Hidden Split in a Caucasus Rove Beetle Reveals Micro-Endemism and a Widespread Wolbachia Infection

Deep in the forest leaf litter of the North-Western Caucasus, a small predatory rove beetle has been quietly keeping a secret. Quedius obliqueseriatus, a wingless montane staphylinid endemic to this global biodiversity hotspot, has long been treated as a single species. A new integrative taxonomic study published in Alpine Entomology shows that this assumption hides a deeper truth: the beetle actually consists of two distinct allopatric lineages, a western and an eastern clade, separated by a mitochondrial COI p-distance of 4.0%. The discovery not only reveals a previously unnoticed micro-endemism pattern within an already endemic species but also uncovered a widespread Wolbachia infection that forced the researchers to design a clever laboratory workaround to recover the beetle’s own DNA barcode.

The North-Western Caucasus covers roughly 87,000 square kilometers of extraordinarily diverse terrain, from the lowland wetlands of the Kuban River to alpine peaks such as Mount Tsakhvoa at 3,346 meters. The region is recognized among the world’s biodiversity hotspots, and its beetle fauna is exceptionally rich in endemics: of the 1,939 beetle species recorded there, around 600 are confined to this area. Many occupy a single mountain range or a system of nearby ranges, with a sister species across a valley. Rove beetles that inhabit soil and leaf litter at various elevations are particularly rich in such narrowly distributed endemics, yet many remain unknown or poorly studied.

Quedius obliqueseriatus belongs to a presumably monophyletic group of apterous, narrowly distributed rove beetle species that also includes Q. humosus, restricted to low-elevation forests near the Black Sea coast, and Q. lgockii, confined to a few alpine localities above the timber line. Among these, Q. obliqueseriatus has the widest distribution, occurring in forest leaf litter from 200 to 600 meters in the foothills up to 1,600 to 1,800 meters at the timber line. But its habitus shows significant morphological variation: beetles differ in body size and in the coloration of the pronotum, elytra and abdomen, ranging from pale reddish to dark blackish. Even the aedeagus, the genitalic structure that clearly separates the three species of the group, displays seemingly continuous variation in the pattern of sensory peg setae on the paramere and in the shape of the median lobe apex.

Prompted by this unusually large distribution and pronounced morphological variation, the researchers set out to test the integrity of Q. obliqueseriatus as a species using both DNA and morphology. They examined 93 specimens, 55 males and 38 females, sampled from 29 localities across nearly the entire range of the species, drawing on collections of the Zoological Institute of the Russian Academy of Sciences in St. Petersburg and the University of Tyumen, along with a special 2022 field trip to the mountain foothills along the Russian Black Sea coast. Following the established practice in staphylinid species delimitation, which favors combining mitochondrial and nuclear gene fragments and checking their congruence with morphology, they targeted the mitochondrial COI barcode together with the nuclear markers H3, ITS1, ITS2, Wg and 28S.

The molecular work soon hit an unexpected obstacle. For 23 of the specimens, the standard COI barcoding primers HCO 3198 and LCO 1490, which amplify a 658-base-pair fragment of the cytochrome oxidase I gene, repeatedly picked up a sequence that BLAST searches identified as belonging to Wolbachia rather than to the beetle. Wolbachia are maternally inherited obligate intracellular alpha-proteobacteria of the order Rickettsiales and the most abundant and widespread intracellular bacteria on Earth. They manipulate host reproduction in ways that favor infected females, most famously through sperm-egg cytoplasmic incompatibility, in which matings between infected males and uninfected females produce few or no offspring while all other crosses remain fertile. Because Wolbachia is transmitted via the egg cytoplasm, the host’s maternally inherited mitochondrial DNA can hitchhike with the spreading bacterium, replacing mitochondrial haplotypes and typically lowering mitochondrial diversity in infected populations.

To confirm the infection, the team amplified a roughly 550-base-pair fragment of the Wolbachia surface protein gene wsp using the specific primer pair wsp81F and wsp691R in 13 of the infected specimens. But recovering the beetle’s own COI barcode from infected individuals required a more inventive approach. Comparing Wolbachia and Q. obliqueseriatus COI sequences with NEBcutter V2.0, the researchers found a SspI restriction site present in the Wolbachia sequence but absent in the beetle’s. They treated the cleaned PCR product with the SspI endonuclease restriction enzyme, which cuts the Wolbachia amplicon at the AATATT site while leaving the beetle fragment intact. Control electrophoresis on a 1% agarose gel yielded one clear band of approximately 600 base pairs, representing the uncut beetle COI, alongside two bands of roughly 300 base pairs from the severed Wolbachia fragment. Excising and sequencing the 600-base-pair band freed six infected specimens from the bacterial amplicon, bringing the total number of beetle COI sequences to 28.

The phylogenetic analysis of the COI data, performed under the GTR+G substitution model selected by the Akaike information criterion and run as a Bayesian analysis in MrBayes 3.2.7a for 10 million generations, revealed with strong statistical support that Q. obliqueseriatus consists of two clades, designated western and eastern. The two lineages are genetically divergent with a p-distance of 4.0%, a level of mitochondrial divergence comparable to interspecific distances reported in other rove beetle studies. Populations of the western clade stretch from around the villages of Pshada and Moldavanovka in the west to about Mount Semashkho and the villages of Shepsi and Druzhba in the east, while the eastern clade occupies the territory from around Vardane and Solokh-Aul to Vardane Verino and Krasnaya Polyana. Strikingly, the nuclear markers sampled from specimens of both clades showed no variation whatsoever, and the variation of non-genitalic morphological characters such as body size and coloration is continuous across both lineages.

Morphology did, however, offer a subtle clue to the hidden split. Most specimens of the western clade have the sensory peg setae on the underside of the parameral apex arranged in two irregular longitudinal groups with a distinct setae-free area between them, at least apically, whereas all sequenced specimens of the eastern clade show these rows indistinct, with the peg setae strongly diffuse and at most an unclear setae-free zone. The subapical tooth of the median lobe also tends to sit slightly farther from the apex in the western clade and slightly closer in the eastern, though this character varies more and does not show a distinct gap between the clades. These aedeagal differences, overlooked in the previous revision of the species, allowed additional specimens without COI data to be assigned to either clade by the shape of the paramere alone, although some specimens remain difficult to place morphologically.

Wolbachia infection proved to be widespread across the entire range of Q. obliqueseriatus, affecting specimens of both clades and both sexes with no appreciable associated morphological trait. Overall, 24% of the examined specimens were infected, comprising 13 males and 10 females. The researchers refrained from describing a new species for the eastern clade, which they consider a potential new species, because of the shortage of DNA-grade material from the geographic area where the western and eastern lineages meet, including localities such as Tatyanovka, Temnolesskaya, Vardane, Solokh-Aul and Babuk-Aul. There may be a zone of transition or co-occurrence between the two forms, and denser sampling is needed before species boundaries can be drawn with confidence. The unclear precise origin of the nineteenth-century type material collected by Hans Leder in Circassia further complicates the decision, though the lectotype’s clearly divided peg setae suggest it belongs to the western clade.

Beyond the practical laboratory lesson in removing a bacterial amplicon that hinders barcoding, the discovery carries a broader message about the patterns and drivers of endemism in the arthropods of the North-Western Caucasus. The ranges of the two clades roughly coincide with two geobotanical provinces of the western Transcaucasia, a drier Mediterranean-aspect western province and a more humid subtropical eastern one, hinting at an ecological dimension to their diversification. The high infection rate also raises the question of whether Wolbachia could have acted as a trigger for speciation, a process discussed for other infected insects in which symbiont strains encoding cytoplasmic incompatibility can create barriers to gene flow. The authors propose screening other narrowly distributed Caucasian endemics for Wolbachia and comparing infection rates with those of widespread species, a research program that may illuminate how much of the region’s celebrated micro-endemism still awaits discovery beneath the surface of morphology alone.

Subject of Research: Evolutionary biology, entomology, integrative taxonomy, Wolbachia, Caucasus biodiversity

Article Title: Micro-endemism pattern and Wolbachia infection of Quedius obliqueseriatus ( Coleoptera , Staphylinidae ), a montane rove beetle endemic of the North-Western Caucasus

Article References: Gebremeskel, A., Salnitska, M., Krivosheeva, V., & Solodovnikov, A. (2023). Micro-endemism pattern and Wolbachia infection of Quedius obliqueseriatus (Coleoptera, Staphylinidae), a montane rove beetle endemic of the North-Western Caucasus. Alpine Entomology, 7, 153-166. https://doi.org/10.3897/alpento.7.111214

Image Credits: AI Generated

DOI: 10.3897/alpento.7.111214

Keywords: Quedius obliqueseriatus, Wolbachia, micro-endemism, North-Western Caucasus, integrative taxonomy, COI barcoding, Staphylinidae, rove beetles, biodiversity hotspot, species delimitation, pattern, infection