Rerouting of the lignin biosynthetic pathway by inhibition of cytosolic shikimate recycling in transgenic hybrid aspen.

Lignin is a phenolic polymer deposited in the plant cell wall, and is mainly polymerized from three canonical monomers (monolignols), i.e. p-coumaryl, coniferyl and sinapyl alcohols. After polymerization, these alcohols form different lignin substructures. In dicotyledons, monolignols are biosynthesized from phenylalanine, an aromatic amino acid. Shikimate acts at two positions in the route to the lignin building blocks. It is part of the shikimate pathway that provides the precursor for the biosynthesis of phenylalanine, and is involved in the transesterification of p-coumaroyl-CoA to p-coumaroyl shikimate, one of the key steps in the biosynthesis of coniferyl and sinapyl alcohols. The shikimate residue in p-coumaroyl shikimate is released in later steps, and the resulting shikimate becomes available again for the biosynthesis of new p-coumaroyl shikimate molecules. In this study, we inhibited cytosolic shikimate recycling in transgenic hybrid aspen by accelerated phosphorylation of shikimate in the cytosol through expression of a bacterial shikimate kinase (SK). This expression elicited an increase in p-hydroxyphenyl units of lignin and, by contrast, a decrease in guaiacyl and syringyl units. Transgenic plants with high SK activity produced a lignin content comparable to that in wild-type plants, and had an increased processability via enzymatic saccharification. Although expression of many genes was altered in the transgenic plants, elevated SK activity did not exert a significant effect on the expression of the majority of genes responsible for lignin biosynthesis. The present results indicate that cytosolic shikimate recycling is crucial to the monomeric composition of lignin rather than for lignin content.

Quantum approach for nanoparticle fluorescence by sub-ns photon detection.

Well defined detection and analysis of nanoparticle-sized samples such as extracellular vesicles or viruses may be important for potential disease diagnostics. However, using conventional flow-cytometry optical methods to evaluate such small particles is quite challenging. The reason is that the particle is smaller than the diffraction limit, making detection difficult. An alternative approach is fluorescence detection via conjugated fluorochromes attached to the nanoparticles; the challenge in this case is the limitation imposed upon detection of a very small number of emitted photons buried in high background photon counts. Emitted fluorescence is described by the well-known equation kf = σa I Q, which describes the emitted fluorescence rate (kf) (photons/s) as the multiplication of molecular absorption cross section(σa), excitation intensity (I), and quantum yield (Q). In addition, the excitation rate is equal to 1/t, which is the inverse of the lifetime of several ns representing the most typical conjugated fluorescent molecules used in flow cytometry. We recently developed a sub-ns photon sensor that is faster than most fluorescence lifetimes, since sub-ns speed is a critically important parameter for the separation of individual emitted photons. Based on our observation of fluorescence and background levels on typical commercial flow cytometers it is evident that a significant component of the background is induced by water-molecular vibrations. Therefore, understanding what constitutes all the components that contribute to the signals we measure in flow cytometry would help in defining what we currently call "background signals." We attempted to define a theoretical model to try to unravel these issues. This model was based on use of a reflective dry surface in the absence of water molecules. Our objective was to determine if it is possible to minimize background and enhance signal, and to provide valuable information on the contributing components of the signals collected. In order to test this model, we tested a single dried particle 50 nm in diameter on a reflective surface with minimum background. While this is clearly not a standard biological system, our results suggest that this quantum approach closely follows established photon base theory. Our goal was to define the parameters for practical nanoparticle-fluorescence analysis while enhancing our knowledge of the contribution of background properties.

Genetic Diversity of Ligidium Isopods in Hokkaido and Niigata, Northern Japan, Based on Mitochondrial DNA Analysis.

The genetic diversity of the genus Ligidium in Hokkaido and Niigata, northern Japan, was investigated by analyzing the cytochrome c oxidase subunit 1 (CO1) region in the mitochondrial DNA (mtDNA). The genetic diversity in Hokkaido was much lower than that in Niigata. Nine different operational taxonomic units (OTUs) were identified. Only a single OTU, most likely Ligidium japonicum, was found in Hokkaido, whereas all nine OTUs were found in Niigata. Using the mtDNA evolutionary rate determined for the marine invertebrate Haptosquilla pulchella (Miers, 1880), population expansion for OTU1 in Hokkaido was estimated to have occurred at 12,600 years BP, suggesting that Ligidium underwent a bottleneck due to glacial cooling, and the population then expanded after postglacial warming. Assuming that the expansion of the OTU1 population occurred at 9600 years BP, when the sea surface temperature rose offshore of Tokachi in the Northwestern Pacific, the evolutionary rate (µ) of the mtDNA CO1 region in Ligidium is calculated as: 0.087 (95% confidence intervals: min: 0.042-max: 0.12) (substitutions/site/million years). The presence of a haplotype common to Hokkaido and Niigata implies that the haplotype migrated across the Tsugaru Strait. Considering that geological evidence indicates that the Tsugaru Strait was continuously present even during the last glacial maximum when the sea level was at its lowest, accidental transport by human beings or animals might have been critical to the migration of Ligidium.

Phylogenetic diversity and distribution of bacterial and archaeal amoA genes in the East China Sea during spring.

Microbial nitrification is a key process in the nitrogen cycle in the continental shelf ecosystems. The genotype compositions and abundance of the ammonia monooxygenase gene, amoA, derived from ammonia-oxidizing archaea (AOA) and bacteria (AOB) in two size fractions (2-10 and 0.2-2 µm), were investigated in the East China Sea (ECS) in May 2008 using PCR-denaturing gradient gel electrophoresis (DGGE) and quantitative PCR (qPCR). Four sites were selected across the continental shelf edge: continental shelf water (CSW), Kuroshio branch water (KBW), transition between CSW and KBW (TCSKB) and coastal KBW (CKBW). The gene copy numbers of AOA-amoA were higher than those of AOB-amoA in ECS. The relative abundance of amoA to the total 16S rRNA gene level reached approximately 15% in KBW and CKBW for the free-living fraction of AOA, whereas the level was less than 0.01% throughout ECS for the AOB. A cluster analysis of the AOA-amoA-DGGE band pattern showed distinct genotype compositions in CSW in both the size fractions and in the surface of the TCSKB and KBW. Sequences of the DGGE bands were assigned to two clades. One of the clades exclusively consisted of sequences derived from the 2-10-µm fraction. This study revealed that AOA-amoA abundance dominated over AOB-amoA throughout the ECS, whereas the genotype composition of AOA-amoA were distributed heterogeneously across the water masses. Additionally, this is the first report showing the distribution of AOA-amoA genotypes characteristic to particle-associated AOA in the offshore of the East China Sea.

Identification and field attraction of the female sex pheromone of a kiwifruit pest, Nokona feralis (Lepidoptera: Sesiidae).

Female sex pheromone of a clearwing moth Nokona feralis (Leech) (Lepidoptera: Sesiidae), a pest of kiwifruit, was identified to be a 7:3 mixture of (3E,13Z)-3,13-octadecadienyl acetate (E3,Z13-18:OAc) and (3E,13Z)-3,13-octadecadien-1-ol (E3,Z13-18:OH) by GC-EAD and GC/MS analyses. Males were attracted to wide-range mixtures of E3,Z13-18:OAc and E3,Z13-18:OH, and a 7:3 mixture of those two compounds strongly attracted the males in the field.

Estimation of Evolutionary Rates of Mitochondrial DNA in Two Japanese Wood Mouse Species Based on Calibrations with Quaternary Environmental Changes.

Reliable estimates of evolutionary rates of mitochondrial DNA might allow us to build realistic evolutionary scenarios covering broad time scales based on phylogenetic inferences. In the present study, we sought to obtain estimates of evolutionary rates in murine rodents using calibrations against historical biogeographic events. We first assumed that land-bridge-like structures that appeared intermittently at glacial maxima with 100,000-year intervals shaped the divergence patterns of cytochrome b (Cytb) sequences (1140 bp) of the larger Japanese wood mouse Apodemus speciosus. The comparison of sequences from peripheral remote islands that are separated from one another by deep straits allowed us to estimate mitochondrial DNA evolutionary rates (substitutions/site/million years) to be 0.027 to 0.036, with presumed calibrations from 140,000, 250,000, 350,000, and 440,000 years ago. Second, we addressed rapid expansion events inferred from analyses of the Cytb sequences of the lesser Japanese wood mouse A. argenteus. We detected five expansion signals in the dataset and established three categories based on the expansion parameter tau values: 3.9, 5.6-5.7, and 7.8-8.1. Considering that the climate became warmer 15,000, 53,000, and 115,000 years ago after preceding periods of rapid cooling, we calculated evolutionary rates to be 0.114, 0.047, and 0.031, respectively. This preliminary concept of the evolutionary rates on a time scale from 15,000 to 440,000 years ago for the wood mouse should be refined and tested in other species of murine rodents, including mice and rats.

Hyperspectral cytometry.

Hyperspectral cytometry is an emerging technology for single-cell analysis that combines ultrafast optical spectroscopy and flow cytometry. Spectral cytometry systems utilize diffraction gratings or prism-based monochromators to disperse fluorescence signals from multiple labels (organic dyes, nanoparticles, or fluorescent proteins) present in each analyzed bioparticle onto linear detector arrays such as multianode photomultipliers or charge-coupled device sensors. The resultant data, consisting of a series of characterizing every analyzed cell, are not compensated by employing the traditional cytometry approach, but rather are spectrally unmixed utilizing algorithms such as constrained Poisson regression or non-negative matrix factorization. Although implementations of spectral cytometry were envisioned as early as the 1980s, only recently has the development of highly sensitive photomultiplier tube arrays led to design and construction of functional prototypes and subsequently to introduction of commercially available systems. This chapter summarizes the historical efforts and work in the field of spectral cytometry performed at Purdue University Cytometry Laboratories and describes the technology developed by Sony Corporation that resulted in release of the first commercial spectral cytometry system-the Sony SP6800. A brief introduction to spectral data analysis is also provided, with emphasis on the differences between traditional polychromatic and spectral cytometry approaches.

Formation of the ternary inclusion complex of limaprost with α- and ß-cyclodextrins in aqueous solution.

The inclusion mode of Limaprost in the presence of α- and ß-cyclodextrins (CDs) was investigated to gain insight into the stabilization mechanism of Limaprost-alfadex upon the addition of ß-CD in the solid state. The inclusion sites of α- and ß-CDs were studied by NMR spectroscopic and kinetic methods. With the addition of α- and ß-CDs, displacements in (13)C chemical shifts of prostaglandin F2α (PGF2α) were observed in the ω-chain and the five-membered ring, respectively, of the drug. Similar shift changes were observed with the addition of both α- and ß-CDs. In two-dimensional (2D) (1)H-NMR spectra, intermolecular correlation peaks were observed between protons of PGF2α and protons of both α- and ß-CDs, suggesting that PGF2α interacts with α- and ß-CDs to form a ternary complex by including the ω-chain with the former CD and the five-membered ring with the latter. In kinetic studies in aqueous solution, Limaprost was degraded to 17S,20-dimethyl-trans-Δ(2)-PGA1 (11-deoxy-Δ(10)) and 17S,20-dimethyl-trans-Δ(2)-8-iso-PGE1 (8-iso). The addition of α-CD promoted the dehydration to 11-deoxy-Δ(10), while ß-CD promoted the isomerization to 8-iso, under these conditions. In the presence of both α- and ß-CDs, dehydration and isomerization were also accelerated, supporting the formation of the ternary Limaprost/α-CD/ß-CD complex.

Effects of novel cathepsin K inhibitor ONO-5334 on bone resorption markers: a study of four sustained release formulations with different pharmacokinetic patterns.

The purpose of the study was clarify the effect of the cathepsin K inhibitor ONO-5334 on bone resortion markers using sustained release (SR) formulations with different pharmacokinetic (PK) patterns, and identify the optimal SR formulation. The PK profiles and pharmacodynamic effect on bone resorption markers of 4 SR candidates formulations were evaluated in healthy postmenopausal women within a randomized, 2-part, open-label crossover study. In Part A, subject received a single dose of each formulation orally in the fed state. In Part B, two selected formulations were evaluated in the fasted state. From the results from Part A, C(max) was reduced and plasma concentrations of ONO-5334 were sustained with all SR formulations compared with an immediate release tablet. In pharmacodynamics, the level of C-terminal telopeptide of type I collagen (CTX) in serum and urine were inhibited with SR tablets rather than with granules. C max and area under the concentration-time curve from time 0 to the last measurable time point (AUC(0-t)) of SR tablets were higher than those of granules. From Part B, C max in the fasted condition was lower than that in the fed condition with two SR tablets. In contrast, C(24 h) in the fasted condition was slightly higher than that in the fed condition, but AUC(0-t) was similar. The inhibitory effect on CTX in serum and urine may depend on the PK pattern of ONO-5334. The SR tablets was well tolerated in postmenopausal women and has the optimal SR profiles on pharmacodynamics effect on bone resortion markers and PK profile. These results suggest that SR tablets of ONO-5334 are an excellent drug candidate for osteoporosis.

Enantiospecific synthesis and filed evaluation of four stereoisomers of 10,14-dimethyloctadec-1-ene, a sex pheromone component secreted by female moths of the apple leafminer.

All four stereoisomers of 10,14-dimethyloctadec-1-ene, a sex pheromone component of the apple leafminer (Lyonetia prunifoliella: Lepidoptera), were synthesized starting from (R)- and (S)-propylene oxide by applying stereospecific inversion of chiral secondary tosylates as a key step. Field evaluation showed that male moths of the Japanese population were selectively attracted by the (10S,14S)-isomer and that the activity was not inhibited by the enantiomer.

Stabilizing effect of ß-cyclodextrin on Limaprost, a PGE1 derivative, in Limaprost alfadex tablets (Opalmon) in highly humid conditions.

Stabilization against humidity of Limaprost (a prostaglandin E1 derivative), which is currently marketed as Opalmon, was undertaken using ß-cyclodextrin (ß-CD). Aqueous solutions of Limaprost alfadex/dextran 40 were lyophilized with and without ß-CD. Limaprost alfadex lyophilized with ß-CD was more chemically stable in humid conditions than that without ß-CD. Moreover, the addition of ß-CD as an excipient to tablets of these lyophilized composites remarkably improved the stability of Limaprost, and Limaprost in this moisture-resistant formulation was chemically stable for 19 weeks at 30°C, 75% relative humidity (R.H.). Chemical analysis of Limaprost and its degradation products indicated that degradation proceeded in the inclusion form (i.e., within the CD cavity). Solid (2)H-NMR spectroscopic studies showed that ß-CD constrained the molecular mobility of water in the solid state. These results suggested that the stabilization of Limaprost by ß-CD was at least partly due to the restricted molecular mobility of water, which acted as a catalytic species for the degradation, and also to the protection of the five-membered ring of Limaprost from water catalytic dehydration through inclusion complex formation with ß-CD.

DeepLabCut-based daily behavioural and posture analysis in a cricket.

Circadian rhythms are indispensable intrinsic programs that regulate the daily rhythmicity of physiological processes, such as feeding and sleep. The cricket has been employed as a model organism for understanding the neural mechanisms underlying circadian rhythms in insects. However, previous studies measuring rhythm-controlled behaviours only analysed locomotive activity using seesaw-type and infrared sensor-based actometers. Meanwhile, advances in deep learning techniques have made it possible to analyse animal behaviour and posture using software that is devoid of human bias and does not require physical tagging of individual animals. Here, we present a system that can simultaneously quantify multiple behaviours in individual crickets - such as locomotor activity, feeding, and sleep-like states - in the long-term, using DeepLabCut, a supervised machine learning-based software for body keypoints labelling. Our system successfully labelled the six body parts of a single cricket with a high level of confidence and produced reliable data showing the diurnal rhythms of multiple behaviours. Our system also enabled the estimation of sleep-like states by focusing on posture, instead of immobility time, which is a conventional parameter. We anticipate that this system will provide an opportunity for simultaneous and automatic prediction of cricket behaviour and posture, facilitating the study of circadian rhythms.

Mass trapping of the diamondback moth (Plutella xylostella L.) by a combination of its sex pheromone and allyl isothiocyanate in cabbage fields in southern Vietnam.

A lure composed of (Z)-11-hexadecenal, (Z)-11-hexadecenyl acetate, and (Z)-11-hexadecen-1-ol at a ratio of 5 : 5 : 1 at a dose of 0.01 mg was optimal for the attraction of the Vietnamese strain of the diamondback moth (DBM). The combination of the sex pheromone with a plant volatile, allyl isothiocyanate, significantly increased the attraction of the pheromone trap. Females were also attracted, but they were only about 2% of all moths captured. In plots with 120-130 traps per ha, mass trapping with the combined lures reduced the DBM larval densities in cabbage fields as effectively as the spraying of insecticides 6 to 8 times. The weekly trap catches indicated that DBM adult densities in the mass-trapping fields were low until 28 days after transplantation, and then were kept to a modest increase until day 49. This field study also shows that the trap catches were well correlated with the DBM larval densities.

Bulk and In Situ Quantification of Coniferaldehyde Residues in Lignin.

Lignin is a group of cell wall localised heterophenolic polymers varying in the chemistry of the aromatic and aliphatic parts of its units. The lignin residues common to all vascular plants have an aromatic ring with one para hydroxy group and one meta methoxy group, also called guaiacyl (G). The terminal function of the aliphatic part of these G units, however, varies from alcohols, which are generally abundant, to aldehydes, which represent a smaller proportion of lignin monomers. The proportions of aldehyde to alcohol G units in lignin are, nevertheless, precisely controlled to respond to environmental and development cues. These G aldehyde to alcohol unit proportions differ between each cell wall layer of each cell type to fine-tune the cell wall biomechanical and physico-chemical properties. To precisely determine changes in lignin composition, we, herein, describe the various methods to detect and quantify the levels and positions of G aldehyde units, also called coniferaldehyde residues, of lignin polymers in ground plant samples as well as in situ in histological cross-sections.

Characterization of posticlure and the structure-related sex pheromone candidates prepared by epoxidation of (6Z,9Z,11E)-6,9,11-trienes and (3Z,6Z,9Z,11E)-3,6,9,11-tetraenes.

trans-11,12-Epoxy-(6Z,9Z)-6,9-henicosadiene (posticlure) has been identified from a pheromone gland of the lymantriid species, Orgyia postica. Since the diversity of Lepidoptera suggests that some species utilize the structure-related epoxy compound as a sex pheromone component, epoxydienes and epoxytrienes derived from (6Z,9Z,11E)-6,9,11-trienes and (3Z,6Z,9Z,11E)-3,6,9,11-tetraenes with a C19-C21 chain were systematically synthesized and the chemical data were accumulated in order to contribute to a new pheromone research. Peracid oxidation of each triene and each tetraene produced, respectively, a mixture of three epoxydienes (cis-6,7-epoxy-9,11-diene; cis-9,10-epoxy-6,11-diene; and trans-11,12-epoxy-6,9-diene) and four epoxytrienes (cis-3,4-epoxy-6,9,11-triene; cis-6,7-epoxy-3,9,11-triene; cis-9,10-epoxy-3,6,11-triene; and trans-11,12-epoxy-3,6,9-triene). While the 9,10-epoxy compounds were unstable and, interestingly, converted into 9-ketone derivatives after chromatography over SiO2, each positional isomer was isolated by HPLC equipped with an ODS column, and the chemical structure was determined by NMR analysis. On the GC-MS analysis with a DB-23 column, the positional isomers were also eluted separately and characteristic mass spectra were proposed. By comparing the spectral data of the epoxy compounds with a different carbon chain, diagnostic fragment ions reflecting the chemical structure were determined as follows: m/z 79, 109, 113, and M-114 for the 6,7-epoxydienes; m/z 69, 97, 111, 139, and M-111 for the 9,10-epoxydienes; m/z 57, 79, 109, 136, M-151, and M-111 for the 11,12-epoxydienes; m/z 79, 91, 105, and 119 for the 3,4-epoxytrienes; m/z 79, 124, M-124, M-96, and M-69 for the 6,7-epoxytrienes; m/z 79, 95, 109, 137, and M-108 for the 9,10-epoxytrienes; and m/z 79, 134, M-149, M-109, and M-95 for the 11,12-epoxytrienes.

Identification of the sex pheromone secreted by a nettle moth, Monema flavescens, using gas chromatography/fourier transform infrared spectroscopy.

The nettle moth Monema flavescens (Limacodidae) is a defoliator of fruit trees, such as Chinese plum and persimmon. The larvae of this species have spines containing a poison that causes serious irritation and inflammation in humans. Coupled gas chromatography-electroantennogram detection and gas chromatography/mass spectrometry analyses of a crude pheromone extract, combined with derivatization, indicated that female moths produced 8-decen-1-ol and 7,9-decadien-1-ol at a ratio of approximately 9:1. The E configuration of the double bonds was assigned for both components from infrared spectra, recorded on a gas chromatograph/Fourier transform-infrared spectrophotometer equipped with a zinc selenide disk cooled to -30 °C. The monoenyl and dienyl alcohols had absorptions characteristic of E geometry at 966 and 951 cm(-1), respectively. A band chromatogram at 951 cm(-1) was useful for distinguishing geometric isomers, because terminal conjugated diene are difficult to resolve, even on high polarity columns. Furthermore, we identified the Z configuration of the same 7,9-dienyl alcohol secreted by another nettle moth, Parasa lepida lepida, through the absence of this absorption. In field trials, lures baited with a 9:1 mixture of (E)-8-decen-1-ol and (E)-7,9-decadien-1-ol attracted M. flavescens males. Furthermore, the field trials indicated that contamination with the (Z)-diene reduced catches to the pheromone mixture more than did contamination with the (Z)-monoene.

Sex pheromones of three citrus leafrollers, Archips atrolucens, Adoxophyes privatana, and Homona sp., inhabiting the Mekong Delta of Vietnam.

Archips atrolucens, Adoxophyes privatana, and Homona sp. are serious defoliators of citrus trees in the Mekong Delta of Vietnam. In order to establish a sustainable pest-management program for the three species, their female-produced sex pheromones were investigated by GC-EAD and GC-MS analyses, and the following multi-component pheromones were identified: (Z)-11-tetradecenyl acetate (Z11-14:OAc), (E)-11-tetradecenyl acetate (E11-14:OAc), and tetradecyl acetate (14:OAc) in a ratio of 64:32:4 for A. atrolucens; Z11-14:OAc and (Z)-9-tetradecenyl acetate (Z9-14:OAc) in a ratio of 92:8 for A. privatana; and Z11-14:OAc and (Z)-9-dodecenyl acetate (Z9-12:OAc) in a ratio of 96:4 for Homona sp. Each lure baited with synthetic components as a mimic of the natural pheromone attracted males of the target species specifically, indicating that each monounsaturated minor component plays a significant role for mating communication and reproductive isolation of the three species inhabiting the same citrus orchards. In an extract of the pheromone glands of A. atrolucens females, the content of 14:OAc was very low, but a synergistic effect was observed clearly when the saturated compound was mixed at the same level as the E11-14:OAc. The synthetic lures will provide useful tools for monitoring flights of adults of the three species.

Defects in D-rhamnosyl residue biosynthetic genes affect lipopolysaccharide structure, motility, and cell-surface hydrophobicity in Pseudomonas syringae pathovar glycinea race 4.

D-rhamnose (D-Rha) residue is a major component of lipopolysaccharides (LPSs) in strains of the phytopathogen Pseudomonas syringae pathovar glycinea. To investigate the effects of a deficiency in GDP-D-rhamnose biosynthetic genes on LPS structure and pathogenicity, we generated three mutants defective in D-Rha biosynthetic genes, encoding proteins GDP-D-mannose 4,6-dehydratase (GMD), GDP-4-keto-6-deoxy-D-mannose reductase (RMD), and a putative α-D-rhamnosyltransferase (WbpZ) in P. syringae pv. glycinea race 4. The Δgmd, Δrmd, and ΔwbpZ mutants had a reduced O-antigen polysaccharide consisting of D-Rha residues as compared with the wild type (WT). The swarming motility of the Δgmd, Δrmd, and ΔwbpZ mutant strains decreased and hydrophobicity and adhesion ability increased as compared with WT. Although the mutants had truncated O-antigen polysaccharides, and altered surface properties, they showed virulence to soybean, as WT did.

History of the terrestrial isopod genus Ligidium in Japan based on phylogeographic analysis.

BACKGROUND: Phylogeographical approaches explain the genetic diversity of local organisms in the context of their geological and geographic environments. Thus, genetic diversity can be a proxy for geological history. Here we propose a genus of woodland isopod, Ligidium, as a marker of geological history in relation to orogeny and the Quaternary glacial cycle. RESULTS: Mitochondrial analysis of 721 individuals from 97 sites across Japan revealed phylogenetic divergence between the northeastern and southwestern Japan arcs. It also showed repeated population expansions in northeastern Japan in response to Quaternary glacial and interglacial cycles. Genome-wide analysis of 83 selected individuals revealed multiple genetic nuclear clusters. The genomic groupings were consistent with the local geographic distribution, indicating that the Ligidium phylogeny reflects its regional history. CONCLUSION: Ligidium DNA sequence analysis can provide insight into the geological, geographical, and paleoenvironmental history of the studied region.