Based on a dataset of 585 samples originating from across the Palearctic region and four genes (COI, CAD, Ca-ATPase, and 28S) for a subset of these samples, we studied the phylogenetic relationships within and between the Old World Swallowtail (Papilio machaon Linnaeus, 1758) and its closest relatives in the Palearctic region. We used Machine Learning (ML) to investigate the concordance of morphological characteristics with the molecular data. Our phylogenetic analyses showed that the Palearctic taxa everesti, archias and hippocrates formed strongly supported sister-group relationships with P. machaon. Within the Palearctic machaon a few distinct mitochondrial linages were observed, including one from the Himalayas and Central Asia, and another one that included all of the north African samples (taxa mauretanica, saharae and neosaharae) without any distinction. ML clustering was supportive of the haplotype and geographic analyses and a positive correlation was measured between average genetic phylogenetic and machine learnt specimen-image distances. Screening for Wolbachia revealed infection only in females of P. archias. We present a biogeographic scenario for the evolution of the P. machaon species group in the Palearctic region.

In the last decade, much attention is given to study of physiological amyloid fibrils. These structures include A-bodies, which are the nucleolar fibrillar formations that appear in response to acidosis and heat shock, and disassemble after the end of stress. One of the proteins involved in the biogenesis of A-bodies, regardless of the type of stress, is Von-Hippel Lindau protein (VHL). Known also as a tumor suppressor, VHL is capable to form amyloid fibrils both in vitro and in vivo in response to environment acidification. As for majority of amyloidogenic proteins fusion with different tags is used for increase of VHL solubility. Here, we first performed AFM-study of fibrils formed by VHL protein and by VHL fused with GST-tag (GST-VHL) at acidic conditions. It was shown that formed by full-length VHL fibrils are short heterogenic structures with persistent length of 2.400 nm and average contour length of 409 nm. GST-tag catalyzes VHL amyloid fibril formation, superimpose chirality, increases length and level of hierarchy, but decreases rigidity of amyloid fibrils. The obtained data indicate that tagging can significantly affect the fibrillogenesis of the target protein.

Natural aging and age-related diseases involve the acceleration of replicative aging, or senescence. Multiple proteins are known to participate in these processes, including the promyelocytic leukemia (PML) protein, which serves as a core component of nuclear-membrane-less organelles known as PML nuclear bodies (PML-NBs). In this work, morphological changes in PML-NBs and alterations in PML protein localization at the transition of primary fibroblasts to a replicative senescent state were studied by immunofluorescence. The fibroblasts were obtained from both healthy donors and donors with premature aging syndromes (ataxia-telangiectasia and Cockayne syndrome). Our data showed an increase in both the size and the number of PML-NBs, along with nuclear enlargement in senescent cells, suggesting these changes could serve as potential cellular aging markers. Bioinformatic analysis demonstrated that 30% of the proteins in the PML interactome and ~45% of the proteins in the PML-NB predicted proteome are directly associated with senescence and aging processes. These proteins are hypothesized to participate in post-translational modifications and protein sequestration within PML-NBs, thereby influencing transcription factor regulation, DNA damage response, and negative regulation of apoptosis. The findings confirm the significant role of PML-NBs in cellular aging processes and open new avenues for investigating senescence mechanisms and age-associated diseases.

The explosive growth in the number of works addressing the phase separation of intrinsically disordered proteins has driven both the development of new approaches and the optimization of existing methods for biomolecular condensate visualization. In this work, we studied the potential use of the fluorescent dye ANS as a sensor for liquid-liquid phase separation (LLPS), focusing on visualizing condensates formed by the stress-granules scaffold protein G3BP1. Using fluorescence lifetime imaging microscopy (FLIM), we demonstrated that ANS can accumulate in RNA-induced G3BP1 condensates in aqueous solutions, but not in G3BP1 condensates formed under macromolecular crowding conditions in highly concentrated PEG solutions. We showed that the experimentally determined limiting fluorescence anisotropy (r0'), which characterizes the amplitude of high-frequency intramolecular mobility of ANS in aqueous solutions containing RNA-induced G3BP1 condensates, is half the value observed for ANS in aqueous G3BP1 solutions. Our results demonstrate the feasibility of using time-resolved fluorescence spectroscopy and microscopy of ANS for detecting LLPS of intrinsically disordered proteins in aqueous solutions.

Renicolids are parasites of aquatic birds. Their species identification based on morphological characters is problematic. Here, we revised the composition of Renicola spp. parasitising anatids in nearshore areas of northern seas using integrated morphological and molecular data. We redescribed Renicola somateria and verified the diagnosis of R. mediovitellata. We established that the first intermediate host (FIH) of R. somateria is the mollusc Buccinum undatum, while the FIHs of R. mediovitellata are Nucella spp. molluscs. We described the intramolluscan stages of both species. Renicola somateria and R. mediovitellata formed a separate clade in the molecular trees of the Renicolidae. This finding confirms the existence of three main phylogenetic branches of renicolids, differing in the structure of adults, type of cercariae, and host range. Molecular data demonstrated an amphiboreal distribution of both R. somateria and R. mediovitellata. The former is represented by a single population in Europe and the North Pacific, while the latter forms separate populations in these regions. This may be because R. somateria actually uses not only B. undatum but also some other buccinid species with similar circum-Arctic ranges as the FIH. We discuss the roles played in the formation of digenean ranges by the vagility of the definitive host, the lifespan of the adults, and the distribution of the FIH.

The genus Colias Fabricius, 1807 includes numerous taxa and forms with uncertain status and taxonomic position. Among such taxa are Colias mongola Alphéraky, 1897 and Colias tamerlana Staudinger, 1897, interpreted in the literature either as conspecific forms, as subspecies of different but morphologically somewhat similar Colias species or as distinct species-level taxa. Based on mitochondrial and nuclear DNA markers, we reconstructed a phylogeographic pattern of the taxa in question. We recover and include in our analysis DNA barcodes of the century-old type specimens, the lectotype of C. tamerlana deposited in the Natural History Museum (Museum für Naturkunde), Berlin, Germany (ZMHU) and the paralectotype of C. tamerlana and the lectotype of C. mongola deposited in the Zoological Institute, Russian Academy of Sciences, St. Petersburg, Russia (ZISP). Our analysis grouped all specimens within four (HP_I–HP_IV) deeply divergent but geographically poorly structured clades which did not support nonconspecifity of C. mongola–C. tamerlana. We also show that all studied females of the widely distributed haplogroup HP_II were infected with a single Wolbachia strain belonging to the supergroup B, while the males of this haplogroup, as well as all other investigated specimens of both sexes, were not infected. Our data highlight the relevance of large-scale sampling dataset analysis and the need for testing for Wolbachia infection to avoid erroneous phylogenetic reconstructions and species misidentification.

At the molecular level, aging is often accompanied by dysfunction of stress-induced membrane-less organelles (MLOs) and changes in their physical state (or material properties). In this work, we analyzed the proteins included in the proteome of stress granules (SGs) and P-bodies for their tendency to transform the physical state of these MLOs. Particular attention was paid to the proteins whose gene expression changes during replicative aging. It was shown that the proteome of the studied MLOs consists of intrinsically disordered proteins, 30-40% of which are potentially capable of liquid-liquid phase separation (LLPS). Proteins whose gene expression changes during the transition of human cells to a senescent state make up about 20% of the studied proteomes. There is a statistically significant increase in the number of positively charged proteins in both datasets studied compared to the complete proteomes of these organelles. An increase in the relative content of DNA-, but not RNA-binding proteins, was also found in the SG dataset with senescence-related processes. Among SGs proteins potentially involved in senescent processes, there is an increase in the abundance of potentially amyloidogenic proteins compared to the whole proteome. Proteins common to SGs and P-bodies, potentially involved in processes associated with senescence, form clusters of interacting proteins. The largest cluster is represented by RNA-binding proteins involved in RNA processing and translation regulation. These data indicate that SG proteins, but not proteins of P-bodies, are more likely to transform the physical state of MLOs. Furthermore, these MLOs can participate in processes associated with aging in a coordinated manner.

Phytoparasitic mites of the superfamily Eriophyoidea Nalepa live and feed on mature leaf surfaces, between leaf bud scales, and (though less commonly) on flowers or fruits. In this study, we focused on the seasonal associations of two eriophyoid species, Shevtchenkella serrata (Nalepa 1892) with the Norway maple tree (Acer platanoides L.), and Brevulacus reticulatus Manson 1984 with the common oak (Quercus robur L.). These species have complex life cycles with two morphologically different, seasonal female forms, the protogyne and deutogyne. In B. reticulatus, both forms retain all the major generic characteristics but in S. serrata only the protogynes conform to the diagnosis of Shevtchenkella, whereas the deutogynes have the typical traits of Anthocoptes. We confirmed the conspecificity of the protogynes and deutogynes of both eriophyoid species by sequencing a barcode fragment of the Cox1 gene from which we obtained four pairwise identical sequences: ON920305/ON920306 (S. serrata) and ON920307/ON920308 (B. reticulatus). In addition, taxonomical studies on Shevtchenkella and Brevulacus resulted in new synonymies and combinations: (1) Oxypleurites obtusus Roivainen 1947 is considered a deutogyne of S. serrata and treated as a junior synonym of S. serrata; (2) two rhyncaphytoptine species from North America are transferred from the genus Rhyncaphytoptus to Brevulacus: B. albus (Keifer 1959) comb. nov. and B. atlanticus (Keifer 1959) comb. nov.; and (3) one species, B. salicinus Soika et al. 2017, is excluded from Brevulacus and transferred to Rhyncaphytoptus: Rhyncaphytoptus salicinus (Soika et al. 2017) comb. nov. Apart from distinct morphological deuterogyny in S. serrata and B. reticulatus, we observed the persistent association of S. serrata with the generative organs of the maple tree, A. platanoides, leading to transmission to the next host generation via the seed-containing winged fruits (samaras) and subsequent colonization of seedlings. In B. reticulatus, similar synchronization with host-plant dispersal was not detected; however, in mid-summer, temporary colonization of immature acorns and feeding was observed. Additional studies conducted in various ecosystems and including different ecological groups of plants, especially anemochorous plants, are needed to estimate the frequency of the association of eriophyoids with plant generative organs, seeds and seedlings to better understand what role in mite ecology such associations may play.

Eriophyoid mites of the genus Trisetacus Keifer are widespread parasites of conifers. A new oligophagous species, T. indelis n. sp., was discovered severely damaging seeds of North American junipers (Juniperus osteosperma, J. occidentalis, and J. californica) in the western USA. It has two codon deletions in the mitochondrial gene Cox1 rarely detected in Eriophyoidea and includes distinct morphological dimorphism of females. A phylogenetic analysis based on amino acid alignment of translated Cox1 sequences using a large set of out-groups (a) determined that two North American congeners, T. batonrougei and T. neoquadrisetus, were the closest known relatives of T. indelis n. sp., and (b) indicated that Old and New World seed-inhabiting Trisetacus from junipers do not form a distinct clade, suggesting a possible independent transition to living in seeds of junipers in America and Eurasia by Trisetacus spp. Our analysis produced a new topology consistent with a scenario assuming gradual reduction of prodorsal shield setation in Eriophyoidea and an ancient switch from gymnosperms to other hosts. Additionally, our analysis did not support monophyly of Trisetacus; recovered a new host-specific, moderately supported clade comprising Trisetacus and Nalepellinae (Nalepella + Setoptus) associated with Pinaceae; and questioned the monophyly of Trisetacus associated with Cupressaceae.

In this work, we performed a comparative study of the formation of PML bodies by full-length PML isoforms and their C-terminal domains in the presence and absence of endogenous PML. Based on the analysis of the distribution of intrinsic disorder predisposition in the amino acid sequences of PML isoforms, regions starting from the amino acid residue 395 (i.e., sequences encoded by exons 4–6) were assigned as the C-terminal domains of these proteins. We demonstrate that each of the full-sized nuclear isoforms of PML is capable of forming nuclear liquid-droplet compartments in the absence of other PML isoforms. These droplets possess dynamic characteristics of the exchange with the nucleoplasm close to those observed in the wild-type cells. Only the C-terminal domains of the PML-II and PML-V isoforms are able to be included in the composition of the endogenous PML bodies, while being partially distributed in the nucleoplasm. The bodies formed by the C-terminal domain of the PML-II isoform are dynamic liquid droplet compartments, regardless of the presence or absence of endogenous PML. The C-terminal domain of PML-V forms dynamic liquid droplet compartments in the knockout cells (PML−/−), but when the C-terminus of the PML-V isoform is inserted into the existing endogenous PML bodies, the molecules of this protein cease to exchange with the nucleoplasm. It was demonstrated that the K490R substitution, which disrupts the PML sumoylation, promotes diffuse distribution of the C-terminal domains of PML-II and PML-V isoforms in endogenous PML knockout HeLa cells, but not in the wild-type cells. These data indicate the ability of the C-terminal domains of the PML-II and PML-V isoforms to form dynamic liquid droplet-like compartments, regardless of the ordered N-terminal RBCC motifs of the PML. This indicates a significant role of the non-specific interactions between the mostly disordered C-terminal domains of PML isoforms for the initiation of liquid–liquid phase separation (LLPS) leading to the formation of PML bodies.

Paramecium (Ciliophora) systematics is well studied, and about twenty morphological species have been described. The morphological species may include several genetic species. However, molecular phylogenetic analyses revealed that the species diversity within Paramecium could be even higher and has raised a problem of cryptic species whose statuses remain uncertain. In the present study, we provide the morphological and molecular characterization of two novel Paramecium species. While Paramecium lynni n. sp., although morphologically similar to P. multimicronucleatum, is phylogenetically well separated from all other Paramecium species, Paramecium fokini n. sp. appears to be a cryptic sister species to P. multimicronucleatum. The latter two species can be distinguished only by molecular methods. The number and structure of micronuclei, traditionally utilized to discriminate species in Paramecium, vary not only between but also within each of the three studied species and, thus, cannot be considered a reliable feature for species identification. The geographic distribution of the P. multimicronucleatum and P. fokini n. sp. strains do not show defined patterns, still leaving space for a role of the geographic factor in initial speciation in Paramecium. Future findings of new Paramecium species can be predicted from the molecular data, while morphological characteristics appear to be unstable and overlapping at least in some species.

Plant photobodies are the membrane-less organelles (MLOs) that can be generated by protein-protein interactions between active form of phytochrome B (phyB) and phytochrome-interacting factors (PIFs). These organelles regulate plant photomorphogenesis. In this study, we developed two chimeric proteins with fluorescent proteins, phyB fused to EGFP and PIF6 fused to mCherry, and investigated their exogenous expression in mammalian cells by confocal fluorescence microscopy. Results showed that irradiation with diffused 630-nm light induced formation and subsequent increase in sizes of the MLOs. The assembly and disassembly of the photo-inducible MLOs in the mammalian cell cytoplasm obeyed the laws inherent in the concentration-dependent phase separation of biopolymers. The sizes of MLOs formed from phyB and PIF6 in mammalian cells corresponded to the sizes of the so-called “early” photobodies in plant cells. These results suggested that the first step for the formation of plant photobodies might be based on the light-dependent liquid-liquid phase separation of PIFs and other proteins that can specifically interact with the active form of phyB. The developed chimeric proteins in principle can be used to control the assembly and disassembly of photo-inducible MLOs, and thereby to regulate various intracellular processes in mammalian cells.

Natural hybridization is rather widespread and common in animals and can have important evolutionary consequences. In terms of taxonomy, exploring hybridization and introgression is crucial in defining species boundaries and testing taxonomic hypotheses. In the present paper, we report on natural hybrid specimens between Ahlbergia frivaldszkyi (Lederer, 1853) and Callophrys rubi (Linnaeus, 1758). To test the hypothesis of their hybrid origin, we employed the molecular mitochondrial (COI gene) and nuclear (wingless, RPS5, and Ca-ATPase genes) markers commonly used in phylogenetic studies and explored the morphology of the specimens. Our analysis revealed that hybrids bear mitochondrial haplotypes of C. rubi, while nuclear fragments are heterozygous, sharing a combination of A. frivaldszkyi and C. rubi lineages. The hybrid specimens combine morphological characters of both genera. Our results for the first time empirically demonstrate the possibility of genetic introgression between these species and between the genera Callophrys and Ahlbergia on the whole.

We report on a new phytoptid mite species, Oziella viscida n. sp., collected in Western Crimea from sea rush, Juncus maritimus (Juncaceae), and give supplementary descriptions of two rarely encountered nalepellid species of the genus Trisetacus from pines: T. confusus Livshits & Vasilieva, 1982 (in Vasilieva et al. 1982) from needle sheaths of Pinus nigra ssp. pallasiana (Pinaceae), an endemic subspecies restricted to Crimea, and T. brevisetus Livshits & Sekerskaya, 1982 (in Vasilieva et al. 1982) from needle sheaths of Pinus brutia ssp. pityusa (Steven) Silba, a relatively isolated subspecies of Turkish pine (P. brutia Tenore) growing in Georgia, Caucasus and Crimea. Oziella viscida n. sp. is remarkable in that most specimens were found inhabiting the basal part of leaves and stems of J. maritimus, an area covered by a transparent, sticky exudate apparently secreted by the plant epidermis. The mites were completely embedded in this substance and, rather than crawling with their legs, were observed moving through the viscous material while bending their opisthosoma in a serpentine or wormlike manner—an adaptation that appears to be currently unreported in eriophyoids and possibly reminiscent of locomotion of ancestral “protoeriophyoids” associated with soil. In comparison to females, males of O. viscida n. sp. and T. confusus have a more distinct prodorsal shield pattern consisting of a larger number of longer lines. Three new barcode gene sequences were obtained: MZ220550 (Cox1, O. viscida n. sp., 1159 bp), MZ224497 (18S, 2012 bp, T. brevisetus), and MZ224498 (18S, 2013 bp T. confusus). A BLAST search of the 18S sequences of T. brevisetus and T. confusus shows them as slightly closer to other 18S sequences of Trisetacus from Pinaceae (95.5%–96.3% identity) than to Trisetacus from Cupressaceae (93.6%–94.0% identity). Comparison of sequences of nalepellids currently present in GenBank suggest that a complete 18S sequence KJ841938.1 (2252 bp) from China belongs to an identified Trisetacus from Pinaceae rather than to Setoptus koraiensis as labelled, highlighting the necessity to review carefully the sequences of Eriophyoidea prior to using them in phylogenetic analyses, as well as the need to recollect and resequence S. koraiensis to clarify the nature of the problematic data from GenBank assigned to this species.

We report on one new species, Aceria asphodelinis n. sp., from inflorescences of Asphodeline taurica (Pall. ex Bieb.) Kunth (Xanthorrhoeaceae), one new combination, Paraphytoptus chondrillae (Canestrini 1891) n. comb. (transferred from Aceria), from deformed inflorescences of Chondrilla juncea L. (Compositae), and six new records of eriophyine mites (Eriophyoidea, Eriophyidae, Eriophyinae) in southeast Crimea: Aceria peucedani (Canestrini 1891) from flowers of Seseli tortuosum L. (Apiaceae), Aceria salviae (Nalepa 1891) from inflorescences of Salvia tesquicola Klokov & Pobed. (Lamiaceae), Aceria erinea (Nalepa 1891) and Aceria tristriata (Nalepa 1890) from leaf galls of Juglans regia L. (Juglandaceae), Eriophyes calycobius (Nalepa 1891) from buds of Crataegus monogyna Jacq. (Rosaceae), Eriophyes pyri (Pagenstecher 1857) from parenchymatous leaf galls (“blisters”) of Pyrus communis L. (Rosaceae). Partial mitochondrial Cox1 sequences (1155 bp, 385 amino acids) of six Aceria and Eriophyes species are provided (GenBank accession numbers: MW691978–MW691983; MW851284). Aceria salviae, previously reported as a gall-former, was found to be vagrant, causing no visible damage to the host plant. Data from the literature suggests that it may be quite common for conspecific eriophyoids to differ in their ability to cause galls on closely related hosts. We provide several hypotheses that may explain this phenomenon and highlight the need for testing them via field experiments as well as molecular methods. We also advocate for broader usage of barcoding techniques in the taxonomy of Eriophyoidea and underline the importance of increasing the number of publicly available barcodes for various eriophyoid taxa.

In this work, we put forward a hypothesis about the decisive role of multivalent nonspecific interactions in the early stages of PML body formation. Our analysis of the PML isoform sequences showed that some of the PML isoforms, primarily PML-II, are prone to phase separation due to their polyampholytic properties and the disordered structure of their C-terminal domains. The similarity of the charge properties of the C-terminal domains of PML-II and PML-VI isoforms made it possible for the first time to detect migration of PML-VI from PML bodies to the periphery of the cell nucleus, similar to the migration of PML-II isoforms. We found a population of “small” (area less than 1 µm2) spherical PML bodies with high dynamics of PML isoforms exchange with nucleoplasm and a low fraction of immobilized proteins, which indicates their liquid state properties. Such structures can act as “seeds” of functionally active PML bodies, providing the necessary concentration of PML isoforms for the formation of intermolecular disulfide bonds between PML monomers. FRAP analysis of larger bodies of toroidal topology showed the existence of an insoluble scaffold in their structure. The hypothesis about the role of nonspecific multiple weak interactions in the formation of PML bodies is further supported by the change in the composition of the scaffold proteins of PML bodies, but not their solidification, under conditions of induction of dimerization of PML isoforms under oxidative stress. Using the colocalization of ALT-associated PML bodies (APBs) with TRF1, we identified APBs and showed the difference in the dynamic properties of APBs and canonical PML bodies.

We report on one new cecidophyine species, Cecidophyes fibigiae n. sp. from Fibigia clypeata (L.) Medik. (Brassicaceae) and three new combinations and seven new records of phyllocoptine mites in Crimea: Abacarus hystrix (Nalepa 1896) from Elytrigia elongata (Host) Nevski (Poaceae), Aculodes cf holcusi Skoracka 2004 from Aegilops biuncialis Vis. (Poaceae), Aculops coronillae (Canestrini & Massalongo, 1893) n. comb. from Securigera varia (L.) Lassen (Fabaceae), Aculops longifilis (Canestrini 1891) n. comb. from Onobrychis miniata (Kit.) DC. (Fabaceae), Aculus sp. from Galium humifusum M.Bieb. (Rubiaceae), Anthocoptes recki (Bagdasarian, 1972) from Pistacia atlantica subsp. mutica (Fisch. & C.A. Mey.) (Anacardiaceae), and Anthocoptes thymi (Nalepa) n. comb. from Thymus callieri Borb. ex Velen. (Lamiaceae). Partial mitochondrial Cox1 sequences (about 1150 bp) of all found species were obtained (GB accession numbers MW439274–MW439282).

Phytoptidae s.str. is a lineage of eriophyoid mites associated with angiosperms. Based on representative taxon sampling and four gene markers (COI, HSP70, 18S, and 28S), we inferred the molecular phylogeny of this group and performed comparative analyses of cuticle-lined female internal genitalia. Although basal relationships were unclear, several well supported clades were recovered. These clades were supported by geography, host associations, and female genital anatomy, but contradicted the current morphology-based systematics. The monophyly of each of five conventional supraspecific groupings (Fragariocoptes, Phytoptus, Phytoptinae, Sierraphytoptinae, and Sierraphytoptini) is rejected based on a series of statistical tests. Additionally, four morphological characters (the absence of tibial solenidion φ and opisthosomal seta c1, presence of telosomal pseudotagma, and 'morphotype') were found to be homoplasies that cannot be used to confidently delimit supraspecific lineages of phytoptids. However, our molecular topology was highly congruent with female genital characters. Eight molecular clades were unambiguously supported by the shapes and topography of the spermathecal apparatus and genital apodemes. This suggests that the female genital anatomy could be an important factor affecting cladogenesis in Phytoptidae, a conclusion contrasting with the general expectation that host characteristics should be a major macroevolutionary force influencing the evolution of host-specific symbionts. Indeed, despite the high host-specificity, there were no apparent cophylogenetic patterns. Furthermore, we show that gall-inducing ability evolved multiple times in phytoptids. Because gall formation creates nearly instantaneous niche partitioning and the potential loss or reduction of gene flow, we hypothesize that it could be an important evolutionary factor affecting speciation within different host-associated clades of phytoptid mites.

We analyzed the phylogenetic relationships of the genus Trisetacus using two genes [cytochrome c oxidase subunit I (COI) and D1-D2 region of 28S rDNA (D1-D2 28S)], a representive taxon sampling (nearly 40% of known diversity), and a large set of close and distant outgroups. Our analyses suggest the presence of a dichotomy between Trisetacus associated with Cupressaceae and Pinaceae. The following smaller molecular clades were found: Pin-1 (bud mites, twig sheath mites, bark gall mites, and endoparasitic mites from pinaceans), Pin-2 (needle sheath mites from pines), Pin-2a (putative Nearctic group of needle sheath mites), Pin-2b (putative Palearctic group of needle sheath mites), Cup-1 and 2 (bud, cone, seed mites and mites living under bark scales from cupressaceans). The monophyly of the recently proposed subgenus Brevithecus nested within clade Cup-2 was confirmed. Ancestral character reconstruction analyses recovered: (1) Pinaceae as the ancestral hosts of Nalepellidae and Trisetacus, (2) repetitive reductions of the spermathecal tube independently occurred in two lineages of Trisetacus from Cupressaceae, and (3) several mite habitats on host (galls, cones, twig sheaths, seeds, inside leaves, and under scales) are evolutionarily derived states, whereas living in buds or needle sheaths are ancestral states for Trisetacus clades Cup and Pin. Using confocal microscopy, we identified six basic types of the female internal genitalia of Trisetacus based on shapes of the spermatheca and spermathecal tube. These genitalic types are strongly correlated with lineages recovered by molecular phylogenetic analyses, suggesting that the female genital morphology is both evolutionarily conserved and is a factor influencing macroevolutionary patterns in this group of mites.