean fauna. The combined evidence confirms the widespread conservation both of the visual system and of the behavior regulated by the biological clock in cave species. The data from over 40 vertebrate and arthropod species further reveal that the absence of activity rhythms is correlated with the regression of the visual system. At the same time, the network of biological clock genes is likely to be generally conserved in cave species, regulating rhythmic behaviors in response to non-visual cycling variables as well as organismal homeostasis. Arrhythmic, eyeless species of cave beetles emerge as the most stringent choice for using deep-sequencing approaches to test and explore the conservation of the biological clock, independently of the visual system.

Structural and functional organization of the visual system in the microphthalmic cave beetle Ptomaphagus hirtus

Structural and functional organization of the visual system in the microphthalmic cave beetle Ptomaphagus hirtus

Coarazuphium tapiaguassu (Coleoptera: Carabidae: Zuphiini), a new Brazilian troglobitic beetle, with ultrastructural analysis and ecological considerations

Coarazuphium tapiaguassu sp. n. was collected in caves SL 30, SL 31 and SL 35 located in Curionópolis (Pará, Brazil). C. tapiaguassu is readily differentiated by the absence of setae on the dorsal surface of the head close to the posterior margin, as the other species of the gens show one, two or tree pairs of setae. Taxa of Coarazuphium exhibit advanced troglobiomorphic characters in comparison to other Brazilian cave beetles. Increased extra-optic sensory structures, presence of particular sensilla, and sensory and gustatory receptors are characters not detected under routine microscopy and thus require ultrastructural methods for their study. Similar analyses are needed in other epigean Zuphiini species for a better interpretation of their functional meaning.

Ultrastructural analysis of Coarazuphium formoso (Coleoptera: Carabidae, Zuphiini), a new Brazilian troglobitic beetle

It is described Coarazuphium formoso sp. n., from male and female specimens collected in Barriguda cave and Calor de Cima cave, both in the municipality of Campo Formoso (Bahia, Brazil). The most striking difference between C. formoso and other species of the genus is the presence of three pairs of setae on the dorsal surface of the head close to the posterior margin, while the other species have only one or two pairs. Species from this genus showed advanced troglobiomorphic characters in comparison to other Brazilian cave beetles. Characters as increased extra-optic sensory structures, and the presence of particular sensilla, and sensory and gustatory receptors were not detected under routine microscopy but required ultrastructural scrutiny. Similar analyses are needed in other epigean Zuphiini species for better interpreting their functional meaning.

Egg gigantism in some Australian earth‐borer beetles (Coleoptera: Geotrupidae: Bolboceratinae) and its apparent association with reduction or elimination of larval feeding

AbstractField and laboratory observations have revealed that four Western Australian bolboceratine beetle species (Blackburnium reichei (Guérin‐Méneville), Blackbolbus frontalis (Guérin‐Méneville), Bolborhachium inclinatum Howden and Bolborhachium trituberculatum (Bainbridge)) develop and lay one relatively gigantic egg at a time. Eggs of Bbu. reichei weighed up to 56% as much as the females that laid them. These observations accord with earlier reports of egg gigantism in two other Bolborhachium species. However, while those earlier reports noted that the eggs were deposited in large brood cells provisioned with a ‘humus‐like material’, no comparable provision was found in the present study (with the possible exception of Bbo. frontalis). Larvae of Bbu. reichei, Brh. trituberculatum and Blackbolbus hoplocephalus (Lea) and the pupa of the latter species are briefly described and illustrated for the first time. The final instar larva of Bbo. hoplocephalus was unusual in its globose body form, vestigial appendages and complete immobility. Evidence is presented which suggests that females of at least Bbu. reichei (and probably other species) deposit their eggs in comparatively small, bare, earthen cells and development to adulthood takes place in the absence of larval feeding. Comparisons are made with similar biology reported for some European cave beetles.

Baronniesia delioti gen. n. sp. n., a new subterranean Leptodirini from the French Pyrenees (Coleoptera: Leiodidae: Cholevinae)

Baronniesia delioti Fresneda, Bourdeau & Faille gen. n. & sp. n., tribe Leptodirini (Coleoptera, Leiodidae, Cholevinae), is described from the French central Pyrenees (Hautes-Pyrénées, Esparros, subterranean river of Artigaléou-Arodets). The genus is remarkable for the large body size and the strong sexual dimorphism in antennae of its type species. The structure of the aedeagus and the inner sac, with two additional strongly curved feather-like structures, forming an inverted “V”, and the lateral styles with only three spines without other structures, indicates affinities between the new genus and the Antrocharis group, diversified in the eastern part of the Pyrenean range. The limestone area in the central Pyrenees where this new genus was discovered appears to be a contact area between the eastern and western faunas of the Pyrenean cave beetles, both for Leiodidae: Leptodirini and Carabidae: Trechini.

Higher-order repeats in the satellite DNA of the cave beetle Pholeuon proserpinae glaciale (Coleoptera: Cholevidae).

The present study characterizes the satellite DNA of the cave beetle Pholeuon proserpinae glaciale which represent about 3-5 % of its genome, and which is composed of monomers of 266 bp and 70.5 % A-T. Concerted evolution seems to act on a higher-order repeat, a dimer, composed of two types of 266-bp monomers that differ in three diagnostic sites. These dimers show a striking nucleotide identity (98.7 % similarity) suggesting strong homogenization processes. The presence of particular mutations shared by several dimers represents an early expansion of these types of repeats as proposed by the molecular drive model. Moreover, evidence of gene conversion tracts in P. proserpinae glaciale, which also could be the result of unequal sister chromatid exchange, would suggest that recombination is involved in the homogenization of stDNA sequences. The presence of a 17-bp-motif repeated six times, along another one of 31-bp repeated twice which also have embedded one 17-bp-motif, suggest that monomers have originated from those basic motifs.