Plesiochorus irwinorum n. sp. (Trematoda: Gorgoderidae) from the urinary bladder of the hawksbill turtle, Eretmochelys imbricata (Testudines: Cheloniidae), off the east coast of Australia

Plesiochorus Looss, 1901 is a genus of Gorgoderidae infecting the urinary bladders of marine turtles globally. Currently, just two morphologically similar species are recognised, Plesiochorus cymbiformis (Rudolphi, 1819) Looss, 1901 and Plesiochorus elongatus Pigulevsky, 1953, which have been distinguished by molecular data and subtle morphological differences. Here we describe a new species, Plesiochorus irwinorum n. sp., infecting hawksbill turtles (Eretmochelys imbricata (L.)), which is primarily distinguished from the other two species of Plesiochorus on the basis of ITS2, cox1 and 28S sequence data. Morphometric data for specimens examined during this study overlap between P. cymbiformis and P. irwinorum n. sp. for every measured feature, rendering them functionally cryptic. However, principal components analysis clearly distinguishes the two species. Additionally, we report new specimens of P. cymbiformis, and provide new sequence data for specimens from Australian loggerhead (Caretta caretta (L.)) and hawksbill turtles. There is little understanding of the host-specificity or geographical distribution of the three species of Plesiochorus, and it remains possible that some of the previously reported sequences have been attributed to the wrong species.


Introduction
The Gorgoderidae Looss, 1899 is a relatively large family of trematodes that infect a wide range of freshwater and marine vertebrates as definitive hosts, including amphibians, chondrichthyans, teleosts and reptiles (Campbell, 2008). In chondrichthyans, gorgoderids infect the body and pericardial cavities, whereas those of amphibians, teleosts, and reptiles are typically reported from the urinary bladder or rarely, the gall bladder. Only two genera, Bicornuata Pearse, 1949 andPlesiochorus Looss, 1901, have been reported from marine turtles. The sole species of the monotypic genus Bicornuata, Bicornuata caretta Pearse, 1949, infects the gall bladder of Caretta caretta (L.). The two known species of Plesiochorus infect the urinary bladder of marine turtles. Plesiochorus cymbiformis (Rudolphi, 1819) Looss, 1901 is reported from C. caretta, Chelonia mydas (L.), This article was registered in the Official Register of Zoological Nomenclature (ZooBank) as urn:lsid:zoobank.org:F8BA990D-D9E2-4C06-B764-F3FFD4A3CAA8. This article was published as an Online First article on the online publication date shown on this page. The article should be cited by using the doi number.
Plesiochorus has been shown to be a phylogenetically stable genus within the subfamily Anaporrhutinae Looss, 1901, with the two known species forming a well-supported monophyletic clade based on ITS2 data (Werneck et al., 2019). The type species, P. cymbiformis, was originally described from the urinary bladder of C. caretta from the Adriatic Sea (Rudolphi, 1819) and has subsequently been reported from off Australia, Brazil, Costa Rica, Cuba, Florida, USA, Gulf of Panama, India, the Mediterranean Sea, Mexico, Morocco, New Guinea, Pakistan, Puerto Rico, and the Red Sea (see Blair & Limpus, 1982;Santoro & Morales, 2007;Werneck et al., 2019). Until recently, P. elongatus, had been considered a subspecies of P. cymbiformis, recognised as Plesiochorus cymbiformis elongatus Pigulevsky, 1953. However, recent molecular studies provided evidence to suggest that P. elongatus is a distinct species, known from off the coasts of Brazil and Egypt (Werneck et al., 2019). The morphological differences between P. cymbiformis and P. elongatus are subtle; P. elongatus is reported to have a generally more elongate body than P. cymbiformis, and uterine coils which are closer to the body margin (Werneck et al., 2019). However, gorgoderids have been reported to exhibit striking morphological variation, naturally and as a result of different fixing methods, leading to difficulties in the delineation of species on the basis of morphology alone (Bakke, 1988;Namuleno & Scholz, 1994;Cutmore et al., 2010).
Here, we report gorgoderids infecting C. caretta and E. imbricata from off the east coast of Australia, describe a new species of Plesiochorus, and comment on the issues of morphological variation within the genus.

Specimen collection
Between December 2019 and October 2021, the urinary and gall bladders of specimens of deceased turtles (C. caretta, C. mydas, E. imbricata and L. olivacea) were examined during necropsies at Australia Zoo Wildlife Hospital (Beerwah, QLD, Australia) and Dolphin Marine Conservation Park (Coffs Harbour, NSW, Australia). Turtles were collected from multiple localities along the east coast of southern Queensland and northern New South Wales, Australia before transportation to rehabilitation facilities (specific localities listed in relevant taxonomic summaries). Urinary bladders were removed from the host, placed in a Petri dish with 0.85% saline solution, split with a single incision and grossly examined for the presence of trematodes. Gall bladders were examined in combination with liver examination, in which the liver and gall bladder were sliced into sections *5cm thick, placed into a container and washed with 0.85% saline solution. The solution was left to settle, and three-quarters of the supernatant was discarded. If needed, clean 0.85% saline solution was added to the wash container, left to settle, and the supernatant was discarded again. When sufficiently clean, the sediment was examined in a Petri dish under a dissection microscope. Trematodes were killed and fixed without pressure in near boiling 0.85% saline solution and immediately transferred to 70% ethanol for parallel morphological and molecular characterisation.

Morphological analysis
Specimens for morphological analysis were washed with fresh water, stained with Mayer's haematoxylin, destained in 1.0% HCl solution, neutralised in 0.5% NH 3 (aq), dehydrated in a graded ethanol series, cleared with methyl salicylate, and permanently mounted in Canada balsam on microscope slides. Measurements of specimens were taken using cellSens Standard imaging software (Olympus, Tokyo, Japan) with an Olympus SC50 digital camera mounted on an Olympus BX-53 compound microscope. All measurements are in micrometres (lm) and are presented as a range with the mean in parentheses. Drawings of specimens were completed with the aid of a drawing tube mounted to the same Olympus BX-53 compound microscope, and digitalised with Adobe Illustrator CC 2022.
Morphometric data for samples collected in the present study were natural log-transformed and examined using a principal components analysis (PCA) of the covariance matrix in RStudio v.1.3.959 (http:// www.rstudio.com/), and visualised using the package ggfortify (Tang & Horikoshi, 2016). Features included in the PCA are noted in Table 1. Specimens were assigned to a species in the PCA by either i) genetic data from hologenophores included in the PCA or ii) whole worms from C. caretta were inferred to represent P. cymbiformis as extensive sequencing showed just one species present in that host.
Species delineation was based on an integrative interpretation of morphological and genetic data, following the criteria of trematode species recognition proposed by Bray et al. (2022) (i.e. reciprocal monophyly in the most discriminating available molecular marker (here, ITS2 and cox1) ? distinction in morphology and/or host range).

Phylogenetic analysis
Newly generated cox1 and ITS2 sequences were aligned separately with sequences from GenBank (Table 2) using MUSCLE in MEGAX (Kumar et al., 2018), with UPGMA clustering for iterations 1 and 2. Indels greater than three base pairs affecting [5% of sequences were removed. To determine the correct reading frame in the cox1 dataset, the aligned data was translated in MESQUITE v.3.6 (Maddison & Maddison, 2021) using the echinoderm/flatworm mitochondrial code, inspected for stop codons and trimmed to start on position one. All three codons were tested for substitution saturation in DAMBE7 (Xia et al., 2003;Xia & Lemey, 2009;Xia, 2018), with no significant substitution saturation detected. Neighbour-joining (NJ) analyses were conducted independently on the aligned cox1 and ITS2 datasets in MEGA X to determine species identities. The parameters for the NJ analyses were: ''test of phylogeny = bootstrap'', ''no. of bootstrap replications = 10,000'', ''model/ method = No. of differences'', ''substitutions to include = d: Transitions ? Transversions'' and ''rates among sites = Uniform rates''. Pairwise distance matrices were generated in MEGA X to determine intra-and interspecific variation within the cox1 and ITS2 datasets. The parameters used for the pairwise distances matrices were the same as those used in the NJ analyses. Phyllodistomum pacificum Yamaguti, 1951 and Phyllodistomum vaili Ho, Bray, Cutmore, Ward & Cribb, 2014 were designated as the function outgroup in the ITS2 NJ analysis based on a preliminary analysis of the 28S dataset.

Data accessibility
The aligned and trimmed ITS2, cox1 and 28S datasets, raw morphometric data, and r code used in this study have been uploaded to the Mendeley data repository at https://doi.org/10.17632/j4hzhpjct9.1.

General results
The urinary and gall bladders of seven C. caretta, 76 C. mydas, 11 E. imbricata and one L. olivacea were examined. Specimens conforming morphologically to the genus Plesiochorus were found in the urinary bladders of three C. caretta and two E. imbricata. No gorgoderids were recovered from the urinary bladders of C. mydas or L. olivacea, nor were they recovered from any of the examined gall bladders. ITS2 sequence data were generated for nine individuals from two of the three infected C. caretta. All newly generated sequence data are 447 bp in length, identical, match sequence data available in GenBank reported for P. cymbiformis from the USA (KC494054), and are similar to sequence data available in GenBank for P. cymbiformis reported from Brazil (KC578463, differing by a single bp) (Fig. 1A). The aligned and trimmed ITS2 dataset yielded 401 bp for analysis. ITS2 sequence data generated for five individuals collected from the two infected E. imbricata form two clades in the NJ analysis, differing at eight bp (1.7%). One clade matches the new and existing sequence data generated for individuals infecting C. caretta, identified as P. cymbiformis; the second clade does not match any available ITS2 sequence data. ITS2 sequence data for new collections of P. cymbiformis differ from that available in GenBank for P. elongatus by 10 bp (4.1%), while those for the second clade differ from P. elongatus by 14 bp (5.8%).
cox1 sequence data were generated for 10 individuals collected from all three of the infected C. caretta. The 10 sequences are 474 bp in length, vary by up to five bp, and do not match any sequence data available in GenBank. cox1 sequence data generated for 11 individuals collected from the two infected E. imbricata again are 474 bp in length, form two distinct clades in the NJ analysis (Fig. 1B), differing at up to 8% (38 bp), neither of which match sequence data available in GenBank. Two sequences form a strong clade with the newly generated sequences from C. caretta, here identified as P. cymbiformis. The second, more common clade, has intraclade variation of just one base pair. Considering the consistent differences in both the ITS2 and cox1 data, we consider the second clade to represent a species new to science and formally describe it below. Additionally, we report the type-species of Plesiochorus, P. cymbiformis, from Australian C. caretta and E. imbricata.
Family Representative DNA sequences: cox1 mtDNA: nine sequences (two submitted to GenBank ON062315-062316); ITS2 rDNA: four sequences (one submitted to GenBank ON062960); 28S rDNA: one sequence (submitted to GenBank ON062963) ZooBank registration: The Life Science Identifier (LSID) for Plesiochorus irwinorum n. sp. is urn:lsid:zoobank.org:act:43312A28-C4B9-4E3E-B7ED-7AF0458E262F Etymology: The specific name irwinorum is in honour of the Irwin family of Australia Zoo, Queensland, Australia, who established the Australia Zoo Wildlife Hospital to support animal welfare, conservation, rehabilitation, and scientific research.
Testes two, large, deeply lobed, generally of similar size, opposite, in middle of body. Vas efferens long, slender, leading from centre of testes to posterior end of seminal vesicle, following margin of ventral sucker. Vas deferens absent. Seminal vesicle elongate, convoluted, anterodorsal to ventral sucker, leading to prostatic chamber. Prostatic chamber a distinct widening of seminal vesicle, surrounded by prostatic cells, ventral to seminal vesicle. Cirrus-sac absent. Ejaculatory duct short. Genital pore medial, ventral, close to anterior margin of ventral sucker.
Ovary deeply lobed, submedial, amphitypic to midline, ventral to uterus and testes when overlapping, mostly anterior to testes, posterior to ventral sucker. Vitellarium in two, symmetrical, deeply lobed, pretesticular masses, generally of similar size, mostly posterior to ventral sucker, slightly anterior to ovary, ventral to caeca. Mehlis' gland ovoid, medial to submedial at level of ovary, ventral to uterus. Seminal receptacle roughly spherical to ovoid, anterodorsal to ovary, amphitypic to midline, immediately posterior to ventral sucker. Laurer's canal dorsal to seminal receptacle, leading to pore on dorsal surface of body.
Uterus extensive, filling posterior third of body, intraand extracaecal posterior to testes, intracaecal anterior to and at level of testes, reaches close to margin of body in large specimens. Eggs ovoid, smallest at formation, gradually increasing in size along uterus, largest just posterior to genital pore.
Plesiochorus cymbiformis (Rudolphi, 1819) Looss, 1901 Synonyms Voucher-material: Six specimens deposited in the QM, four from Caretta caretta (G240071-240074) and two from E. imbricata (G240069-240070) Representative DNA sequences: cox1 mtDNA: 12 sequences (eight submitted to GenBank; six from Caretta caretta ON062308-062313; two from Eretmochelys imbricata ON062307 and ON062314); ITS2 rDNA: 10 sequences (two submitted to GenBank; one from Caretta caretta ON062958; one from Eretmochelys imbricata ON062959); 28S rDNA: two sequences (both submitted to GenBank; one from b Figure 1 Phylogenetic relationships between anaporrhutine gorgoderids sequenced during this study and those available on GenBank, inferred from (A) rooted neighbour-joining analysis of the ITS2 dataset and (B) unrooted neighbour-joining analysis of the cox1 dataset. (A) Taxa in bold represent sequences generated during this study; (B) all sequences in phylogram generated during this study. Bootstrap support is shown above each node; only values [80 are shown. Scale-bars: number of base pair differences. OUT, functional outgroup.
Caretta caretta ON062962; one from Eretmochelys imbricata ON062961) Description (Fig. 3A, B, C) [Based on 12 unflattened, gravid, hologenophores from C. caretta and E. imbricata, and 10 unflattened, gravid, whole worms from C. caretta; measurements in Table 1.] Functionally cryptic species, qualitatively not morphologically distinguishable from P. irwinorum n. sp. Subtle differences seen only in uterus; does not reach close to body wall. Remarks: Plesiochorus cymbiformis was described by Rudolphi (1819) as Distoma cymbiforme Rudolphi (1819), infecting the urinary bladder of C. caretta from the Adriatic Sea. The original description was vague and contained no morphometric data. Looss (1901) later recombined the species as Plesiochorus cymbiformis but did not provide a redescription. The earliest description with morphometric data appears to be that of Pratt (1914) (see Table 1). Subsequent redescriptions of P. cymbiformis are broadly consistent with those provided by Pratt (1914), although overall body size (and features expected to correlate with body size) vary.

Morphometric results
Preliminary analyses of the morphometric data generated for hologenophores from C. caretta and E. imbricata and whole worms from C. caretta during this study suggests that the two species of Plesiochorus examined in this study are morphologically indistinguishable, as the two forms are highly similar and all measured features have at least some overlap (Table 1). PCA was employed to determine if the novel species, P. irwinorum n. sp., detected via molecular data, can be distinguished from P. cymbiformis on the basis of morphology. The two species form two distinct clusters in this analysis (Fig. 4). Principal component (PC) 1 explains 60.86% of the variation in the dataset, with testes length and width acting as the key explanatory variables for this PC, but it should be noted that overall body length, and features strongly correlated with body length (i.e. ventral sucker length and width, oral sucker length and width, post-testicular region, post-caecal length, etc.), also contribute to PC1, although the overall size of the specimens relating to the two species is strikingly similar. PC2 explains 12.73% of the variation present in the dataset, and is largely explained by the distance between the uterus and the body margin, with some additional minor contribution from ovary length and width. However, when examined in isolation, these features (testis length, testis width, distance between uterus and body margin, ovary length, and ovary width) all have overlapping ranges between the two species examined during this study, and further overlap with measurements of P. cymbiformis and P. elongatus from previous studies (see Table 1). When examined as a proportion of body length, the distance between the uterus and the body margin almost consistently differentiates the two species, except for the smallest specimen of P. irwinorum n. sp., which overlaps with the measurements of P. cymbiformis collected during this study. All other features examined as a proportion of body length overlap between the two species.

Molecular taxonomy
Both ITS2 and cox1 sequence data have been widely and reliably used in the delineation of marine and freshwater gorgoderid species. Closely related, morphologically distinguishable, species of Phyllodistomum Braun, 1899 (Gorgoderinae Looss, 1899), such sequence data. Although the differences between P. irwinorum n. sp. and P. cymbiformis are not large (8-9 bp), the presence of two ITS2 genotypes in complete sympatry (same individual turtle) suggests there is a barrier to genetic exchange between the two species.
cox1 mtDNA has also been used for the delineation of gorgoderid species; interspecific variation within morphologically distinct species of the subfamily Gorgoderinae is reported to be as low as 6.2% (19 bp), between Phyllodistomum spinopapillatum Pérez-Ponce de León, Pinacho-Pinacho, Mendoza-Garfias and García-Varela, 2015, and Phyllodistomum inecoli Razo-Mendivil, Pérez-Ponce de León and Rubio-Godoy, 2013(see Pérez-Ponce de León et al., 2015b. However, cox1 has been shown to vary significantly within species, suggesting that some gorgoderids may have distinct populations, or undescribed cryptic species. Rosas-Valdez et al. (2011) provided evidence that specimens of Phyllodistomum lacustri (Loewen, 1929) Lewis, 1935 show significant genetic variation (up to 7.8%) based on geographic and host species separation. They suggested that these data may represent cryptic speciation. The data generated during this study differs from previous reports, in that it explores specimens with highly similar morphology in complete sympatry. The specimens examined during this study, representing Plesiochorus cymbiformis and P. irwinorum n. sp., differ by 8% (38 bp). cox1 has limited capacity to recombine (unlike ITS2 and 28S regions, which readily recombine), and genetic differences can accumulate rapidly when two groups of organisms become isolated from each other. This phenomenon can pose a challenge when examining cox1 datasets in isolation, particularly when studying parasites which infect hosts with complex movement patterns and populations (Jensen et al., 2019), such as marine turtles. However, when examined in the light of recombining gene data (like ITS2), cox1 data can be used to further support taxonomic inferences (Cutmore et al., 2021). Following the species recognition criteria of Bray et al. (2022), the presence of two genetic clades (in the 28S, cox1 and ITS2 datasets) from the same geographic region and host species, and indeed in the same host individual, with morphological differentiation (supported by the PCA), does not support the interpretation of a single species with multiple populations, but rather two distinct species, P. cymbiformis and P. irwinorum n. sp.

Morphological taxonomy
Based on morphological data in isolation, gorgoderid specimens infecting E. imbricata during this study appeared to represent a single species, conforming to descriptions of P. cymbiformis. However, molecular data revealed sufficient genetic differences to consider the specimens to represent two species. The genetic differences are supported by the PCA, indicating the two species can be differentiated on the basis of morphometric data, although the differences do not translate into readily recognisable distinctions.
Significant morphological variation has been documented for some gorgoderids, explored in detail for S. cymatodes (see Cutmore et al., 2010), P. folium (see Namuleno & Scholz, 1994), and P. umblae (see Bakke, 1988). This phenomenon has also been noted for P. cymbiformis, and to a smaller extent, P. elongatus (see Werneck et al., 2019). The most dramatically variable feature for species of Plesiochorus is the overall body size, and by extension, features which correlate with it; P. cymbiformis has been reported between 2.5-15.7 mm in body length, and 1.21-5.4 mm in body width (see Table 1). Specimens collected during the present study also have a significant level of variation within both species, and as a result, measurements of all features overlap between P. cymbiformis and P. irwinorum n. sp.
Two lines of morphological data were explored to determine if P. cymbiformis and P. irwinorum n. sp. can be morphologically distinguished. The first, morphometric data in isolation, indicates that the two species are morphologically indistinguishable, as all measured features have overlap between the two species, even when examined as a proportion of body length. The second, PCA, unambiguously distinguishes the two species. A key driving feature for the separation of the two species in the PCA is the distance between the body margin and the uterus, which is, on average, less for P. irwinorum n. sp. (43-87 lm) than for P. cymbiformis (79-220 lm), especially when examined as a proportion of body length (0.48-1.46% vs 0.92-3.9%, respectively). However, there is still overlap in this feature so that it does not reliably distinguish the two species. The PCA also suggests that testes size may distinguish the two species, with P. irwinorum n. sp. generally possessing larger testes as a proportion of body length than P. cymbiformis; however, there is also overlap between the two species for this feature. Although the PCA did not provide a definitive means of differentiating the two species, the presence of the two, separate clusters further supports the presence of two species. Werneck et al. (2019) also noted that only subtle differences distinguish P. cymbiformis and P. elongatus, including a slightly more elongate body and the uterus being closer to the body margin for P. elongatus. Thus, despite the evidence from the PCA that P. irwinorum n. sp. and P. cymbiformis are morphologically distinct, the only reliable way to differentiate the three species of Plesiochorus is currently by molecular means.
Additional issues surrounding the morphological variation of species of Plesiochorus arise when we consider that individuals continue to grow after reaching maturity (as indicated by the possession of a gravid uterus), and the position of various organs (e.g., testis, ovary, uterus) change allometrically. The description of P. irwinorum n. sp. in the present study further shows that morphological differentiation of species within the genus Plesiochorus is difficult, ultimately rendering the three species functionally morphologically cryptic. Due to the increasing unreliability of morphological data for the identification of species of Plesiochorus, we recommend that future reports be accompanied by molecular data (either ITS2 or 28S, and cox1) when possible.
Notably, there are differences between the description of P. elongatus provided by Werneck et al. (2019) and Pigulewsky (1953), relating mainly to egg size and body shape. Pigulewsky (1953) described specimens infecting C. caretta from the Adriatic Sea as lanceolate and possessing eggs 38-40 lm in length and 34-52 lm in width (the reporting of eggs being wider than long seems anomalous). The figure of P. elongatus infecting C. caretta from Brazil provided by Werneck et al. (2019) is spatulate, rather than lanceolate, and the provided measurements state that they possess eggs almost twice the size (76-100 9 52-85 lm) of those reported by Pigulewsky (1953). However, we think these differences may be sufficient to consider the specimens examined by Werneck et al. (2019) distinct from those reported by Pigulewsky (1953), and warrant further investigation of the voucher material relating to both reports.

Host specificity
Prior to the present study, no species of Plesiochorus has been shown to infect more than one host species on the basis of molecular data; all previous reports of P. cymbiformis infecting C. mydas, E. imbricata, and L. olivacea are based entirely on morphological data. These reports were all prior to the resurrection of P. elongatus and occurred when Plesiochorus was regarded as monotypic. Given that the three species of Plesiochorus are functionally morphologically indistinguishable, we hypothesise that at least some of the previous reports may represent species other than P. cymbiformis, but without corresponding sequence data, we cannot be certain about their true identities. Nevertheless, the present study provides the first definitive evidence that a single species of Plesiochorus can infect two host genera, and we think it is likely that additional sequence data for gorgoderids from C. mydas and L. olivacea will provide additional evidence for low host specificity for species of Plesiochorus.
Although sequence data are available for all three species of Plesiochorus, there is none for the type-host and type-locality for either P. cymbiformis and P. elongatus (C. caretta from the Adriatic Sea). Instead, the available sequence data are from Brazil, the USA (for both P. cymbiformis and P. elongatus) and Australia (for P. cymbiformis and P. irwinorum n. sp.). Thus, we cannot be certain about the genetic identity of P. cymbiformis, or by extension, P. elongatus. The certainty of true species identity is further reduced by the fact that both P. cymbiformis and P. elongatus infect C. caretta, and that the sequence data representing both species were generated from this turtle species. It is conceivable that, given the morphological similarities between the two species, the sequence data generated for P. cymbiformis actually represents P. elongatus, and vice versa, or possibly either or both represent additional, undescribed species. This uncertainty surrounding the true identity for the species of Plesiochorus poses an issue when making taxonomic inferences about the genus, and assessing host specificity and geographic distribution. A thorough molecular and morphology-based study of the gorgoderid fauna of C. caretta of the Adriatic Sea is needed to disentangle the issues surrounding the species identity for species of Plesiochorus. Regardless of the true identity of P. cymbiformis and P. elongatus, we are confident of the identification of P. irwinorum n. sp., as the current evidence suggests that it is restricted to E. imbricata, and is not found in the type-host for P. cymbiformis and P. elongatus. providing constructive feedback throughout the preparation of this manuscript.
Funding Open Access funding enabled and organized by CAUL and its Member Institutions. We acknowledge the Australian Biological Resources Study (ABRS) for their continued support. This work was funded ABRS National Taxonomy Research Grant RG19-37, awarded to SCC.

Declarations
Conflict of interest The authors declare that they have no conflict of interest.
Ethical approval All applicable institutional, national and international guidelines for the care and use of animals were followed.
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