Archigetes Leuckart, 1878 (Cestoda, Caryophyllidea): diversity of enigmatic fish tapeworms with monoxenic life cycles

The caryophyllidean genus Archigetes Leuckart, 1878 is unique among all tapeworms in that its species can mature in invertebrate hosts (Oligochaeta), i.e., have a monoxenic (direct) life cycle. All five species were described as progenetic plerocercoids in oligochaetes and two of them also as adults from cypriniform fishes. Two species, A. sieboldi Leuckart, 1878 and A. iowensis Calentine, 1962, were found in North America in non-native common carp (Cyprinus carpio). A molecular study of caryophyllideans from the southern United States has revealed the occurrence of three new species in native freshwater fishes (Catostomidae, Ictiobinae): Archigetes loculotruncatus n. sp. from Ictiobus bubalus, I. niger and Carpiodes cyprinus is the largest representative of the genus and differs by a loculotruncate scolex. Archigetes megacephalus n. sp. from Ictiobus niger, I. bubalus and I. cyprinellus is characterised by a prominent, bothrioloculodiscate scolex. Archigetes vadosus n. sp. from I. bubalus is typified by a globular scolex with very shallow loculi; it differs from the closely related A. sieboldi in the shape of the body, with a distinct neck region and a scolex wider than the remaining body. Archigetes iowensis Calentine, 1962 becomes a junior synonym of Paraglaridacris limnodrili (Yamaguti, 1934). The generic diagnosis of Archigetes is amended and a key to identification of North American taxa is provided. Species of Archigetes and Paraglaridacris differ from each other most conspicuously in the structure of the ovary, which is follicular in Archigetes versus compact in Paraglaridacris.


Introduction
The members of the genus Archigetes Leuckart, 1878 are extraordinary among tapeworms due to their direct (monoxenic) life cycle, i.e., the lack of an intermediate host, with sexual maturation in the invertebrate host. Since its erection by Leuckart [25], the genus has been the subject of intense speculation about the origin of the tapeworm life cycle [28,29]. The monoxenic life cycle, with the production of eggs by progenetic plerocercoids in the body cavity of oligochaetes, and excluding fish as a typical caryophyllidean definitive host, was considered by some authors to be an ancestral type of the cestode life cycle, assuming that invertebrates were primary hosts of the most ancient tapeworms [1,5,11,52].
This monoxenic cycle was found in all five species of the genus Archigetes recognised as valid [5,19]. Accidentally infected freshwater teleosts most likely served as a postcyclic host, which may have resulted in the life cycle being extended to two host, i.e., heteroxenic (indirect) life cycle [11]. An alternative view is that some Archigetes species evolved by progenesis, i.e., early development of the reproductive system leading to sexual maturity (including the production of eggs) in a larval stage [36]. This scenario is supported by some morphological and developmental data [41,42].
Molecular data have clearly shown that Archigetes is neither the most basal lineage within all cestodes nor within the order Caryophyllidea [36,50]. This means that sexual maturity in the invertebrate host is unlikely to have an impact on understanding of the protocestode state [36]. In contrast, these molecular phylogenetic studies show that at least three Archigetes species are members of the most recently diverging caryophyllidean lineage, which comprises almost entirely Nearctic caryophyllideans [50]. The available data thus support the hypothesis that the monoxenic life cycle of Archigetes is a secondary abbreviation of the developmental cycle, similar to some spathebothriideans, such as Cyathocephalus truncatus (Pallas, 1781), Diplocotyle olrikii Krabbe, 1874 or Spathebothrium simplex Linton, 1922, whose maturation and egg production may occur in gammarid amphipods as their intermediate host [20,35,46].
In addition to controversies over the position of Archigetes and the evolutionary origin of its monoxenic life cycle, there is much confusion about the species composition of the genus. Kennedy [19] considered the following five species as valid: A. sieboldi Leuckart, 1878 (type species); A. brachyurus Mrázek, 1908; A. cryptobothrius Wisniewski, 1928; A. limnodrili (Yamaguti, 1934)  ; and A. iowensis. However, no taxonomical revision based on properly fixed material and molecular data has been carried out. Moreover, the taxonomic position of some caryophyllideans placed in the genera Biacetabulum Hunter, 1927, Brachyurus Szidat, 1938 and Paraglaridacris Janiszewska, 1950 is also problematic, as they morphologically resemble the species of Archigetes.
The genus Archigetes was first reported from North America by Ward [57] who found unidentified tapeworms resembling those of Archigetes spp. known from Europe in a fish from the Illinois River at Havana. The genus was for a long time represented in North America by only two species, A. iowensis Calentine, 1962, described from non-native common carp (Cyprinus carpio L.) and Limnodrilus hoffmeisteri Claparède [5], and A. sieboldi, reported from the same hosts [4]. Recently, Scholz and Pérez-Ponce de León [49] reported three morphotypes of Archigetes tapeworms, which may represent putative new species, from eastern shiners, Notropis spp. (Cypriniformes: Leuciscidae), and silverside, Chirostoma humboldtianum (Valenciennes) (Atheriniformes: Atherinidae) in the Nearctic part of Mexico. The latter fish is the first representative of the order Atheriniformes that has been reported as the host of caryophyllidean tapeworms [49].
Recent studies on North American caryophyllideans have revealed high diversity of these tapeworms, previously only partly described. Two new genera were erected and nine new species of the genera Biacetabulum Hunter, 1927, Promonobothrium Mackiewicz, 1968 and Isoglaridacris Mackiewicz, 1965 described [37,39,40,49,[54][55][56]. In the present paper, three new species of the enigmatic Archigetes are described from catostomid fishes from the southern United States (USA). In addition, species diversity of the genus is discussed together with the assessment of their phylogenetic relationships. The diagnosis of Archigetes is amended and a key to identification of North American taxa, including A. iowensis transferred to Paraglaridacris, is provided. Taxonomic status of morphologically similar species of the latter genus, which may also mature in oligochaetes, is discussed based on material from North America (USA), Asia (Japan) and Europe (Czech Republic, Russia and Slovakia).

Materials and methods
The specimens studied were newly collected by the present authors and their collaborators in Mississippi (USA), in 2012 and 2019. In addition, specimens from the private collection of the late John S. Mackiewicz (USA) donated to the senior author (T.S.) and vouchers deposited in the Helminthological Collection of the Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, České Budějovice (IPCAS) were examined.
Newly collected tapeworms were obtained from the intestines of freshly killed fish; they were rinsed with saline and fixed in hot, nearly boiling, 4% formaldehyde for morphological studies [38]. Some worms were fixed completely in 96% molecular-grade ethanol or in hot saline and then placed in 80% molecular-grade ethanol for DNA sequencing (see below). For light microscopy, specimens were stained in Mayer's carmine, dehydrated in an ethanol series, cleared with clove oil (eugenol), and mounted in Canada balsam. Line drawings were made using a Leica DM 5000B light microscope (Leica Microsystems, Wetzlar, Germany). For scanning electron microscopy, selected specimens were examined using a Jeol I AM 6510LV and a Jeol JSEM 7401F electron microscope (Jeol Ltd., Tokyo, Japan) according to the procedure described by Oros et al. [38].
The phylogenetic relationships of the studied tapeworms were assessed based on the partial (D1-D3 region) nuclear ribosomal large subunit rRNA gene (lsrDNA) sequences. A list of sequenced samples and sequences used in the phylogenetic analysis is provided in Table 1. Genomic DNA was isolated using the Monarch Genomic DNA Purification Kit (New England Biolabs, Inc., USA), following the manufacturer's instructions. A 1420 nucleotide (nt) long fragment of the 28S rRNA gene was amplified following the protocol described by Brabec et al. [3] or Scholz et al. [51]. An ExoSAP-IT PCR Cleanup enzymatic kit from Thermo Fisher Scientific, Inc.
(Waltham, MA, USA) was used to purify the PCR products, following the manufacturer's protocol. PCR amplicons were thereafter sequenced from both strands using the PCR primers and additional internal sequencing primer 300F and ECD2 [26]. Sequences were assembled and edited using Geneious version 11 (Biomatters, Auckland, New Zealand). The new lsrDNA sequences were aligned with the sequences of Archigetes spp. and Biacetabulum spp. available in GenBank using ClustalW implemented in Geneious ver. 11 (Biomatters, Auckland, New Zealand). The length of the final alignment was 1329 nucleotides (nt). Bayesian inference (BI) and maximum likelihood (ML) methods were used to assess phylogenetic relationships within the dataset. The best-fitting model for the analyses, GTR + I + G, was estimated using jModelTest 2.1.2 [10]. MrBayes software (ver. 3.2.3) [45] was used to perform the BI analysis. Markov chain Monte Carlo analyses were run for 10,000,000 generations, log-likelihood scores were plotted, and only the final 75% of trees were used to build the consensus tree. ML analysis was performed using PhyML version 3.0 [13] with nonparametric bootstrap validation based on 100 pseudoreplicates. FigTree ver. 1.4 software [44] was used to visualise the phylogenetic trees. To calculate genetic distances (uncorrected p-distance), a separate alignment including only sequences of Archigetes spp. (1356 nt) was used in MEGA ver. X [24].
In addition to IPCAS (see above), the studied specimens are deposited in the National Helminthological Collection of Mexico, Instituto de Biología, Universidad Nacional Autónoma de México, Mexico City, Mexico (CNHE), Harold W. Manter Laboratory, University of Nebraska, Lincoln, USA (HWML), and the National Museum of Natural History, Washington, D.C., USA (USNM). The terminology of the scolex morphology follows the proposal of Mackiewicz [30] and Oros et al. [39]. Terminology of microtriches follows Chervy [9]. The scientific and common names of fish hosts follow FishBase [12]. In descriptions, measurements are given in micrometres unless otherwise noted.
Host code numbers correspond to a unique individual fish host examined and small letters specify individual tapeworm (s) found in this host, i.e., correspond to morphological voucher(s). See Table 1.

Molecular phylogeny
Molecular phylogenetic analysis, which included sequences of nine newly recovered isolates of Archigetes and sequences of this genus available in GenBank, revealed a well-supported monophyletic group (Fig. 1). Archigetes spp. were positioned in three clades. Clade I contained isolates of two morphologically distinct species, i.e., the type species of the genus, A. sieboldi, from Europe and Japan and a putative new species from I. bubalus collected in North America. The genetic divergence within this clade was 0-5 nt (0-0.37%). In contrast to conspicuous morphological differences (see below), the interspecific divergence between A. sieboldi and this new species was rather low and ranged between 1 and 5 nt (0.08-0.37%). Clades II and III contained isolates representing two species from Ictiobus spp. in North America that were recognised as species new to science. Sequences of Clade II were identical. The three new species are described below. The nucleotide divergence of sequences of each isolate is summarised in Supplementary Table S1.

Species diversity of Archigetes Leuckart, 1878
Several taxa have been placed in Archigetes, but only the four following species were recently recognised as valid [48]: A. sieboldi Leuckart, 1878 (type species); A. brachyurus Mrázek, 1908 (both species found in oligochaetes and fish [43]); A. cryptobothrius Wisniewski, 1928 (known only from oligochaetes); and A. iowensis (species reported from oligochaetes and fish). Scholz and Oros [48] erroneously listed Paraglaridacris limnodrili (Yamaguti, 1934) Mackiewicz, 1994 also among valid species of Archigetes. The validity of two species, namely A. brachyurus and A. cryptobothrius, which have been reported only from Europe [19,43], could not be critically assessed because of the lack of properly fixed material and molecular data.
Archigetes sieboldi and A. iowensis also occur in North America and their taxonomic status and biogeography are briefly discussed below. In addition to the three unidentified morphotypes of Archigetes recently reported from Mexico [49], another three new species found in catostomids from the southern USA are described below based on morphological and molecular data. Material studied: Four slides with eight whole-mounted specimens and five slides with sagittal and cross sections of another specimen from I. bubalus (US 257a and 260b -PBI464); five slides with nine specimens (one incomplete) and five slides with longitudinal and cross sections of another specimen from I. niger (US 244b); one slide with one wholemounted specimen from C. cyprinus (host code No. US 262b), all specimens from Chotard Lake near Vicksburg, Representative DNA sequences and phylogenetic relationships: Sequences of one individual from I. bubalus (US 260d/PBI-464) in Mississippi were provided by Scholz et al. [50]: ssr DNA (GenBank Accession Nos. MW027441), lsr DNA (MW027502) and rrnL (MW027379); Scholz et al. [50]). This species differs most conspicuously from all other sequenced species in lsrDNA sequences by 16-20 nt (1.18-1.48%) (Supplementary Table S1).
Etymology: The species name loculotruncatus refers to the loculotruncate scolex, which is a unique characteristic of this new species among other members of the genus Archigetes.
Uterus forms several loops, with single loop extending slightly anterior to cirrus-sac (Figs. 2C, 3C); uterine glands welldeveloped, absent only in distal and proximal parts of uterus. Eggs operculate, without fully formed oncosphere in utero.

Differential diagnosis
The new species is the largest member of the genus with a total body length of 6.0-8.3 mm vs 1.1-3.5 mm in the other five valid species. It also differs from all valid species of Archigetes, including the other two new species described below, in possessing a loculotruncate (Figs. 2B; 3B, 3C; 4A-4E; 5A-5C), rather than a bothrioloculodiscate or bulboloculate scolex as reported in other species (for types of scolecessee Mackiewicz [30]).
The new species is also distinguished from the five previously known species by (i) a relatively long neck, whose length represents 10-15% of the total body length and which is absent or indistinct in the other species; (ii) a butterfly-shaped ovary in the new species vs dumb-bell-shaped ovary; (iii) an elongate seminal vesicle, which is longer than the diameter of the cirrus-sac in the new species vs spherical and smaller than the cirrus-sac in the other valid species.

Remarks
The new species is placed in the genus Archigetes based on morphological and molecular data (see Fig. 1 in Scholz et al. [50]), but differs from nominal species in having a distinct, long neck and a butterfly-shaped ovary. Archigetes loculotruncatus has been found in two species of Ictiobus and in Carpiodes cyprinus, i.e., suckers of the subfamily Ictiobinae. Most specimens were found in Chotard Lake, a former branch of the Mississippi River on the Mississippi-Louisiana state line. However, some specimens have also been found in the quillback from the Pascagoula River near Benndale, in southeastern Mississippi, in black buffalo from the Sunflower River in Indianola, east of the Mississippi River in central Mississippi, and smallmouth buffalo from Reelfoot Lake in Tennessee. This indicates a relatively large range for the new species in the southern USA.   Table S1).
Etymology: The species name, megavery big; cephalushead or scolex, refers to a conspicuously large scolex, the width of which considerably exceeds that of the neck.
Uterus forms several loops, one loop extending slightly anterior to cirrus-sac (Fig. 6C, 8C); uterine glands welldeveloped, absent only in distal and proximal parts of uterus. Eggs operculate, without fully formed oncosphere in utero.

Differential diagnosis
The new species differs from other congeneric species, including Archigetes loculotruncatus n. sp., by the shape and relative size of the scolex, which is bothrioloculodiscate and conspicuously (> 2Â) wider than the body (Figs. 6A, 6B; 7A-7D; 8A, 8B). The new species can also be distinguished from A. loculotruncatus n. sp. by the presence of vitelline follicles nearly always present alongside the ovarian wings, whereas only some specimens of A. loculotruncatus n. sp. have a few follicles laterodorsal to the ovary and they never form a continuous row as in A. megacephalus n. sp. (compare Figs. 2C, 3C and 6C). In addition, the new species can be distinguished by some subtle biometrical differences (see also Table 2).

Remarks
Archigetes megacephalus n. sp. was found in all three species of buffalo (Ictiobus spp.) examined from Chotard Lake (western part of Mississippi State), and in smallmouth buffalo from Reelfoot Lake, Tennessee. The new species was found sympatric with A. loculotruncatus n. sp. in the anterior part of the intestine in three fish individuals, one black buffalo (US 244) and two smallmouth buffalo (US 257; RF3/490). These new species can be distinguished from each other mainly by the shape and size of the scolex and body length, but live cestodes are highly mobile and change shape, including the scolex. For this reason, identification of live specimens with the naked eye can be difficult in mixed infections. Specimens should be fixed with hot fixative so that they retain their natural shape and do not shrink unnaturally, as is typical for specimens fixed with unheated fixative.    Etymology: The species name vadosus (= shallow in Latin) refers to the unusually shallow pair of median loculi.
Uterus forms several loops, one loop extending anterior to cirrus-sac (Fig. 9C); uterine glands well-developed, absent only in distal and proximal parts of uterus. Eggs operculate, without fully formed oncosphere in utero. The new species differs from the five previously known species of Archigetes by having an elongate body with a distinct neck, and from the two new species by possessing a bulboloculate scolex with shallow median loculi, almost indistinct lateral loculi and indistinct apical disc (Figs. 9A, 9B; 10B-10D). Archigetes vadosus n. sp. can also be distinguished from most congeners by the complete absence of vitelline follicles alongside the ovarian wings (present in other species). Other biometrical differences from A. loculotruncatus n. sp. and A. megacephalus n. sp. are obvious in Table 2. The new species is closely related to A. sieboldi (Fig. 1), but differs considerably by body shape, especially the presence of a distinct neck region which separates the scolex from the remaining body, a butterfly-, rather than dumb-bell-shaped ovary, and the external seminal vesicle longer than the diameter of the cirrus-sac.

Remarks
Archigetes vadosus n. sp. was found in only one species of buffalo, I. bubalus, in the Pascagoula River, neighbouring Bluff Creek, Mississippi, and Reelfoot Lake, Tennessee. One smallmouth buffalo from Reelfoot Lake harboured all three new species, one A. loculotruncatus, five A. megacephalus, and eight A. vadosus. Interestingly, A. vadosus n. sp. was not found in Chotard Lake, where two other new species commonly occurred in two smallmouth buffaloes examined.
Uterus forms several loops, with distal part of uterus not extending farther anteriorly than cirrus-sac (Fig. 11). Uterine glands well-developed, absent only in distal and proximal parts of uterus. Eggs operculate, without fully formed oncospheres in utero.

Remarks
Calentine [5] described A. iowensis based on adults found in common carp and progenetic plerocercoids in the naidid L. hoffmeisteri from the Iowa River. The same author [6] studied the life cycle of this species in laboratory conditions. Thereafter, the species was found in common carp from the Red Cedar River in Wisconsin by D.R. Sutherland in 1979 (voucher deposited as USNM 1397329), but this record does not seem to have been published. Williams [58] also found A. iowensis in L. hoffmeisteri in Wisconsin (USNM 1370351, 1370352), but designated his specimens as "Archigetes limnocesti", which is a non-existing name (nomen nudum).
Comparison of North American specimens of A. iowensis from cypriniforms and oligochaete host with those of Paraglaridacris limnodrili from the Palearctic region has revealed that these species can be distinguished from each other neither by their morphology nor measurements [18,43,61].
Both species are typified by (i) a compact, dumb-bellshaped ovary; (ii) a small body separated by a short, but distinct neck, which is slightly narrower than the scolex, anterior-most testes being always anterior to anterior-most vitelline follicles; (iii) largely lateral preovarian vitelline follicles, with a very few median folllicles (Calentine [5] originally the preovarian vitelline follicles were reported as exclusively lateral, but Mackiewicz [30] corrected this characteristic); (iv) vitelline follicles uninterrupted alongside (dorsolateral) the ovarian wings, thus making bands of pre-and postovarian follicles continuous; (v) the uterus reaching anteriorly only to the anterior margin of the cirrus-sac; and (vi) external seminal vesicle shorter than the diameter of the cirrus-sac. Archigetes iowensis and P. limnodrili have been found as adults in cypriniforms and as progenetic plerocercoids in oligochaetes [5,61]. Based on the rule of priority, A. iowensis becomes a junior synonym of P. limnodrili and the number of valid species of Archigetes is reduced to six (three new species, A. sieboldi, A. brachyurus and A. cryptobothrius).

Remarks
Calentine and DeLong [4] and Calentine [7] studied the life history of A. sieboldi from the Kinnickinnic River in Wisconsin. The authors concluded that gravid stages occur in oligochaetes and occasionally in fish because a high prevalence of infection with A. sieboldi in oligochaetes compared to its rare occurrence in fish indicates that A. sieboldi is primarily a parasite of oligochaetes. Wiśniewski [60] and Nybelin [34] in Europe failed to infect cyprinids, but Kulakovskaya [23] successfully infected cyprinids (tench -Tinca tinca (L.)) with A. sieboldi from oligochaetes.
The specimen from Chirostoma humboldtianum in Mexico, designated as Archigetes sp. 3 by Scholz and Pérez-Ponce de León [49] (CNHE 6801), corresponds in its morphology to A. sieboldi found by Calentine and DeLong [4] in the USA, including overall shape of the body, with the scolex of a similar width as the remaining body, a more anterior position of vitelline follicles compared to that of the anterior-most testes, follicular, dumb-bell-shaped ovary, preovarian vitelline follicles lateral and median, with follicles lacking at the ovarian level, thus making pre-and postovarian vitelline follicles separated. Therefore, this specimen is considered to belong to A. sieboldi from C. humboldtianum in Mexico and represents a new definitive host and new geographical area, but this fish species should be confirmed as a true host of A. sieboldi.

Unidentified species of Archigetes
Scholz and Pérez-Ponce de León [49] reported three morphotypes of Archigetes from two species of shiners, Notropis spp., and Chirostoma humboldtianum in the Nearctic part of Mexico. These specimens differ conspicuously from the newly described species from the USA in their much smaller size (total length up to 2.50 mm), dumb-bell-shaped ovary, uterine loops reaching far anterior to the cirrus-sac, and the external seminal vesicle smaller than the cirrus-sac.
All the three morphotypes can be distinguished from Paraglaridacris limnodrili (syn. A. iowensis) by the lateral and median position of preovarian vitelline follicles (follicles form two lateral bands only, with only very few median follicles in A. iowensis), and by much shallower loculi on the scolex in Mexican specimens (conspicuous, deep loculi in A. iowensis). The third morphotype from C. humboldtianum (Archigetes sp. 3) resembles in its morphology A. sieboldi found by Calentine and DeLong [4] in North America and is considered conspecific (see above).
Key to the identification of species of Archigetes Leuckart, 1878 and Paraglaridacris Janiszewska, 1950

in North America
Because of the lack of newly collected or museum material of A. brachyurus and A. cryptobothrius, the identification key includes only five species of both morphologically similar genera that occur in North America. Morphotypes Archigetes sp. 1 and sp. 2 from Mexico are not included (see above).
1a. Ovary compact; anterior-most testes anterior to anteriormost vitelline follicles; median vitelline follicles almost completely absent (a few follicles may be present medially

Discussion
Previously, only two nominal taxa of Archigetes have been reported in North America [4][5][6][7]58]. New data greatly expand the known spectrum of definitive hosts for Archigetes species that now include also catostomids (suckers). Three new species were found in the southern part of the USA, whereas Paraglaridacris limnodrili (new syn. A. iowensis) and A. sieboldi were previously known only from the Midwest (Iowa and Wisconsin). The present study, together with previous studies by the present authors, clearly demonstrates that the Nearctic fauna of freshwater fish tapeworms is still severely underexplored and insufficiently known [21,47].
All new North American species of Archigetes have an elongate body with a distinct, relatively long neck (usually absent or indistinct in species described in Europe), butterflyshaped ovary, and a conical to globular scolex wider than the neck. The new species also share the presence of a small, shallow genital atrium to which the male and female (uterovaginal canal) gonopores open separately, thus not forming a single hermaphroditic duct. The presence of a single common gonopore or two separate gonopores has been considered taxonomically important at the genus-level. Mackiewicz [30] distinguished four types, but some of them are difficult to differentiate from each other, depending in part on the contraction or relaxation of the worm.
Species of Archigetes and the closely related genus Biacetabulum Hunter, 1927 should have a single gonopore according to Mackiewicz [30]. However, recent studies of Biacetabulum spp. have shown that the male and female gonopores are separate and do not form a hermaphroditic duct, but open into a shallow genital atrium (see [54,55]). Thus, using the terminology of Mackiewicz [30] and his key to the genera of the Caryophyllidea, all species of these two genera would be classified as having separate gonopores, but in fact have a single porea mouth of the common genital atrium. This situation is consistent with observations of Yamaguti [61] and Protasova et al. [43].
Johnston and Muirhead [17] described, based on a single specimen, Biacetabulum tandani Johnston and Muirhead, 1950 from the plotosid catfish Tandanus tandanus (Mitchell) in Australia. The authors placed the species in Biacetabulum because of the anterior extent of the uterus far anterior to the cirrus-sac and the presence of two acetabulum-like loculi on the scolex. Scholz and Oros [48] recognised this species as valid, but Uhrovič et al. [56] questioned its generic assignment. However, B. tandani is provisionally retained in Biacetabulum until new material is available. The most intriguing features of this poorly known species are its occurrence in a very distant zoogeographical distribution from that of species of both genera, which occur in the Palaearctic and Nearctic zoogeographical regions, and the presence of an external seminal vesicle, which is a morphological characteristic present only in most species of the Capingentidae, including Archigetes and Biacetabulum (see [50]). A feasible explanation of the occurrence of B. tandani in Australia could be possible introduction of this species with imported common carp to Australia and subsequent host switch to local plotosid catfish which may represent an accidental host. However, there is no supporting evidence for this assumption because no data are available on the occurrence of caryophyllidean tapeworms in introduced common carp in Australia.
One of the interesting results of molecular analysis is that both European isolates of A. sieboldi fall within the same clade as A. vadosus n. sp. However, these tapeworms are not considered conspecific because they differ considerably from each other in their morphology, including shape of the ovary (butterfly-shaped in the new species versus dumb-bell-shaped in A. sieboldi), different spectrum of fish hosts (Catostomidae versus Cyprinidae) and partly zoogeographical distribution (southern USA versus Holarctic, with most records in Europe). Comparison of sequence data from A. sieboldi from North America with A. vadosus could be quite instructive, but no ethanol-fixed material of A. sieboldi from North America is available. Another obstacle to better understand relationships between caryophyllidean tapeworms is the existence of multiple mt haplotypes that most likely represent paralogs (i.e. numts), which were detected by Brabec et al. [3] in two mitochondrial genes (cox1 and nad3).
Molecular phylogenetic analysis also revealed that four North American species of Archigetes form a monophyletic group nested within the clade composed of Biacetabulum spp. (Fig. 1), which is consistent with the results in Scholz et al. [50]. The species of Archigetes form a sister lineage to the clade composed of the three so-called long-necked, recently described species of Biacetabulum from redhorses and spotted suckers [54].
The close relationship between the species of Archigetes and Biacetabulum is not surprising, and some species have been placed in both genera, such as Caryophyllaeus appendiculatus Ratzel, 1868 (probably larva of Caryophyllaeus laticeps (Pallas, 1781)) in Archigetes and Biacetabulum [19]. Hunter [14] mentioned close morphological similarity between these genera when he erected Biacetabulum and differentiated them only by exclusive egg production of progenetic plerocercoids in oligochaetes for species of Archigetes versus full maturation exclusively in teleosts for species of Biacetabulum.
Based on the ICZN [15] priority rule, Archigetes has priority and all species of Biacetabulum would be transferred to this genus, provided that both genera are considered synonymous. Unfortunately, no molecular data are available for the type species of B. infrequens Hunter, 1927. Therefore, the synonymy of the two genera is not yet proposed. However, these two genera could be distinguished from each other by a somewhat smaller body in Archigetes (less than 8 mm, usually less than 5 mm in Archigetes vs 4-16 mm in Biacetabulum), an H-shaped ovary in Biacetabulum vs dumb-bell-shaped ovary in Archigetes, the extent of vitelline follicles, which may be present alongside the ovary in Archigetes but are absent in Biacetabulum, and uterine loops extending slightly anterior to the cirrus-sac in Archigetes vs uterine loops extending far anterior to the cirrus-sac in Biacetabulum. The addition of three new species with a larger body and a butterfly-shaped ovary implies that the generic diagnosis of the genus should be amended:

Archigetes Leuckart, 1878
Amended generic diagnosis: Caryophyllidea: Capingentidae sensu Scholz et al. [50]. Body small (less than 10 mm, usually only a few mm), with maximum width most commonly at middle part of body. The taxonomy of the genus Archigetes is still insufficiently clear because European taxa were described superficially, without any type or voucher specimens deposited in collections.
Archigetes brachyurus and A. cryptobothrius were described in the first third of the 20th century from oligochaetes L. hoffmeisteri from the Czech Republic and Poland, respectively [33,59]. Both species seem to differ from each other especially in the shape of the scolex, with A. brachyurus having a hexagonal scolex with deep loculi and a distinct, narrow neck (see [33]), whereas A. cryptobothrius has a short scolex, only slightly wider than a wide, almost indistinct neck, and bears shallow median loculi (see [59]).
Kennedy [19] differentiated both species only by the number of testes, which in fact overlaps: 120-159 in several rows of 30-40 each in A. brachyurus, versus 154 in several rows of 18-26 each, in A. cryptobothrius. In addition, this differential criterion is questionable because of difficulties in reliably counting numerous testes and their aggregation in rows. Nevertheless, both species are considered valid by most authors [19,43,48]. Protasova et al. [43] reported several cyprinids as hosts of A. brachyurus, namely Abramis brama (L.), Barbus barbus (L.), Barbus petenyi Heckel, Gobio gobio, and Vimba vimba (L.). However, no details on geographical origin (Dniester, Prut or Northern Doniets basins) and morphology of these specimens were provided. Protasova et al. [43] illustrated only progenetic plerocercoids from L. hoffmeisteri but these larvae identified as A. brachyurus differ from each other considerably in the shape of the body and scolex, and in the number of testes. Archigetes cryptobothrius has been reported only once since its original description, in L. hoffmeisteri from the Susaa River at Møllebro and Nymøllebro in Denmark [2].
Mackiewicz [30] proposed Paraglaridacris Janiszewska, 1950 as the first available name for Brachyurus Szidat, 1938, which was preoccupied by Brachyurus Fischer-Waldheim, 1813 (a genus of rodents). Szidat [53] erected Brachyurus, apparently unaware that this generic name was preoccupied, to accommodate B. gobii Szidat, 1938, which he found in G. gobio from small fishpond around the former Prussian town of Rossitten (now Rybachy in the Kaliningrad enclave of Russia) in Curonian Spit. Kennedy [19] synonymised this species with Yamaguti's [61] Glaridacris limnodrili as A. limnodrili. Mackiewicz [30] did not question conspecificity of both taxa, but considered G. limnodrili to belong to Brachyurus Szidat, 1938, nec Fischer-Waldheim, 1813 (= Paraglaridacris sensu Mackiewicz, 1994). Comparison of A. iowensis specimens with Paraglaridacris limnodrili did not reveal any difference in their morphology and measurements in the present study ( Table 3). As a result, both taxa are considered conspecific, with A. iowensis becoming a new junior synonym of P. limnodrili.
The second species, Paraglaridacris silesiacus Janiszewska, 1950, was described based on a single decomposed specimen from common bream, A. brama, in Poland [16]. The author even considered the new species description as provisional ("Comprenant qu'il s'agit d'une espèce, probablement assez rare, je résolus d'en donner une description provisoire."). Morphological description of this species was not detailed and Janiszewska [16] did not recognise the presence of postovarian vitelline follicles, which was later corrected by Mackiewicz [27], who studied the holotype of P. silesiacus.
The validity of Paraglaridacris sensu Mackiewicz, 1994 could not be confirmed using molecular data, because no ethanol-fixed material was available. However, validity of the genus is supported by the following morphological characteristics, in which this genus differs from Archigetes: (i) compact ovary (versus follicular in Archigetes); (ii) largely lateral preovarian vitelline follicles, with only very few follicles median (median follicles are common in Archigetes); (iii) more anterior position of the first testes compared to vitelline follicles (versus the testes usually posterior or at same level as the anterior-most vitelline follicles). The difference in the structure of the ovary is conspicuous and enables easy differentiation between the specimens of both genera. Therefore, Paraglaridacris is provisionally considered a valid genus. It should also be mentioned here that two of the present authors contributed to the existing confusion as to nomenclature of these tapeworms [48].
The host specificity of most species of Archigetes and P. limnodrili was previously considered narrow, especially in North America. Calentine [6] examined 975 fishes representing 20 species from the same locality and only the common carp was infected, as in experimental infections. He also examined four species of oligochaetes but only L. hoffmeisteri was infected, with the same results of experimental infections of oligochaetes. A similarly narrow host range was reported for A. sieboldi in North America [4,7], but a presumably conspecific tapeworm was found in Chirostoma humboldtianum from Mexico by Scholz and Pérez-Ponce de León [49]. The present record of A. iowensis in smallmouth buffalo, i.e., fish of a different family (Catostomidae), also challenges the abovementioned assumption about strict host specificity of these species at the level of fish definitive host. Indeed, a broader host spectrum has been reported for A. sieboldi and P. limnodrili in the Palaearctic region ( [43,21], present study) and for two of the three new species of Archigetes described herein, because they occur in three species of fish hosts.