Phylogenetic Considerations Research Articles

Nectary diversity in Brassicaceae in the context of phylogenetic considerations

Nectary diversity in Brassicaceae in the context of phylogenetic considerations

The Characterization of the Mitochondrial Genome of Fulgoraria rupestris and Phylogenetic Considerations within the Neogastropoda.

Fulgoraria rupestris is a predatory marine gastropod belonging to Neogastropoda and possessing considerable taxonomic significance. However, research on this species remains limited. We acquired the complete mitochondrial genome of F. rupestris through second-generation sequencing and conducted an analysis of its genome structural features. The mitochondrial genome of F. rupestris spans a total length of 16,223 bp and encompasses 37 genes (13 protein-coding genes (PCGs), 22 transfer RNAs, and 2 ribosomal RNAs). Notably, most tRNAs exhibit the typical cloverleaf structure, but there is an absence of the Dihydrouridine (DHU) arm in the trnS1 and trnS2 genes. The A + T content is 68.67%, indicating a pronounced AT bias. Additionally, we conducted a selection pressure analysis on the mitochondrial genomes of four species within Volutidae, revealing that all PCGs are subjected to purifying selection. In comparison to other species within Neogastropoda, F. rupestris shares an identical gene arrangement. Additionally, based on mitochondrial genome sequences of the 13 PCGs from 50 species within Neogastropoda, we constructed a phylogenetic tree. The phylogenetic tree indicates F. rupestris forms a clade with species within the family Volutidae (Cymbium olla, Neptuneopsis gilchristi, and Melo melo). This study serves as a valuable reference for future research on F. rupestris, offering insights for the upcoming phylogenetic and taxonomic classification within Neogastropoda. Furthermore, the findings provide valuable information for the development of genetic resources in this context.

Cofactor maturase NifEN: A prototype ancient nitrogenase?

Nitrogenase plays a key role in the global nitrogen cycle; yet, the evolutionary history of nitrogenase and, particularly, the sequence of appearance between the homologous, yet distinct NifDK (the catalytic component) and NifEN (the cofactor maturase) of the extant molybdenum nitrogenase, remains elusive. Here, we report the ability of NifEN to reduce N2 at its surface-exposed L-cluster ([Fe8S9C]), a structural/functional homolog of the M-cluster (or cofactor; [(R-homocitrate)MoFe7S9C]) of NifDK. Furthermore, we demonstrate the ability of the L-cluster-bound NifDK to mimic its NifEN counterpart and enable N2 reduction. These observations, coupled with phylogenetic, ecological, and mechanistic considerations, lead to the proposal of a NifEN-like, L-cluster-carrying protein as an ancient nitrogenase, the exploration of which could shed crucial light on the evolutionary origin of nitrogenase and related enzymes.

Larval ground plan of Platynectes Régimbart, 1879 reconsidered: description of the larvae of three species with phylogenetic considerations (Coleoptera: Adephaga, Dytiscidae)

In this contribution we describe and illustrate for the first time the larvae of three species of Platynectes Régimbart, 1879 (P. agallithoplotes Gustafson, Short & Miller, 2016, P. bakewelli (Clark, 1863), and P. reticulosus (Clark, 1863)) including detailed morphometric and chaetotaxic analyses of selected body structures in order to discover useful characters for distinguishing Platynectes larvae from those of other known Agabinae (Coleoptera: Dytiscidae). A parsimony analysis based on larval characteristics of 19 Agabinae species in six genera (Agabus Leach, 1817, Agabinus Crotch, 1873, Hydrotrupes Sharp, 1882, Ilybiosoma Crotch, 1873, Platynectes, and Platambus Thomson, 1859) was conducted using the program TNT. The results support Platynectes as a natural group whilst the six species studied split into two well supported clades: P. agallithoplotes + P. decemnotatus (Aubé, 1838) and P. curtulus (Régimbart, 1899) + P. davidorum Hájek, Alarie, Šťastný & Vondráček, 2019 + P. reticulosus + P. bakewelli. Larvae of P. agallithoplotes have proven to be characterized by a large number of unique character states, several of which were never observed until now in the Agabinae. Evidence is provided to the effect that the Neotropical species of Platynectes s. str. evolved as a unique lineage within Platynectes.

Erythrobacter oryzae sp. nov., isolated from rice paddy soil.

Two novel bacterial strains, designated as COR-2T and CR-8, were isolated from paddy soil. These isolates were aerobic, Gram-stain-negative, non-spore-forming, non-motile, rod-shaped, and formed orange-coloured colonies. Phylogenetic analysis based on 16S rRNA gene sequences showed that two strains formed a clear phylogenetic lineage with the genus Erythrobacter. Strains COR-2T and CR-8 showed 99.9 % 16S rRNA gene sequence similarity. Both strains had the highest 16S rRNA gene similarity of 99.1-99.7 % to Erythrobacter colymbi TPW-24T, Erythrobacter donghaensis SW-132T and Erythrobacter tepidarius DSM 10594T, respectively. The genome of strain COR-2T comprised 3 559 918 bp and the genomic DNA G + C content was 67.7 mol%. The average nucleotide identity and digital DNA-DNA hybridization values between strain COR-2T and its closely related species of the genus Erythrobacter were 79.3-85.5% and 24.1-29.1 %, respectively. The major respiratory quinone was Q-10, while the major fatty acids were C18 : 1 ω7c and C17 : 1 ω6c. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, two unidentified phospholipids and eight unidentified lipids. Based on phylogenetic and phenotypic considerations, the two strains [COR-2T (type strain; = KACC 22941T=JCM 35529T) and CR-8 (= KACC 22945=JCM 35530)] are considered to represent novel species of the genus Erythrobacter, for which the name Erythrobacter oryzae sp. nov. is proposed.

Dawn of the Delphinidans: New Remains of Kentriodon from the Lower Miocene of Italy Shed Light on the Early Radiation of the Most Diverse Extant Cetacean Clade.

Nowadays, the infraorder Delphinida (oceanic dolphins and kin) represents the most diverse extant clade of Cetacea, with delphinids alone accounting for more than 40% of the total number of living cetacean species. As for other cetacean groups, the Early Miocene represents a key interval for the evolutionary history of Delphinida, as it was during this time span that the delphinidans became broadly distributed worldwide, first and foremost with the widespread genus Kentriodon and closely related forms. Here, we report on a new odontocete find from Burdigalian (20.4-19.0 Ma) deposits of the Friulian-Venetian Basin of northeastern Italy, consisting of the partial cranium of a small delphinidan with associated ear bones (right periotic, stapes, malleus and tympanic bulla). Osteoanatomical considerations and comparisons allow us to assign the studied specimen to the genus Kentriodon. This is the first confirmed record of Kentriodon from Europe as well as from the whole proto-Mediterranean region. Stratigraphic and phylogenetic considerations suggest that our new specimen may represent the geologically oldest member of Kentriodon. The evolutionary success of Kentriodon may correlate with the emergence of narrow-band high-frequency echolocation as a possible strategy to escape acoustic detection by large marine predators such as the squalodontids. In addition, the relatively high encephalization quotient of Kentriodon spp. may have provided these early dolphins with some kind of competitive advantage over the coeval non-delphinidan odontocetes.

Characterization of Lophiotoma leucotropis Mitochondrial Genome of Family Turridae and Phylogenetic Considerations within the Neogastropoda.

Neogastropoda is a group of marine organisms with an extremely wide distribution that is rich in species and economic and ornamental values, the classification of species in this order has been ongoing for a long time, but there is still a great controversy about whether this order is monophyletic. In this study, we obtained the complete mitogenome of Lophiotoma leucotropis by next-generation sequencing and analyzed the basic structural features of the genome, and we found that the number of genes was consistent with that of most of the Neogastropoda snails, containing 37 genes, including 13 protein-coding genes (PCGs), 2 rRNAs, and 22 tRNAs. Analyzing base content, amino acid content, codon usage preference, and tRNA structure, the mitogenomes of eight species of Turridae were selected for analysis of selection pressures, and it was found that the evolution of species in this family was affected by purifying selection. In addition, by analyzing the rearrangement characteristics, it was found that the sequence of L. leucotropis was consistent with the Conoidea consensus order, and four of the eight species involved in the analysis showed rearrangements. Finally, we constructed a phylogenetic tree by combining PCGs of 60 species within Caenogastropoda and found Neogastropoda to be a monophyletic group, validating the results of morphological classification. The results will provide more references for the classification and species evolution of Neogastropoda, as well as phylogenetic analysis.

Description of eight new mitochondrial genomes for the genus Neoarius and phylogenetic considerations for the family Ariidae (Siluriformes).

The genus Neoarius, known as marine catfish, is a group of the family Ariidae, composed of 10 species found in Oceania. None of the species in this genus have their mitochondrial genome described, which is highly valuable in phylogenetic and molecular evolution studies. For the present work, eight species from the Neoarius genus were selected: Neoarius utarus, Neoarius midgleyi, Neoarius graeffei, Neoarius leptaspis, Neoarius berenyi, Neoarius paucus, Neoarius pectoralis, and Neoarius aff. graeffei. DNA sequences of the eight species were obtained through the NCBI Sequence Read Archive (SRA) database, and the mitochondrial genomes were assembled using the NOVOplasty tool on the Galaxy platform, subsequently annotated with the MitoAnnotator tool. We then utilized the protein-coding genes from the mitogenomes to estimate the phylogenetic relationships within the group, including seven additional mitogenomes available in the NCBI. In all species, the mitochondrial genomes presented 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and 1 D-loop.

An updated floristic map of the world

Floristic regions reflect the geographic organization of floras and provide essential tools for biological studies. Previous global floristic regions are generally based on floristic endemism, lacking a phylogenetic consideration that captures floristic evolution. Moreover, the contribution of tectonic dynamics and historical and current climate to the division of floristic regions remains unknown. Here, by integrating global distributions and a phylogeny of 12,664 angiosperm genera, we update global floristic regions and explore their temporal changes. Eight floristic realms and 16 nested sub-realms are identified. The previously-defined Holarctic, Neotropical and Australian realms are recognized, but Paleotropical, Antarctic and Cape realms are not. Most realms have formed since Paleogene. Geographic isolation induced by plate tectonics dominates the formation of floristic realms, while current/historical climate has little contribution. Our study demonstrates the necessity of integrating distributions and phylogenies in regionalizing floristic realms and the interplay of macroevolutionary and paleogeographic processes in shaping regional floras.

Novel Chaphamaparvovirus in Insectivorous Molossus molossus Bats, from the Brazilian Amazon Region.

Chaphamaparvovirus (CHPV) is a recently characterized genus of the Parvoviridae family whose members can infect different hosts, including bats, which constitute the second most diverse order of mammals and are described worldwide as important transmitters of zoonotic diseases. In this study, we identified a new CHPV in bat samples from the municipality of Santarém (Pará state, North Brazil). A total of 18 Molossus molossus bats were analyzed using viral metagenomics. In five animals, we identified CHPVs. These CHPV sequences presented the genome with a size ranging from 3797 to 4284 bp. Phylogenetic analysis-based nucleotide and amino acid sequences of the VP1 and NS1 regions showed that all CHPV sequences are monophyletic. They are also closely related to CHPV sequences previously identified in bats in southern and southeast Brazil. According to the International Committee on Taxonomy of Viruses (ICTV) classification criteria for this species (the CHPV NS1 gene region must have 85% identity to be classified in the same species), our sequences are likely a new specie within the genus Chaphamaparvovirus, since they have less than 80% identity with other CHPV described earlier in bats. We also make some phylogenetic considerations about the interaction between CHPV and their host. We suggest a high level of specificity of CPHV and its hosts. Thus, the findings contribute to improving information about the viral diversity of parvoviruses and show the importance of better investigating bats, considering that they harbor a variety of viruses that may favor zoonotic events.

Protecting biodiversity via conservation networks: Taxonomic, functional, and phylogenetic considerations

A key element of conservation action involves the incorporation of sites into networks of protected areas. Historically, most network-creation strategies have been based on considerations of species richness and site complementarity. Nonetheless, phylogenetic or functional biodiversity may be more critical to the maintenance of ecosystem resilience or functioning than is the number of species. Therefore, we explore the efficacy of three strategies (i.e., random, sequential, and simultaneous inclusion of sites into conservation networks of particular sizes) to maximize species richness in a network, and explore associated consequences to aspects of functional and phylogenetic biodiversity. We do so for passerines in Connecticut, bats in Paraguay, and trees in North Carolina, which differ in β, functional, and phylogenetic biodiversity. The efficacy of sequential and simultaneous strategies for conserving species richness are similar at all network sizes and represent improvements over random strategies for each of the three taxa, conserving all species in as few as 35 % of the sites required based on a random strategy. For aspects of functional and phylogenetic biodiversity, metrics converged on the value of the entire biota, even when networks contained as few as five sites, suggesting that richness-based approaches can be effective in guiding conservation action from multiple perspectives. Evaluation of networks intended to conserve biodiversity at spatial extents that include more complex environmental gradients than the examples presented here, or that comprise more heterogenous environments than those represented in our analyses, are needed to more fully explore the generality of our conclusions.

Characteristics of the complete mitochondrial genome of Douhua chicken (Gallus gallus) and phylogenetic considerations.

Douhua chicken is a unique local breed from Anhui Province, China. This study aimed to illustrate the Douhua chicken mitogenome and clarify its phylogenetic status by sequencing and annotating the complete mitochondrial genome using high-throughput sequencing and primer walking. Phylogenetic analysis through the Kimura 2-parameter model indicated the maternal origin of Douhua chicken. The results revealed that the mitochondrial genome is a closed circular molecule (16,785 bp) that consists of 13 protein-coding genes, 22 transfer RNA (tRNA) coding genes, two ribosomal RNA (rRNA) coding genes, and a control region. The base composition of the Douhua chicken mitogenome contains 30.3% A, 23.7% T, 32.5% C, and 13.5% G, and the haplotype and nucleotide diversity values are 0.829 (Hd) and 0.00441 (Pi), respectively. Furthermore, 10 haplotypes of D-loop sequences among 60 Douhua chickens were identified and distributed into four haplogroups (A, C, D, and E). Overall, the result of the present study indicates that Douhua chicken may have originated from Gallus gallus, and this process was influenced by Gallus gallus spadiceus, Gallus gallus murghi, and Gallus gallus bankiva. This study provides novel mitogenome data to support further phylogenetic and taxonomic studies on Douhua chicken. Additionally, the findings of this study will provide deeper insights for identifying the genetic relationships among populations and tracing maternal origins based on phylogenetic considerations for use in studies on the geographic conservation, utilization, and molecular genetics of poultry species.

The sexual determination system in Salix L. (Salicaceae)

Current knowledge of the sex determination system that has been analyzed in different species of the genus Salix L.is reviewed. In this genus, sexual chromosomes have been recognized and it was proposed that a gene for sexual determination must be present. In the genus Salix there are two systems of sexual determination: one in which the homogametic system is female and the heterogametic is male (XX/XY system) and another in which the homogametic system is male and the heterogametic is female (ZZ/ZW). The evidence is analyzed in relation to phylogenetic considerations and the need to include the analysis to species of other subgenera is raised. The role of the determination system in the hybridization of species with a mixed determination system is discussed, while it is suggested that this characteristic be investigated in the rest of the members of Salicaceae. Both sex determination systems (SDSs) are found in Salix and are of phylogenetic importance because each type of system is found in different phylogenetic groups. The SDS can influence the expression of the sexes in individuals, especially in polyploid species, and at a population level by influencing the sex ratio related to ploidy levels.

Morphology of ctenostome bryozoans: 6. Amphibiobeania epiphylla.

Ctenostome bryozoans are unmineralized and mostly marine. Their lack of calcified skeletal features requires other characters to be considered for systematic and phylogenetic considerations. As a continuation of an ongoing series of studies, we herein investigate the morphology of Amphibiobeania epiphylla, a unique bryozoan inhabiting mangrove leaves that are highly exposed to tidal cycles and regular dry events according to the tidal cycle. Besides this interesting mode of life, the species was originally interpreted to be a weakly mineralized cheilostome bryozoan, whereas molecular data place it among ctenostome bryozoans. To elucidate the systematic and phylogenetic position of the genus and also find morphological adaptations to an extreme habitat, we investigated the morphology of A. epiphylla in detail. Zooids show a lophophore with eight tentacles and a simple gut with a prominent caecum, lophophoral anusand most notably a distinct gizzard in the cardiac region. Gizzard teeth are multiple, simple homogeneous cuticular structures. The cuticle of the zooid is rather uniform and shows no respective thickenings into opercular flaps or folds. Likewise, apertural muscles are represented by a single pair of muscles. There are no specific closing muscles in the apertural area like the operculum occlusors of cheilostomes. Most prominent within zooids is a spongiose tissue filling most of the body cavity. Although not properly understood, this tissue may aid in keeping animals moist and hydrated during prolonged dry times. In summary, all morphological characters support a ctenostome rather than a cheilostome affinity, possibly with Vesicularioidea or Victorelloidea. In addition, we provide new molecular data that clearly supports such a closer relationship.

Correction to: Littoraria flava (Gastropoda: Littorinidae) mitogenome: phylogenetic considerations within the Caenogastropoda and evidence of microscale local adaptation

Correction to: Littoraria flava (Gastropoda: Littorinidae) mitogenome: phylogenetic considerations within the Caenogastropoda and evidence of microscale local adaptation

Comparative Mitogenomics of Two Sympatric Catfishes of Exostoma (Siluriformes: Sisoridae) from the Lower Yarlung Tsangpo River and Its Application for Phylogenetic Consideration.

The genus Exostoma is a group of stenotopic and rheophilic glyptosternine catfishes distributed in South and Southeast Asia. So far, comprehensive studies on mitogenomics referring to this genus are very scarce. In this study, we first sequenced and annotated the complete mitochondrial genomes of Exostoma tibetanum and Exostoma tenuicaudatum—two sympatric congeners from the lower Yarlung Tsangpo River, Tibet, China. The mitogenomes of both species contained 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes, one light-strand origin of replication, and one control region, with lengths of 16,528 bp and 16,533 bp, respectively. The mitogenome architecture, nucleotide composition, and codon usage of protein-coding genes were almost identical between the two Exostoma species, although some estimated parameters varied. Phylogenetic analysis strongly supported the monophyly of Exostoma in the subfamily Glyptosternae, and Exostoma tibetanum had the closest relationship to Exostoma tenuicaudatum. The divergence time estimation demonstrated that these two species diverged approximately 1.51 Ma during the early Pleistocene, which was speculated to be triggered by the river system changes caused by the uplift of the southeastern Tibetan Plateau. Selection pressure analyses indicated that all protein-coding genes of Exostoma species underwent a strong purifying selection, while minority positive sites from NADH dehydrogenase complex genes were detected. These findings are expected to promote our understanding of the molecular phylogeny of the genus Exostoma and provide valuable mitogenomic resources for the subfamily Glyptosternae.

Population genetics study of Strongyloides fuelleborni and phylogenetic considerations on primate-infecting species of Strongyloides based on their mitochondrial genome sequences

Strongyloides is a genus of parasitic nematodes of vertebrates comprising approximately 50 documented species, each with various host ranges. Among these, three species (S. stercoralis, S. fuelleborni, and S. cebus) are known to infect primate hosts. S. fuelleborni typically infects non-human primates in the Old World. To complement the existing information on the global genetic structure of this species, we conducted a genotyping study of S. fuelleborni samples collected from rhesus macaques in Myanmar, Japanese macaques in Japan, and some zoo-kept primates. This study identified a novel haplotype group in isolates from the Myanmar rhesus macaques. Subsequently, we obtained the complete or nearly complete mitochondrial genome sequences of S. fuelleborni, S. cebus (Strongyloides of New World monkeys), and S. vituli (Strongyloides of cattle). Phylogenetic analysis based on concatenated mitochondrial protein sequences of various Strongyloides species indicated a close relationship between S. fuelleborni, S. vituli and S. papillosus (Strongyloides in sheep and cattle). S. cebus is quite distantly related to both S. fuelleborni and S. stercoralis, which led to the hypothesis that the three primate Strongyloides species evolved independently as parasites of primates.

Arsenicicoccus cauae sp. nov., isolated from the blood of a pediatric gastroenteritis patient.

A Gram-stain-positive coccus was isolated from the blood of a paediatric patient suffering from gastroenteritis. The taxonomic position of this catalase-positive, non-motile, non-spore-forming facultative anaerobe designated as strain MKL-02T was investigated using a polyphasic approach. Colonies grown on tryptic soy agar with 10 % sheep blood were circular, creamy yellow, and convex. Phylogenetic analysis based on 16S rRNA gene and whole-genome sequences revealed that this strain was most closely related to Arsenicicoccus bolidensis CCUG 47306T within the cluster of the genus Arsenicicoccus. Average nucleotide identity and digital DNA-DNA hybridization values between strain MKL-02T and A. bolidensis DSM 15745T, A. dermatophillus DSM 25571T and A. piscis DSM 22760T were 89.5 and 37.0 %, 79.6 and 22.4 %, and 75.9 and 21.0 %, respectively. The genomic size of strain MKL-02T was 3 423 857 bp with a 72.7 mol% G+C content. Growth was observed at 10-45 °C (optimum, 37-40 °C) and pH 6.0-10.0 (optimum, pH 7.0), in the presence of 0-10 % (w/v) NaCl (optimum, 0.5 %). Cells of strain MKL-02T were non-motile cocci and 0.50-0.60 µm long, as determined by transmission electron microscopy. The strain was catalase-positive and oxidase-negative. The major fatty acid type (>10 % of total) was C15 : 0. The polar lipid profile consisted of two unidentified phospholipids, three unidentified lipids and an unidentified aminophospholipid. The strain contained MK-8 (H4) as the predominant menaquinone. Based on phylogenetic and phenotypic considerations, it is proposed that strain MKL-02T be classified as a new species, named Arsenicicoccus cauae sp. nov. The type strain is MKL-02T (=NCCP 16967T=JCM 34624T).

Second species of Langlieria (Tristichopteridae, Sarcopterygii) from the Upper Devonian Catskill Formation of Pennsylvania, U.S.A., and a new phylogenetic consideration of Tristichopteridae

A new species of Langlieria is described from the Upper Devonian (Frasnian) Irish Valley Member of the Catskill Formation. The type material was collected from a road cut exposure on the north side of the westbound lanes of Pennsylvania Route 322 west of Port Matilda, Centre County, Pennsylvania, U.S.A. The new species of Langlieria, the second from the Catskill Formation, is represented by high quality cranial material including parietal and postparietal shields, cheek, principal gular, and lower jaw; fin material; and body scales. The new species is the fourth tristichopterid to be described from the Catskill Formation (after Hyneria lindae, Langlieria radiatus, and Eusthenodon bourdoni) and the first from the Irish Valley Member. It is also the first species of Langlieria with a known record in the Frasnian. Sedimentological and stratigraphic data suggest that the discovery site represents a distal, tide-dominated location within the Catskill Delta system with a greater marine influence than has been reconstructed for the previously described Catskill Formation tristichopterids. A new phylogenetic analysis of Tristichopteridae is presented that is the first to include the new species of Langlieria described here and E. bourdoni from the Cogan House Exit Ramp locality (Famennian, Duncannon Member) along U.S. Route 15/Interstate 99. The 50% majority-rule consensus tree from the analysis supports a highly-nested clade of tristichopterids that includes all of the considered species from the Famennian, with the exception of Heddleichthys dalgleisiensis, and additionally includes the Frasnian Langlieria species that is described here.

The mitochondrial genome of Tremoctopus violaceus (Octopoda, Tremoctopodidae) and its phylogenetic consideration

The complete mitochondrial genome of Tremoctopus violaceus was sequenced to analyze its organization and phylogenetic status within the order Octopoda. The mitochondrial genome of T. violaceus had a structure and organization similar to that of other Octopoda. The content of the nucleotides A, C, G, and T was 31.68 %, 7.71 %, 20.02 %, and 40.58 %, respectively. All protein-coding genes (PCG) began with the ATG codon, excluding ND4 and ATP6, which began with ATC and ATT, respectively, and terminated with TAG, TAA, TA, or T. Codons for isoleucine were the most used codons, whereas those for arginine were used the least. Two extra tRNAs, trnN and trnL, were found in the control region. These tRNAs have a D-armless structure. The control region had excess A + T content (83.16 %) and a stem-loop structure with two elements, which is reported for the first time in Octopoda by our study. Bayesian inference using 13 PCG revealed that Octopus and Octopodidae were polyphyletic, and that Tremoctopodidae diverged relatively earlier within Octopoda. The mitochondrial genome of T. violaceus and its characteristics may help to understand the evolutionary history of Octopoda and establish a marine biodiversity conservation strategy.