No vagina, one vagina, or multiple vaginae? An integrative study of Pseudaxine trachuri (Monogenea, Gastrocotylidae) leads to a better understanding of the systematics of Pseudaxine and related genera

The presence/absence and number of vaginae is a major characteristic for the systematics of the Monogenea. Three gastrocotylid genera share similar morphology and anatomy but are distinguished by this character: Pseudaxine Parona & Perugia, 1890 has no vagina, Allogastrocotyle Nasir & Fuentes Zambrano, 1983 has two vaginae, and Pseudaxinoides Lebedev, 1968 has multiple vaginae. In the course of a study of Pseudaxine trachuri Parona & Perugia 1890, we found specimens with structures resembling “multiple vaginae”; we compared them with specimens without vaginae in terms of both morphology and molecular characterisitics (COI barcode), and found that they belonged to the same species. We also investigated the male copulatory organ (MCO) of this species, the accuracy of the original description of which is known to be a matter of debate. We found that the genital atrium is armed with 12 hooks arranged as a single circle and a central hollow stylet which is probably involved in traumatic insemination. We redescribed Pseudaxine trachuri based on newly collected specimens from off the coast of Algeria and Museum specimens from off France. Specimens from the type-host, Trachurus trachurus, were found to be similar, for both molecular sequences and morphology, to those found on Boops boops. We can therefore confirm, for the first time with molecular evidence, that B. boops is a host of this parasite. We consider that Pseudaxinoides was erected on the basis of an erroneous interpretation of structures which are not vaginae and, consequently, propose the transfer of most of its species to Pseudaxine, as P. australis (Lebedev, 1968) n. comb., P. bychowskyi (Lebedev, 1977) n. comb., P. caballeroi (Lebedev, 1977) n. comb., P. cariacoensis (Nasir & Fuentes-Zambrano, 1983) n. comb., and P. vietnamensis (Lebedev, Parukhin & Roitman, 1970) n. comb. We also propose Allogastrocotyle dillonhargisorum nom. nov. for Pseudaxine bivaginalis Dillon & Hargis, 1965 to avoid a secondary homonymy.


Introduction
Pseudaxine Parona & Perugia, 1890, Allogastrocotyle Nasir & Fuentes Zambrano, 1983 and Pseudaxinoides Lebedev, 1968 are three genera that belong to the family Gastrocotylidae Price, 1943. Species of these three genera share a general boot-shaped appearance, because of their oblique haptor, and differ mainly in the presence and number of vaginae. The organisation of internal organs is similar, especially the testes and the male copulatory organ (MCO); however, members of Pseudaxine have no vagina, the single member of Allogastrocotyle has two vaginae, and species of Pseudaxinoides are said to have multiple ventro-lateral vaginae [49,60,63,66].
Pseudaxine was established with Pseudaxine trachuri Parona & Perugia, 1890, from the gills of Trachurus trachurus off Genoa, Italy, as the type species [63]. This species was redescribed several times [53,64,67,70,76,77]. One confusing aspect of this parasite was the armature of the genital atrium, which was described as "consisting of two crowns of hooks" [63], whereas a single crown was observed in subsequent redescriptions.
During an ongoing investigation on monogeneans infesting the gills of teleosts off the Algerian coast, we collected gastrocotylids consistent with the diagnosis of P. trachuri from gills of the type-host, the carangid T. trachurus, and also from a sparid, Boops boops. Specimens from both hosts were submitted to molecular analysis. Based on this material, we provide a detailed study of the genital atrium of P. trachuri and describe for the first time in this species a central stylet.
Unexpectedly, we found several specimens with multiple ventro-lateral papillae, without ducts, which mimicked vaginae. According to the diagnostic criteria of gastrocotylid genera [53], these specimens of Pseudaxine trachuri should have been attributed to Pseudaxinoides. We compared measurements and COI sequences of these specimens with "multiple vaginae" with typical specimens of Pseudaxine trachuri without vaginae, and found that they belonged to the same species. This clearly questioned the alleged differences between Pseudaxine and Pseudaxinoides, and we finally propose a series of nomenclatural decisions, including the demise of Pseudaxinoides.
This work is part of a general survey of the monogenean fauna of fishes of the southern shores of the Mediterranean Sea [5, 7-10, 15-18, 22, 44].

Monogeneans Morphological methods
Monogeneans were removed from gills using fine dissection needles, killed in hot seawater, then preserved in 70% ethanol, stained with acetic carmine, dehydrated in ethanol series (70,96 and 100%), cleared in clove oil, and finally mounted in Canada balsam. Drawings were made with the help of an Olympus BH-2 microscope equipped with Differential Interference Contrast (DIC) and a drawing tube. Micrographs were taken with a Leitz microscope. Drawings were scanned and redrawn on a computer with Adobe Illustrator (CS5). Measurements are in micrometres, and indicated as the means, ± the standard deviation if n > 30, and in parentheses, the range and number of measurements.

Molecular methods
To ensure that hosts and parasites were labelled with respect to their host-parasites relationships, we followed recent works dealing with description of new species of Monogenea by using barcoding [5,[8][9][10]43]. For each host fish from which monogeneans were collected, a tissue sample from the gill was taken. Monogeneans were cut into three parts using a scalpel blade. The anterior part (which includes the genital atrium) and posterior part (which includes the haptor) of each specimen were mounted as vouchers (both parts on a single slide) for drawing and deposition in a Museum; their middle part was preserved in absolute ethanol then subjected to molecular analyses. For some specimens, we did not cut them in three part but rather took a lateral part in the middle of the body for the molecular analysis and mounted the rest of the specimen in one part as voucher. Nine specimens were analysed: 2 from the type-host T. trachurus (already published in our study of Allogastrocotyle [10]; 3 typical specimens from the sparid host B. boops, and 4 specimens with "multiple ventro-lateral vaginae" from B. boops.

Molecular barcoding of fish
Total genomic DNA was isolated using a QIAamp DNA Mini Kit (Qiagen, Courtaboeuf, France), as per the manufacturer's instructions. The 5 0 region of the mitochondrial cytochrome c oxidase subunit I (COI) gene was amplified with the primers FishF1 (5 0 -TCAACCAACCACAAAGACATT-GGCAC -3 0 ) and FishR1 (5 0 -TAGACTTCTGGGTGGC-CAAAGAATCA -3 0 ) [75]. PCR reactions were performed in 20 lL, containing 1 ng of DNA, 1Â CoralLoad PCR buffer, 3 mM MgCl 2 , 66 lM of each dNTP, 0.15 lM of each primer, and 0.5 units of Taq DNA polymerase (Qiagen). The amplification protocol was 4 min at 94°C, followed by 40 cycles at 94°C for 30 s, 48°C for 40 s, and 72°C for 50 s, with a final extension at 72°C for 7 min. PCR products were purified (Ampure XP Kit, Beckman Coulter) and sequenced in both directions on a 3730 Â l DNA Analyzer 96-capillary sequencer (Applied Biosystems, Foster City, CA, USA). We used CodonCode Aligner version 3.7.1 software (Codon Code Corporation, Dedham, MA, USA) to edit sequences, compared them to the GenBank database content with BLAST, and deposited them in GenBank under accession numbers MT666082-MT666086. Species identification was confirmed with the BOLD identification engine [68].

COI sequences of monogeneans
Total genomic DNA was isolated using a QIAmp DNA Micro Kit (Qiagen). The specific primers JB3 (=COIASmit1) (forward 5 0 -TTTTTTGGGCATCCTGAGGTTTAT -3 0 ) and JB4.5 (=COI-ASmit2) (reverse 5 0 -TAAAGAAAGAACA-TAATGAAAATG -3 0 ) were used to amplify a fragment of 402 bp of the COI gene [11,55]. PCR reaction was performed in 20 lL of a mixture containing 1 ng of DNA, 1Â CoralLoad PCR buffer, 3 mM MgCl 2 , 0.25 mM dNTP, 0.15 lM of each primer, and 0.5 units of Taq DNA polymerase (Qiagen). Thermocycles consisted of an initial denaturation step at 94°C for 2 min, followed by 37 cycles of denaturation at 94°C for 30 s, annealing at 48°C for 40 s, and extension at 72°C for 50 s. The final extension was conducted at 72°C for 5 min. PCR products were purified (Ampure XP Kit, Beckman Coulter) and sequenced in both directions on a 3730 Â l DNA Analyzer 96-capillary sequencer (Applied Biosystems, Foster City, CA, USA). We used CodonCode Aligner version 3.7.1 software (Codon Code Corporation, Dedham, MA, USA) to edit sequences, compared them to the GenBank database content with BLAST, and deposited them in GenBank under accession numbers MT666075-MT666081 (Table 1).

Trees and distances
Most gastrocotylid sequences available in GenBank were included in a preliminary phylogenetic analysis, with 9 sequences of Pseudaxine trachuri, and sequences of two members of the Gotocotylidae Yamaguti, 1963 used as the outgroup. The trees were inferred by the Neighbour-Joining method and the Maximum Likelihood method using MEGA7 [48]. After examining trees and sequences, we refined our analysis and finally used only 12 sequences, as detailed in the results. For the final analysis, we employed the Neighbour-Joining method and the Maximum Likelihood method using MEGA7 [48]; after selecting the best model with MEGA7, the ML was performed with HKY + G [34]. Genetic distances (p-distance and Kimura-2 parameter distance [45]) were estimated with MEGA7 and all codon positions were used.

Molecular identification of fish
The provisional identification of the fish species using morphological characteristics was confirmed by the DNA barcoding approach. BLAST analysis of the COI sequences of the fish species of the present study with the NCBI and BOLD databases showed sequence similarity values of 100% for B. boops and 99.85% for T. trachurus.

Molecular characterization of monogeneans
A preliminary dataset involved 20 COI sequences, including 9 sequences of P. trachuri and sequences from GenBank. After alignment and trimming, the dataset was 391 bp in length with 211 informative sites. The main result for this preliminary analysis was that our sequences of P. trachuri from off the Algerian coast formed a robust monophylum with the single sequence of P. trachuri available from the type-host T. trachurus off Sète, France (AY009168) [40]; this sequence is therefore confirmed as P. trachuri. However, we noted that sequence AY009167, also from T. trachurus off Sète, France, grouped with P. trachuri, although it was identified as Gastrocotyle trachuri Van Beneden & Hesse, 1863 and labelled as such in GenBank by its authors, Jovelin & Justine [40]; we interpret this as a misidentification, and sequence AY009167 should be considered P. trachuri.
We noticed that several GenBank sequences were unconfirmed and/or unpublished, and there was no certainty in the systematic assignment for some of them (in addition to the obvious error mentioned above); also, some had apparent insertionsdeletions, or ambiguous bases, and the alignment included gaps. Therefore, from the preliminary dataset, we selected only the sequences that had the same span as our sequences and no ambiguous bases. The restricted dataset included only 12 sequences: 9 sequences of P. trachuri (including 7 new), 2 sequences of Allogastrocotyle bivaginalis, and the COI part of the complete mitogenome (NC016950) of the chauhaneid Pseudochauhanea macrorchis Lin, Liu & Zhang in Zhang, Yang & Liu, 2001 [81]. We obtained perfect alignment, 389 bp in length, without any gap or missing data. The tree ( Fig. 1) showed a robust monophylum that includes all sequences of P. trachuri. The P. trachuri clade contained sequences from the type-host and from the sparid host B. boops, confirming the occurrence of P. trachuri on a sparid. Moreover, all specimens of P. trachuri with apparent "multiple ventrolateral vaginae" and specimens without vaginae clustered in the same clade, without having a distinct branch.
Distances were computed using p-distance and the Kimura-2 parameter. All sequences of P. trachuri, whatever host or apparent number of vaginae, were very similar, with less than 2% variation. This suggests that P. trachuri also occurs on B. boops and that specimens with apparent multiple vaginae are conspecific with typical P. trachuri.

Description of specimens from Boops boops from off Algeria
Based on 30 specimens. Measurements in Table 2. Body elongate, typically gastrocotylid. Haptor, oblique, asymmetrical, with single row of clamps. Clamps typically gastrocotylid; ventral arm of median spring Y-shaped; proximal end of ventral arm of median spring narrow; its distal part Y-shaped; dorsal arm of median spring short, has 3-4 pairs of apertures arranged in 2 longitudinal, symmetrical, parallel rows; distal end of dorsal arm with accessory skeletal piece represented by V-shaped process at its distal end (Fig. 4). Dorsal arm of ventral jaw short and curved inwards. Dorsal jaw shorter than ventral. Oblique sclerites with inner ends touching each other in median line. Base of clamp with cuticularised hinge ligament on each side, connecting median spring with base of jaw sclerites (Fig. 4).
Testes postovarian, large and oval, arranged irregularly in intercaecal field in posterior third of body. Vas deferens passes forward along body midline and enlarges to form ejaculatory bulb at level just anterior to genital atrium (Figs. 3A, 3G and 3H).
Ovary pretesticular, in form of inverted "U". Ovary begins at level of anteriormost testes, continues anteriorly in midline, reflexes and passes posteriorly toward haptor to terminate as oviduct. Oviduct arises from distal end of ovary and opens into oötype with Mehlis' gland cells. Uterus, arises from oötype and passes anteriorly. Genito-intestinal canal short, ventral to ovary. Vitellarium follicular, co-extensive with intestinal caeca, extending from level of genital atrium to haptor. Transverse vitelloducts fuse in midline, ventral to ovary, to form Y-shaped vitelline reservoir. Vagina absent (Fig. 2J).

Genital atrium
Genital atrium ventral, with a muscular rim, located in the anterior constriction of the body. The genital atrium includes 2 sclerotised structures: a crown of 12 hooks and a central stylet. Gastrocotylid-like atrial hooks (Fig. 3J) are arranged as a perfect circle; this was seen in some specimens in which the hooks are aligned with the optical axis of the microscope, i.e. perpendicular to the longitudinal body axis. In these polar views, observation with a DIC microscope, which has a very low field depth, enables various optical sections, from distal to proximal: the distal (external) extremity of the hooks, with The stylet is connected basally with an unsclerotised bulb, which is itself connected at its base to the vas deferens (Fig. 3A). In flattened whole-mounts, the stylet is occasionally tilted such that it appears as a pair of slender spines (Fig. 3I).

Vagina and multiple pseudo-vaginae
In most specimens, a vagina was absent; the DIC microscope enables one to examine with precision the surface of the tegument and we did not detect any opening. In a series of specimens from B. boops, we found, however, multiple ventro-lateral papillae which could be interpreted as two rows of lateral vaginae. We tried to find a duct leading from these openings to the posterior parts, but did not detect it; instead, we noticed that flame cells were often close to these apertures. We consider, therefore, that these openings are pores of the excretory system, which mimic lateral vaginae. Figure 5 shows micrographs of the region of the genital atrium and vaginae in P. trachuri (both specimens without a vagina and with alleged multiple vaginae) and in Allogastrocotyle bivaginalis.

Boops boops as a host for Pseudaxine trachuri
Pseudaxine trachuri is a cosmopolitan monogenean, reported from many carangid hosts, mainly Trachurus spp., and from a single sparid, Boops boops (Table 3). In the present study, measurements and counts of P. trachuri from B. boops were within range of previous descriptions (Table 2), and COI sequences of P. trachuri from B. boops differed from those from the type-host by only 1-2%. Differences higher than 4% in COI sequences are generally interpreted as an indication that species are different [4,33,74,75]; in polyopisthocotylean monogeneans, differences in COI sequences reported as intraspecific ranged 0.2-5.6% [8]. The 1-2% difference reported here is well within the intraspecific range. We thus confirm, for the first time with molecular evidence, that P. trachuri uses B. boops as a host. We noted that, in our specimens, the prevalence in B. boops was lower than in T. trachurus (10% vs. 28%), which could be an indication of a lower adaptation to the sparid host, or a recent host-switch.

General structure of the genital atrium of Pseudaxine trachuri
The original description of P. trachuri is apparently very detailed and beautifully illustrated, especially for a paper of the 19th Century [63]. It has been supplemented by redescriptions and more morphometric data by Yamaguti [76,77], Sproston [70], Parukhin [64], Lebedev [53] and Radujkovic & Euzet [67]. However, the illustrations were sometimes poor or redrawn from the original 1890 account of Parona & Perugia [63]. Parona and Perugia indicated that the genital atrium was armed with two crowns of hooks [63]: "L'apertura cloacale [. . .] coronata da un cerchio di 24 uncini minutissimi (Fig. 11), con base allargata e punta arcuata rivolta verso il lume del canale. Inferiormente e nell'interno della cloaca si osserva altro cerchio di aculei (Fig. 12) molto più lunghi dei superiori (0,017) che soprastanno all'apertura del deferente e che si può rigardare quale armatura maschile" which translates as "The cloacal opening [. . .] is crowned by a circle of 24 very fine hooks (Fig. 11), with an enlarged base and the point turned towards the lumen of the canal. Beneath and inside the canal, another circle of spines is observed (Fig. 12), which are much longer than those above (0.017) and are present distal to the aperture of the vas deferens; these can be considered the male armature".
Yamaguti redescribed the species twice, on the basis of two (1938) and one (1942) specimens, from the gills of the typehost off Japan [76,77]. In both descriptions, Yamaguti clearly indicated that the genital atrium was surrounded by a single crown of hooks, but did not comment on the differences in the genital atrium. Sproston (1946) redescribed P. trachuri from its type-host, based on a single specimen collected from off Plymouth, England, and indicated the presence of 16 atrial hooks. However, she reproduced in her paper one of the drawings by Parona & Perugia and commented on the original description: "these authors give a curious figure of the genital corona [. . .] and describe it as having an outer ring of 24 small hooks and 17 larger hooks below at the end of the vas deferens"; she stated "neither the Japanese nor Plymouth specimens from the same host reveal anything like this structure". In this sentence, Sproston mentioned 17 larger hooks, but Parona & Perugia did not provide the number of the larger hooks; a possible explanation is that Sproston confused the length (17 lm) and the number of the larger hooks in the original description in Italian, although the length was provided as "0.017".
Unnithan [73], probably using Sproston's text, wrote in his description of Pseudaxine kurra Unnithan, 1968 "no structure identical to the genital corona having an outer rim of 24 small hooks and 17 larger hooks below it at the end of the vas deferens is observed in the new species".
Lebedev commented "The discussion of Sproston [70] and Unnithan [73] about the allegedly incorrect description of these structures by Parona and Perugia [63] seems unreasonable to us: if we accept that the outer ring of spines in the picture of the copulatory apparatus given in the first description is the apical parts of the hooks, and the inner ring is a stylet, then everything falls into place [53].  Radujkovic & Euzet, on the basis of specimens from off Montenegro, described briefly the genital atrium as having a crown of 12 spines [67].
Our careful examination of new specimens of Pseudaxine trachuri from different hosts and localities showed the presence of a single crown (corona) of 12 hooks. Clearly, the two circles described by Parona & Perugia [63] have never been mentioned in all subsequent descriptions of this species (Table 4). Although Lebedev [53] suggested that this could be considered a minor misinterpretation of the structure, we believe that it is important to clarify this point.
The number of atrial hooks in the material examined in this study and other accounts ( Table 2) varies according to locality. Yamaguti suggested that it was due to intraspecific variability [76,77]. However, the number of hooks given in this study is the same as for the specimens from the Mediterranean described by Radujkovic and Euzet [67]. It may be that specimens from seas other than the Mediterranean correspond to different species; this needs to be investigated using molecular methods. We provisionally consider that all records worldwide correspond to one and the same species.
In none of the previous description was a stylet mentioned. Although Lebedev used this term to explain the drawing by Parona & Perugia, he did not explicitly describe a stylet in his specimens.

The central stylet of the genital atrium
We describe in our specimens of P. trachuri the presence of a conical structure in the centre of the crown of spines of the genital atrium; this is the first time that such as structure has been described in this species. Llewellyn (1983) provided a detailed description of the genital atrium of Gastrocotyle trachuri; he named the hooks forming the crown "ribs" and the central stylet a "penis tube" [56]; he also proposed a hypothesis for sperm transfer in which the genital atrium would work as a sucker and the penis tube would penetrate the tegument to allow the deposition of sperm in the subtegumental tissue of the partner. What could be a stylet-like structure was also described by George (1960) in another gastrocotylid, Engraulicola forcipopenis George, 1960, as a "forceps-like spine at the end of the penis" [31]. In the diclidophorid Diclidophora merlangi (Kuhn, 1829) Krøyer, 1838, which has no vagina, MacDonald & Caley showed that sperm were injected into the tissue of the partner [57]; however, a stylet was not described in this species.
Our observations of the structure of the stylet show that it is hollow and thus could allow sperm to be injected into the tissue of the partner, and the absence of a vagina (see below) suggests that P. trachuri uses traumatic insemination, a process which is not uncommon in polyopisthocotylean monogeneans [41].
Vaginae in specimens of the genus Pseudaxine and allied genera Parona & Perugia, in their original description of P. trachuri, which is interspersed with comparison with another species, clearly stated that there was no vagina "Infatti non travammo traccia di canale e di apertura vaginale", i.e. "In fact we did not find a trace of a canal or of a vaginal opening" [63].
Yamaguti redescribed the species twice [76,77]. In neither description did he mention the presence or absence of a vagina. However, in his "Systema Helminthum" he wrote for the diagnosis of Pseudaxine "vagina opening middorsally" [78].
Dillon & Hargis described Pseudaxine bivaginalis Dillon & Hargis, 1965, and emended Yamaguti's (1963) diagnosis of the genus to include the presence of "a single vagina opening middorsally or paired vaginae opening near lateral margins at or near level of genital atrium" [23].
Unnithan described Pseudaxine kurra Unnithan, 1968, with "vaginal pore unarmed, dorsal, with few gland cells and situated in front of the ovarian loop, slightly shifted to the right side"; apparently unaware of Dillon & Hargis's work (1965), he emended the diagnosis of the genus as "vagina single median dorsal and unarmed" [73]. Lebedev commented on the position of the vagina in Pseudaxine kurra: "Most likely a mistake was made, since the vagina cannot be in this place. In our specimen (not in very good condition) no vagina was found"; and he questioned the validity of the species [53]. Table 4. Descriptions of the genital atrium of Pseudaxine trachuri.

Reference
Host Locality Description of genital atrium Parona & Perugia [63] T. trachurus Off Italy, M.S. Circle of 24 very fine hooks, with enlarged base and point turned towards lumen plus Second circle of longer hooks (17 lm), above aperture of vas deferens Yamaguti [76] T. trachurus Off Japan, P.O. Crown of 13 hooks, each of which is about 20 lm long and has long blade with it tips strongly curved posteriorly and inwards, and with thickening near its base, by which it connects with neighbouring hooks Yamaguti [77] T. trachurus Off Japan, P.O.
Lebedev recorded Pseudaxine trachuri from Trachurus novaezelandiae (which is the type-host of Pseudaxine bivaginalis Dillon & Hargis, 1965) off Australia in 1968, and mentioned the presence of two lateral vaginal openings [49]. Since two vaginae have never been observed in P. trachuri (Table 5), we believe it likely that the gastrocotylids recorded by Lebedev on T. novaezelandiae were in fact P. bivaginalis (see the taxonomic decision made regarding this taxon below).
Yamaguti described Pseudaxine decapteri Yamaguti, 1968 with a "vagina on each side divided into longitudinal row of 5-13 transversely elongated cuticular pits, beginning just behind level of genital pore" [80]. He also claimed "The peculiar structure of the vagina must have been overlooked by Parona and Perugia and subsequent authors because it is recognizable in well preserved specimens alone". Subsequently, Lebedev erected the genus Pseudaxinoides Lebedev, 1968 for monogeneans resembling Pseudaxine spp. but differing from it by the presence of multiple ventro-lateral vaginal papillae [49]. Lebedev (1977) studied descriptions and figures of Pseudaxine decapteri and assigned this species to Pseudaxinoides [51].

Taxonomic decisions
As explained above, three genera of gastrocotylids are very similar in all aspects, except for their (alleged) number of vaginae. Figure 6 shows drawings of, side by side, specimens of Pseudaxine trachuri (no vagina), Pseudaxine trachuri ("multiple vaginae"), and Allogastrocotyle bivaginalis (two vaginae). Figure 5 shows micrographs of the anterior region of similar specimens. Specimens with multiple vaginae would have been attributed to Pseudaxinoides in its traditional concept.
In the present study, we have demonstrated, on the basis of morphology and molecules, that specimens of P. trachuri without a vagina and specimens with apparent multiple lateral vaginae are conspecific. We thus consider that all similar species described with apparent multiple vaginae are based on an erroneous interpretation of the excretory system; this makes the distinction between Pseudaxine and Pseudaxinoides void, and we propose the synonymy of these two genera. Since Pseudaxine has priority over Pseudaxinoides (1890 vs. 1968), we propose to transfer most Pseudaxinoides spp. to Pseudaxine, with the following new combinations: Pseudaxine australis (Lebedev, 1968) n. comb.; Pseudaxine bychowskyi (Lebedev, 1977) n. comb.; Pseudaxine caballeroi (Lebedev, 1977) n. comb.; Pseudaxine cariacoensis (Nasir & Fuentes-Zambrano, 1983) n. comb.; and Pseudaxine vietnamensis (Lebedev, Parukhin & Roitman, 1970) n. comb. [49-52, 54, 60, 80]. Pseudaxine decapteri Yamaguti, 1968 is kept within Pseudaxine. The status of P. kurra is pending [53]. We noticed that Pseudaxinoides pusanowi Lebedev, 1984 has two of its testes that are paraovarian [52], in contrast to species of Pseudaxine in which they are all post-ovarian; Lebedev noted later [53] that is was close to species of Allopseudaxinoides Yamaguti, 1968; a reappraisal of this species is probably necessary and thus we provisionally refrain from transferring it to Pseudaxine.
A comparison of Pseudaxine bivaginalis Dillon & Hargis, 1965 with the single species of Allogastrocotyle, A. bivaginalis Nasir & Fuentes Zambrano, 1984 [10,60], suggests that the former species should be transferred to Allogastrocotyle, as indicated by the presence of two ventrolateral vaginal openings surrounded by muscular tissue near the genital atrium [23,53]. Since the name Allogastrocotyle bivaginalis is already occupied, we propose here the new name Allogastrocotyle dillonhargisorum nom. nov. to avoid homonymy. The new name has been registered in ZooBank as: urn:lsid:zoobank. org:act:1ECC6CF2-4BC4-45E8-9AE7-049C00FC7735.
Considering that in a single species, P. trachuri, we found specimens with and without what look like "multiple lateral vaginae", we believe that the taxa mentioned above, and probably others, should be re-examined, preferably using both morphology and molecular information. More generally, we stress the importance of integrative taxonomy [21] for the taxonomy of parasites, including correct identification of hosts using both morphology and molecular analysis as in the present work and recent studies [5,[8][9][10]43] and, whenever possible, deposition of hosts in curated collections [12,36,39,42].

Conflict of interest
The Editor-in-Chief of Parasite is one of the authors of this manuscript. COPE (Committee on Publication Ethics, http:// publicationethics.org), to which Parasite adheres, advises special treatment in these cases. In this case, the peer-review process was handled by an Invited Editor, Jérôme Depaquit.