Hidden Diversity in European Allocreadium spp. (Trematoda, Allocreadiidae) and the Discovery of the Adult Stage of Cercariaeum crassum Wesenberg-Lund, 1934

Abstract

DNA sequences for adult and larval Allocreadium spp. from their natural fish and molluscan hosts were generated. Phylogenetic analyses based on two molecular markers (ITS2 and 28S rDNA) yielded unexpected results regarding the diversity and life cycles of European species. It was found that specimens morphologically consistent with the concept of Allocreadium isoporum (Looss 1894) form two different species-level genetic lineages. For now, the morphological differences between the specimens belonging to different genetic lineages are not discernible; they can infect the same fish species at the same or different localities. However, the species differ in their life-cycle patterns, specifically in terms of larval stages and first intermediate host specificity. Based on molecular markers, the tailed ophthalmoxiphidiocercaria developing in Pisidium spp. was associated with a sexual adult A. isoporum from Alburnus alburnus, Barbatula barbatula and Rutilus rutilus. Representatives of another genetic lineage, recovered from R. rutilus and Scardinius erythrophthalmus, turned out to be conspecific with the enigmatic European larval trematode Cercariaeum crassum Wesenberg-Lund, 1934, from the sphaeriid bivalve Pisidium amnicum. This finding requires the recognition of the cryptic species Allocreadium crassum.

1. Introduction

Morphological recognition of digenean species can be obscured by age, as well as by host-induced intraspecific phenotypic variation or cryptic speciation (morphologically conservative species). In addition to natural morphological variability, the gross morphology of trematodes is strongly influenced by specimen preparation and fixation methods [1]. Cryptic trematode species are encountered more commonly than other helminth species, and consequently many trematode species requiring proper characterization are likely to remain hidden within previously described taxa [2]. Due to the existence of cryptic species, the identification of larval stages and the elucidation of life cycles also often faces great challenges.

The digeneans of the family Allocreadiidae Looss, 1902, are restricted to freshwater ecosystems [3]. These relatively small digeneans inhabit the digestive system of teleosts. In Lithuania and other European countries, four genera are known in the family Allocreadiidae: Allocreadium Looss, 1900, Bunodera Railliet, 1896, Crepidostomum Braun, 1900 and Stephanophiala Nicoll, 1909. Hidden diversity was uncovered in the genus Bunodera and Crepidostomum during molecular surveys of European species, and new species were described based on molecular findings [4,5,6,7]. Moreover, existing confusion in life cycles due to the incorrect morphological identification of adult and larval stages became apparent in molecular studies of the European species of the genus Bunodera [4].

Trematodes of the genus Allocreadium are widely distributed and are one of the most frequent species of parasites of freshwater fish, especially cyprinids, in the Palearctic region. Allocreadium isoporum (Looss 1894) Looss, 1900, the type species of the genus, was originally described from various cyprinids from Germany. Subsequently, it was recorded not only from a wide range of cyprinids, but also from some other fishes [8,9,10]. According to Moravec [11], it is possible to judge that other congeneric species have also often been included under this name. Individual morphological variability was noticed in this species [11,12], and different morphological forms were described as subspecies of this trematode: A. isoporum dubium Koval, 1957 and A. isoporum macrorchis Koval et Kulakowskaya, 1957. However, according to Moravec [11], A. isoporum dubium and A. isoporum macrorchis are within the limits of individual intraspecific variability and are therefore suppressed.

Relatively few of the large number of species assigned to the genus Allocreadium have had their larval stages described. The larval (asexual) allocreadiid stages use fingernail clams as first intermediate hosts and aquatic arthropods as second intermediate hosts. Known cercariae of this family typically have the form of ophthalmoxiphidiocercariae (i.e., with eye-spots and stylet). The distinguishing characters for cercariae of different allocreadiid genera are weak and rife with homoplasy, and as a result, their morphological demarcation is under discussion [13]. The history of the long-lasting studies on the life cycle of the type species, A. isoporum, is very confusing. Descriptions for the cercaria of A. isoporum were given by Looss [14], Dolfus [15] and Wiśniewski [16]. Conclusions on the life cycle were based on morphological similarities and ecological evidence and, despite the absence of experimental studies or any other more reliable evidence, by common assent, the name A. isoporum was used for this cercaria in numerous subsequent publications. However, comparative analysis of the ITS2 and 28S sequences of the cercaria of A. isoporum sensu Wiśniewski, 1958 (ophthalmoxiphidiocercaria characterized by a tail surrounded by a broad tegumental inflation), developing within rediae in Sphaerium corneum and S. rivicola revealed their identity as adult Bunodera luciopercae from Perca fluviatilis. On the other hand, identical rDNA was revealed for the sexual adult of B. acerinae Roitman and Sokolov, 1999, and cercaria described by Wiśniewski [16] as the larval form of B. luciopercae (see [4]). These findings point to the need to review and reassess established knowledge about even the life cycles of well-known trematode species and, in particular, to determine the larval stages of A. isoporum.

In a continuation of our efforts to uncover the allocreadiid diversity in European freshwater fishes and to clarify their life cycles, a new phylogenetic analysis of the Allocreadiidae was performed using sequences of the ITS2 region and 28S ribosomal rRNA gene of newly sampled adult and larval specimens from our field collections from several localities of Lithuania, Norway, and Slovakia in 2015–2018. In this current study, we report a molecular analysis of Allocreadium species infecting cyprinid fish that morphologically most closely resemble A. isoporum. However, the levels of molecular divergence between the “Allocreadium isoporum” isolates indicate two distinct species.

2. Materials and Methods

2.1. Sample Collection and Morphological Observation

Adult specimens of Allocreadium spp. were recovered from the intestine of common roach, Rutilus rutilus (Linnaeus, 1758), common rudd, Scardinius erythrophthalmus (Linnaeus, 1758) and spined loach, Cobitis taenia Linnaeus, 1758. Respectively, the fish hosts were caught in the Curonian Lagoon, Lake Ilmėdas and Lake Balsys, Lithuania. The collected trematodes were identified in vivo, fixed in 96% ethanol for molecular analysis and stored at −20°C. To confirm morphological identification, living specimens were photographed with the aid of a digital camera on the light microscope Olympus BX51 (Tokyo, Japan). Adult specimens consistent with the species A. isoporum were collected from R. rutilus and S. erythrophthalmus and were identified based on descriptions by Bykhovskaya and Kulakova [8], Moravec [11] and Niewiadomska [10]. Adult specimens collected from C. taenia consistent with the morphological characteristics of A. transversale (Rudolphi, 1802) were identified based on descriptions of Koval [17] and Bykhovskaya and Kulakova [8]. The most conspicuous morphological feature distinguishing our specimens from the morphologically close A. baueri Spassky et Roitman, 1960, was the short oesophagus, bifurcating before the ventral sucker, while A. baueri is characterised by a long oesophagus, bifurcating close to the level of the posterior margin of the ventral sucker [17].

Sphaeriid clams were collected from freshwater bodies in Norway and Slovakia using a hand-net. The clams were dissected with the aid of a stereo microscope and examined for the presence of rediae and cercariae. Photomicrographs of live cercaria were taken with a microscope for measurements and further identification; all measurements are presented in micrometres. Samples of cercariae were fixed in 96% ethanol for molecular analysis.

2.2. DNA Sequencing and Phylogenetic Analysis

Total genomic DNA was extracted from the ethanol-fixed specimens according to Stunžėnas’ protocol [18,19]. Amplification and sequencing of the two rDNA markers, the nuclear second internal transcribed spacer (ITS2 rDNA) and the large (28S) ribosomal subunit RNA coding regions were performed following the protocol used in our previous studies [19,20,21]. DNA fragments spanning the 3′ end of the 5.8S rRNA gene, the complete internal transcribed spacer 2 region (ITS2) and a small section at the 5′ end of the 28S gene were amplified using universal primers for flatworms, the forward primer 3S (5′-CGG TGG ATC ACT CGG CTC GTG-3′) [22] and the reverse primer ITS2.2 (5′-CCT GGT TAG TTT CTT TTC CTC CGC-3′) [23]. The end of the internal transcribed spacer 1 (ITS1), the complete 5.8S rDNA and ITS2, and also a small section at the 5′ end of the 28S gene, were amplified using the forward primer AlJe-F (5′-GTCTGG CTT GGC AGT TCT A-3′) and the reverse primer AlJe-R (5′-CTG CCC AAT TTG ACC AAG C-3′) [6]. A fragment at the 5′ end of the 28S rRNA gene was amplified using the forward primers Digl2 (5′-AAG CAT ATC ACT AAG CGG3-′) or ZX-1 (5′-ACC CGC TGA ATT TAA GCA TAT3-′) [24] and the reverse primers L0 (5′-GCT ATC CTG AG (AG) GAA ACT TCG3-′) [25] or 1500R (5′-GCT ATC CTG AGG GAA ACT TCG3-′) [26,27]. Polymerase chain reaction (PCR) products were purified and sequenced by the Sanger sequencing method in both directions at BaseClear B.V. (Leiden, Netherlands) using the PCR primers. Contiguous sequences were assembled using Sequencher 4.10.1 software (Gene Codes Corporation, Ann Arbor, MI, USA). Estimates of mean evolutionary divergence over sequence pairs within and between groups were calculated using the MEGA v.11.0.11 programme. Newly generated 28S and ITS2 sequences from intramolluscan and adult stages were compared and identical, and similar and related sequences for phylogenetic analyses were found by the “Basic Local Alignment Search Tool” (BLAST) [28]. Both the ITS2 and 28S datasets were aligned independently using ClustalW [29] with an open gap penalty of 15 and gap extension penalty of 6.66. The best-fit model of sequence evolution for phylogenetic analysis was estimated using jModeltest v.0.1.1 software [30]. The maximum likelihood (ML) trees were obtained using the general time reversible model with a gamma distribution rate (GTR + G) for both the ITS2 and the 28S gene datasets. The value for gamma and the number of invariant sites were estimated from the data. Parsimony analysis based on subtree pruning and regrafting (SPR) was used with default parsimony settings. Branch support was estimated by bootstrap analyses with 1000 pseudoreplicates. If two or more sequences belonged to one species, they were collapsed into one branch, except those newly obtained in this study. Estimates of mean evolutionary divergence over sequence pairs within and between groups were calculated using the MEGA v.11.0.11 programme [31]. The newly generated sequences of Allocreadium spp. were deposited on GenBank (see accession numbers in

Table 1. Species subjected to molecular phylogenetic analysis with information on their host, locality and GenBank accession numbers.

Species	Host	Locality	GenBank ID (Reference) **
			28S	ITS2
Allocreadium apokryfi	Labeobarbus aeneus	South Africa: Vaal River	MW907591 [32]
Allocreadium crassum	Rutilus rutilus	Lithuania: Curonian Lagoon	OQ359131, OQ359132	OQ359139, OQ359140
Allocreadium crassum	Scardinius erythrophthalmus	Lithuania: Lake Ilmėdas	OQ359133	OQ359141
Allocreadium crassum (=Cercariaeum crassum) *	Pisidium amnicum	Lithuania: River Ūla	JF261144 [33]	JF261152 [33]
Allocreadium crassum (=Cercariaeum crassum)	Pisidium amnicum	Lithuania: River Žeimena	GU462117, GU462118, [33]	JF261145, JF261148 [33]
Allocreadium crassum (=Cercariaeum crassum)	Pisidium amnicum	Finland: Siilaisenpuro River	JF261141, JF261142 [33]	JF261149, JF261150 [33]
Allocreadium isoporum	Rutilus rutilus	Lithuania: Curonian Lagoon	OQ359125
Allocreadium isoporum *	Pisidium milium	Norway: Lake Hurdalsjøen	OQ359126	OQ359134
Allocreadium isoporum *	Pisidium milium	Norway: Lake Hurdalsjøen		OQ359135
Allocreadium isoporum *	Pisidium sp.	Slovakia: Rivulet in Panovce	OQ359127
Allocreadium isoporum	Alburnus alburnus	Russia: Lake Oster, Karelia	GU462125, GU462126 [4]	FJ874921 [4]
Allocreadium isoporum	Barbatula barbatula	Russia: River Il’d, upper Volga River basin	MH143102 [6]	MH143096 [6]
Allocreadium gotoi	Misgurnus anguillicaudatus	Japan: Nagano, Iiyama, Midori	LC215274 [34]
Allocreadium hemibarbi	Hemibarbus labeo	Russia: Primorsky territory	MK211220, MK211222 [35]
Allocreadium khankaiensis	Phoxinus oxycephalus	Russia: Primorsky territory	MK211211, MK211212 [35]
Allocreadium khankaiensis	Barbatula toni	Russia: Artyomovka River	MZ448168 [35]
Allocreadium khankaiensis	Rhynchocypris lagowskii	Russia: Pavlovka River	MZ448176 [36]
Allocreadium lobatum	Semotilus corporalis	USA: Moosehead Lake, Maine	EF032693 [37]
Allocreadium neotenicum	Hydroporus rufifrons	United Kingdom: Lake District, Cumbria	JX977132 [38]
Allocreadium neotenicum	Oreodytes sanmarkii	Norway: Lake Takvatn	KY513133 [5]
Allocreadium neotenicum *	Pisidium casertanum	Ukraine: River Burulcha, Crimea	MH143103 [6]	MH143075 [6]
Allocreadium neotenicum *	Pisidium casertanum	Norway: Lake Takvatn	MH143104 [6]	MH143076 [6]
Allocreadium neotenicum *	Pisidium sp.	Norway: Lake Nordersjoen	MH143105 [6]	MH143077 [6]
Allocreadium pseudoisoporum	Carassius gibelio	Russia: Primorsky territory	MK258687 [36]
Allocreadium transversale	Cobitis taenia	Lithuania: Curonian Lagoon	OQ359128, OQ359129	OQ359136, OQ359137
Allocreadium transversale	Cobitis taenia	Lithuania: Lake Balsys	OQ359130	OQ359138
Allocreadium sp. C51Ukr (=Crepidostomum sp.) *	Sphaerium corneum	Ukraine: River Belka, Dnieper River basin	GU462121 [4]	FJ874919 [4]
Allocreadium sp. 1	Phoxinus phoxinus	Russia: Nadezhdinsky district, tributary of the River Nezhinka	MK211209, MK211210 [35]
Allocreadium sp. *	Pisidium amnicum	Russia: River Tvertsa, upper Volga River basin		FJ874923 [4]
Bunodera acerinae	Gymnocephalus cernua	Russia: Lake Segozero, Karelia	GU462114 [4]	FJ874914 [4]
Bunodera acerinae *	Pisidium amnicum	Russia: River Tvertsa, upper Volga River basin	GU462112, GU462113, GU462122 [4]	FJ874911 [4]
Bunodera inconstans	Culaea inconstans	Canada: Brokenhead River in Manitoba;	DQ029330 [39]
Bunodera luciopercae	Perca fluviatilis	Lithuania: Curonian Lagoon	MH143101 [6]	MH143097 [6]
Bunodera luciopercae	Perca fluviatilis	Russia: Lake Segozero, Karelia	GU462115 [4]	FJ874917 [4]
Bunodera luciopercae	Perca fluviatilis	Russia: River Tvertsa, upper Volga River basin	GU462123 [4]	FJ874918 [4]
Bunodera luciopercae *	Sphaerium rivicola	Lithuania: dammed up River Nemunas near Kaunas	GU462116 [4]	FJ874916 [4]
Bunodera luciopercae *	Sphaerium rivicola	Ukraine: River Teterev	GU462111 [4]	FJ874915 [4]
Bunodera luciopercae (Neoarctic)	Perca flavescens	Canada: Lake Sasajewun, Ontario	DQ029331 [39]
Bunodera mediovitellata	Gasterosteus aculeatus	Canada: Little Campbell River, British Columbia	DQ029332 [39]
Bunodera mediovitellata	Gasterosteus aculeatus	Canada: BC, Campbell Cr.	EF202573 [40]
Bunodera vytautasi	Pungitius pungitius	Russia: Magadan region	MG262545 [41]
Crepidostomum affine	Hiodon tergisus	USA: Pearl River, Mississippi	KF250358 [42]
Crepidostomum affine	Aplodinotus grunniens	USA: Pearl River, Mississippi		KF356363 [42]
Crepidostomum brinkmanni	Salmo trutta	Iceland: Lake Hafravatn	MT080777 [7]	MT080751 [7]
Crepidostomum brinkmanni (=Crepidostomum sp. 2) *	Pisidium casertanum	Ukraine: River Burulcha, Crimea	MH143117, MH143118, MH143119 [6]	MH143098, MH143099, MH143100 [6]
Crepidostomum brinkmanni (=Crepidostomum sp. 2) *	Pisidium casertanum	Norway: Lake Sagelvvatn	MH143115, MH143116 [6]	MH143087, MH143088, MH143089 [6]
Crepidostomum brinkmanni (=Crepidostomum sp. 2)	Salmo trutta	Norway: Lake Takvatn	KY513154 [5]
Crepidostomum brinkmanni (=Crepidostomum sp. 2) *	Siphlonurus lacustris	Norway: Lake Takvatn	KY513151 [5]
Crepidostomum brinkmanni (=Crepidostomum sp. 2) *	Diura bicaudata	Norway: Lake Takvatn	KY513152 [5]
Crepidostomum cooperi	Percopsis omiscomaycus	Lake Winnipeg, Canada	DQ029328 [40]
Crepidostomum cornutum	Lepomis gulosus	USA: Pascagoula River, Mississippi	EF032695 [37]	KF356374 [42]
Crepidostomum illinoiense	Hiodon alosoides	USA: Red Lake River, Minnesota	KF356372 [42]	KF356364 [42]
Crepidostomum oshmarini	Barbatula barbatula	Russia: River Il’d, upper Volga River basin	MH159990, MH159992 [6]	MH143095 [6]
Crepidostomum oshmarini	Cottus gobio	Russia: River Il’d, upper Volga River basin	MH159989, MH159991 [6]	MH143090, MH143091 [6]
Crepidostomum oshmarini *	Pisidium casertanum	Lithuania: River Nedzingė	MH159993, MH159994 [6]	MH143092, MH143093 [6]
Creptotrema (=Auriculostoma) astyanace	Astyanax aeneus	Costa Rica: Tempisquito River, Guanacaste	HQ833707 [43]
Creptotrema (=Auriculostoma) astyanace	Astyanax fasciatus	Costa Rica: Rio Sapoa, Guanacaste	KF631422 [39]
Creptotrema funduli (species inquirenda)	Fundulus notatus	USA: Mississippi, Biloxi River	JQ425256 [44]
Creptotrema (=Auriculostoma) lobata	Brycon guatemalensis	Mexico: Mangal Lagoon, Tabasco	KX954172 [45]
Creptotrema (=Auriculostoma) totonacapanense	Astyanax aeneus	Mexico: Metzabok, Chiapas	MK648262 [46]
Creptotrema (=Auriculostoma) totonacapanense	Astyanax mexicanus	Mexico: Filipinas, Veracruz	KF631420 [47]
Creptotrematina aguirrepequenoi	Astyanax aeneus	Costa Rica: Rio Tempisquito, Guanacaste	HQ833708 [43]
Margotrema bravoae	Allotoca dugesii	Central Mexico	KT833278 [48]
Megalogonia ictaluri	Ictalurus punctatus	USA	EF032694 [37]
Paracreptotrema blancoi	Priapichthys annectens	Costa Rica: Quebrada Plata, El Aguacate	KT833279 [48]
Paracreptotrema heterandriae	Heterandria bimaculata	Mexico: Agua Bendita, Xico, Veracruz	KF697697 [47]
Pseudoparacreptotrema axtlaensis	Dajaus monticola	Río Axtla, Axtla de Terrazas, San Luis Potosí, Mexico	MT180832 [49]
Pseudoparacreptotrema falciformis	Dajaus monticola	River at Matías Romero, Oaxaca, Mexico	MT180824, MT180828, MT180829 [49]
Pseudoparacreptotrema macroacetabulata	Profundulus candalarius	Central Mexico: Río San Carlos, Chia	KT833305 [48]
Pseudoparacreptotrema macroacetabulata	Profundulus sp.	Central Mexico: Río PuebloViejo, San Gabriel Mixtepec, Oax.	KT833299 [48]
Pseudoparacreptotrema pacificum	Dajaus monticola	Puente Novillero, Chiapas, Mexico	MT180810, MT180819 [49]
Stephanophiala pseudofarionis (=Crepidostomum pseudofarionis)	Salvelinus alpinus	Iceland: Lake Hafravatn	MT080789 [7]	MT080771 [7]
Stephanophiala pseudofarionis (=Crepidostomum sp. 1, =C. pseudofarionis) *	Sphaerium sp.	Norway: Lake Takvatn	KY513149 [5]
Stephanophiala pseudofarionis (=Crepidostomum sp. 1, =C. pseudofarionis) *	Siphlonurus lacustris	Norway: Lake Takvatn	KY513150 [5]
Stephanophiala pseudofarionis (=Crepidostomum sp. 1, =C. pseudofarionis)	Salmo trutta	Norway: Lake Sagelvvatn	MH143111, MH143112 [6]	MH143080, MH143082 [6]
Stephanophiala pseudofarionis (=Crepidostomum sp. 1, =C. pseudofarionis) *	Pisidium casertanum	Norway: Lake Sagelvvatn	MH143113, MH143114 [6]	MH143078, MH143081, MH143086 [6]
Stephanophiala pseudofarionis (=Crepidostomum sp. 1, =C. pseudofarionis) *	Pisidium sp.	Norway: Lake Sagelvvatn	MH143107, MH143108 [6]	MH143084, MH143085 [6]
Stephanophiala pseudofarionis (=Crepidostomum sp. 1, =C. pseudofarionis) *	Sphaerium nitidum	Norway: Lake Kykkelvatn	MH143106, MH143109, MH143110 [6]	MH143079, MH143083 [6]
Stephanophiala farionis (=Crepidostomum farionis) *	Pisidium casertanum	Norway: Lake Takvatn	KY513139 [5]
Stephanophiala farionis (=Crepidostomum farionis) *	Pisidium sp.	Norway: Lake Takvatn	KY513136 [5]
Stephanophiala farionis (=Crepidostomum farionis)	Oncorhynchus masou	Russian Far East	FR821399, FR821402 [50]
Wallinia anindoi	Astyanax aeneus	Mexico: San Juan del Rio, Oaxaca	MH997011 [51]
Wallinia brasiliensis	Astyanax fasciatus	Batalha River, Brazil	MH520995 [52]
Wallinia chavarriae	Bryconamericus scleroparius	Costa Rica	DQ991918 [53]
Wallinia mexicana	Astyanax mexicanus	Covadonga River, Durango, Mexico; Huichihuayan River, San Luis Potosí, Mexico	KJ535505 [54]
Outgroup
Phyllodistomum folium	Gymnocephalus cernua	Lithuania: Curonian Lagoon	KX957729 [55]	KY307885 [55]
Phyllodistomum angulatum	Sander lucioperca	Russia: Rybinsk water reservoir on the Volga river	KX957735 [55]	KJ740511 [55]
Phyllodistomum macrocotyle *	Dreissena polymorpha	Belarus: Lake Lepelskoe	AY288828 [50]	AY288831 [56]
Polylekithum catahoulensis	Ictalurus furcatus	USA: Lake Catahoula		EF032698 [37]
Prosthenhystera oonastica	Ictalurus furcatus	USA: Pearl River, Mississippi	KM871180 [57]

* Larval sequences; ** Sequences generated in the present study are indicated in bold.

). Additional rDNA sequences of allocreadiid species and outgroup taxa (Table 1) used in analyses were downloaded from GenBank.

3. Results

3.1. Molecular Phylogenetic Analysis

The newly generated sequences were aligned and compared with sequences of other allocreadiid taxa available in the GenBank (Table 1) that were not shorter than 805 bp for the 28S alignment and not shorter than 450 bp for the ITS2 alignment. Alignments of the ITS2 and partial 28S data sets yielded 470 and 1104 characters for analysis, respectively. Sequences of the Prosthenhystera oonastica (only for 28S tree), Polylekithum and the Gorgoderidae were used as the outgroup. Analyses of these datasets produced almost identical tree topologies (Figure 1 and Figure 2); all Allocreadium sequences clustered there in one well-supported monophyletic clade. The newly obtained sequences of Allocreadium isoporum from Rutilus rutilus and Pisidium spp. appeared to be identical to previously generated sequences of A. isoporum from Barbatula barbatula and Alburnus alburnus. Some new sequences of specimens from R. rutilus and Scardinius erythrophthalmus, morphologically consistent with the concept of A. isoporum, were identical to sequences of Cercariaeum crassum from Pisidium amnicum. Both of these groups formed a 99% supported clade in 28S (Figure 1). In the ITS2 tree (Figure 2), these groups formed a 96% supported clade together with sequences of Allocreadium neotenicum. The newly obtained sequences of Allocreadium transversale from Cobitis taenia were closest to Allocreadium pseudoisoporum [36] from Carassius gibelio (Figure 1).

Intergeneric divergence among Allocreadium genera in the 28S fragment was 1.4–6.8% of 1104 bp and 1.5–7.13% of 480 bp for the ITS2 fragment. The studied ITS2 and 28S sequences of A. transversale had the greatest divergence from the other Allocreadium: the divergence of the ITS2 fragment from the Allocreadium sp. C51Ukr ITS2 sequence reached 7.13% and the 28S fragment was 6.8% different from the A. isoporum 28S sequence. The intraspecific divergence of the studied 28S sequences of A. isoporum was 1 bp (0.09%), and the ITS2 was more variable and reached 6 bp (1.04%) difference. The 28S and ITS2 differences between A. isoporum and A. crassum were low, occurring at 14, 15 bp (1.27%, 1.36%) and 7, 8, 12 bp (1.46%, 1.67%, 2.5%), respectively. In the ITS2 tree (Figure 2), a sequence of Allocreadium sp. (FJ874923) from P. amnicum grouped with the clade of A. crassum; the difference between them was 2 bp (0.42%) and is within the limits of intraspecific variability, but the available information is insufficient to determine the taxonomic status of this line. It could be A. crassum, but this presumption is based only on the unpredictable variable ITS2 sequence.

Under our current taxon sampling, the monophyly of the Allocreadium and Bunodera is well supported in the 28S and ITS2 trees, at 90%, 96% and 99%, 96%, respectively (Figure 1 and Figure 2). This is in contrast to the species of the genus Crepidostomum, which, as has been repeatedly noted, are not monophyletic and form separate clades including representatives of other genera (Figure 1).

3.2. Descriptions

Family Allocreadiidae Looss, 1902

Genus Allocreadium Looss, 1900

Allocreadium crassum (Wesenberg-Lund, 1934) (Figure 3A)

Synonym (larval stage ex Pisidium amnicum): Cercariaeum crassum Wesenberg-Lund, 1934.

Final hosts: Rutilus rutilus (Linnaeus, 1758), Scardinius erythrophthalmus (Linnaeus, 1758).

Site in host: Intestine.

Localities: Curonian Lagoon and Lake Ilmėdas, Lithuania. Representative DNA sequences: 28S rDNA (OQ359131–OQ359133); ITS2 rDNA (OQ359139–OQ359142).

Molecular vouchers deposited in the Helminthological Collection at the Nature Research Centre under the numbers: EKOI-217A-Lt, EKOI-270-2-Al-Lt, EKOI-227-Lt.

Description (Figure 3A) (based on two mature specimens, measurements are given for both): Body elongated, spindle-shaped, 1890 and 1988 long; widest at level of ventral sucker. Tegument unarmed, on both suckers bearing minute papillae. Both suckers of almost same size; oral sucker ventro-subterminal, muscular, 335 × 285, 343 × 293; ventral sucker 272 × 305, 284 × 312. Prepharynx absent. Pharynx oval, muscular, 110 × 120, 115 × 128. Oesophagus 345, 360 long. Intestinal bifurcation approximately at level of ventral sucker; caeca long, extending to mid-way between posterior testis and posterior extremity of body. Testes two, large, oval, tandem, contiguous, in the middle of body. Anterior testis 190 × 270, 220 × 285; posterior testis 210 × 250, 214 × 265. Cirrus sac large, elongate, antero-dorsal to ventral sucker. Ovary rounded, entire, 190, 205 in diameter, located posterior to the ventral sucker. Uterus between ventral sucker and anterior testis. Eggs 85–97 × 65–70. Vitelline follicles, forming two lateral fields, do not reach the anterior level of the ventral sucker, joining behind the testes in posterior extremity. Excretory vesicle I-shaped, excretory pore terminal.

Allocreadium isoporum (Looss, 1894) Looss, 1900 (Figure 3B)

Syn. Distomum isoporum Looss, 1894; Creadium isoporum Looss, 1900.

Type-hosts: various freshwater cyprinid fishes.

Type locality: Germany.

Final hosts: Rutilus rutilus, Alburnus alburnus, Barbatula barbatula.

Localities: Curonian Lagoon, Lithuania; Lake Oster, Karelia and River Il’d, upper Volga River basin, Russia.

Site in host: Intestine.

Representative DNA sequences: 28S rDNA (GU462125, MH143102, GU462126, OQ359125); ITS2 rDNA (FJ874921, MH143096).

Molecular voucher deposited in the Helminthological Collection at the Nature Research Centre under the number EKOI-163Lt.

Description (based on two mature specimens, measurements are given for both): Body elongated, spindle-shaped, 2500 and 2624 long, 653, 755 wide. Tegument unarmed. Oral sucker rounded, ventrally subterminal, 335 × 330, 340 × 345. Ventral sucker almost the same size as oral, 320 × 333, 326 × 342, muscular, in anterior third of body. Prepharynx not seen. Pharynx oval. Intestinal bifurcation at level of ventral sucker. Caeca long, but not reaching posterior extremity of body. Two testes, large oval, entire, tandem, contiguous. Anterior testis 275 × 352, 282 × 363, posterior 320 × 345, 341 × 350. Cirrus-sac oval, anterio-lateral to ventral sucker, contains seminal vesicle, prostatic complex and ejaculatory duct. Genital pore median, below pharynx level. Ovary rounded, entire, smaller than testes, 205 × 210, 212 × 215, located submedially, postacetabular. Vitellarium follicular; lateral fields anteriorly not reaching ventral sucker, confluent posterior to testes. Uterus coiled between anterior testis and ventral sucker, between intestines, slightly overlapping them. Eggs oval, 75–90 × 53–56. Excretory vesicle I-shaped, excretory pore terminal.

Allocreadium isoporum (Looss, 1894) cercaria (Figure 4)

First intermediate host: Pisidium milium Held, 1836, Pisidium sp. (Bivalvia; Sphaeriidae).

Locality: Lake Hurdalsjøen, Norway; rivulet in Panovce, Slovakia.

Representative DNA sequences: 28S rDNA (OQ359126, OQ359127); ITS2 rDNA (OQ359134, OQ359135).

Molecular vouchers deposited in the Helminthological Collection at the Nature Research Centre under the numbers: EKOI-3Nor, EKOI-126Nor, EKOI-719Slo.

Based on 10 specimens obtained by dissection. Ophthalmoxiphidiocercaria developing in rediae. Body elongate-oval, with maximum width at level of ventral sucker, 313–463 × 141–165; tail approximately equal to the body length, 352–358 × 47–56, with maximum width at base. Tegument unarmed. Tegumental inflations of the tail appeared as tail fins in lateral view. The compact pigment of eye spots 19 × 18, situated lateral slightly below pharynx. Oral sucker rounded 69–75 × 57–63, subterminal, with stylet 16–20 long on anterior lip. Stylet with antero-lateral wings before its pointed termination (Figure 4B). Ventral sucker 56–58 × 50–54, smaller than oral sucker; numerous glandular cells may be seen in ventral sucker. Digestive system with short prepharynx, globular pharynx, about 19 in diameter, and narrow oesophagus. Caeca incompletely developed. In some specimens intestinal bifurcation near anterior margin of ventral sucker and short caeca can be seen. Penetration gland cells were not observed clearly. Cystogenous glands sparse, generally ovoid, concentrated near surface in the posterior part of the body. Flame cell formula difficult to establish due to hardly visible flame cells. The excretory ducts are more visible. Main excretory ducts issue from anterior end of bladder; below posterior margin of ventral sucker they form loops and divide into anterior and posterior collecting ducts. I-shaped excretory vesicle long; the wall of the vesicle thick, composed of large cells (Figure 4C). Excretory pore terminal.

Remarks. To date, no life cycle of any European Allocreadium spp. has been determined and no cercariae have been reported. Taken together, our knowledge of the life history data for allocreadiid species is quite limited. The most detailed study of the development of European allocreadiids was carried out by Wiśniewski [16], who reported results on the experimental study of the development of Bunodera lucioperca and also provided a description of cercaria of “A. isoporum”. However, molecular analysis revealed identical rDNA in adult Bunodera acerinae Roitman and Sokolov, 1999, and larval B. luciopercae described by Wiśniewski [16], while rDNA sequences of larval “A. isoporum” sensu Wiśniewski [16] were identical to those of adult B. luciopercae [4]. Consequently, the life history of A. isoporum has remained inadequately described. On the other hand, Cercariaeum crassum, a larval digenean commonly reported in European sphaeriid bivalve P. crassum, has been shown to be a member of the genus Allocreadium after a fairly long history of investigation, but its adult stage remains unknown [33,58]. Current molecular analyses of a range of larval and adult Allocreadium spp. based on ITS2 and partial 28S rDNA data clearly indicated the identity of C. crassum with adult A. crassum, while sequences of ophthalmoxiphidiocercaria form Pisidium spp. revealed the conspecificity with adult A. isoporum.

The cercaria of A. isoporum resembles species of ophthalmoxiphidiocercariae developing in Sphaerium spp. described by Wiśniewski [16]. It is noteworthy that there are no clear morphological differences that would help to easily differentiate allocreadiid cercaraiae not only of different species, but also of different genera. The cercaria of B. acerinae (=B. luciopercae sensu Wiśniewski [16]) is smaller than that of A. isoporum, described herein, but the metrical data can differ in naturally emerged cercariae and those obtained in the dissection of molluscs. Furthermore, our material collected and analyzed under field conditions was insufficient for a more precise morphological description such as of the excretory system or glandular system. The cercaria of A. isoporum differs from that of B. luciopercae (=A. isoporum sensu Wiśniewski [16]) mainly in the structure of the tail. The tail of the latter is surrounded by a broad tegumental inflation. Allocreadium crassum is distinguished by the life cycle, which may include both tailed cercaria and cercariaeum stages, developing in the same clam [58]. The species is specific to its intermediate host, P. amnicum.

Allocreadium isoporum, the type species of the genus, is the most frequently reported species in the genus. It is widely distributed in European and Asian freshwater cyprinid fish. With our recent findings of distinctions in the molecular markers and biology (life-cycle) of specimens consistent with the concept of A. isoporum, a problem in the understanding of the status of the two species (priority) arises. The molecular analyses show the presence of two strongly supported independent ITS2 clades, as well as 28S clades. The specimens of these two clades are by no means morphologically very similar; there is variation in body shape and/or size of the testes, but the specimens are broadly consistent with each other and the differences fall within the range of intraspecific variability. Morphological comparison of A. isoporum sensu lato from R. rutilus and S. erythrophthalmus from this study, and descriptions by Bykhovskaya and Kulakova [8], Moravec [11] and Niewiadomska [10], indicates that our material fully corresponds to the characteristics of A. isoporum sensu lato. However, it should be noted that measurements of specimens presented by different authors are extremely variable. It is highly probable that the original and subsequent morphological descriptions of A. isoporum given by different authors include individuals of different species. In our previous molecular studies of allocreadiid trematodes, the sequences of specimens consistent with A. isoporum from A. alburnus and B. barbatula were found to be identical [4,6], and no taxonomic problems arose. However, a comparative analysis of additional material revealed a hidden diversity of specimens identified under the name A. isoporum. Since the DNA of some specimens matched that of the larval form known as Cercariaeum crassum Wesenberg-Lund, 1934, we thought it reasonable to name them Allocreadium crassum (Wesenberg-Lund, 1934). It is also worth noting that in the light of the new data, specimens of Allocreadium isoporum sensu lato collected from the same host individual cannot be considered as paragenophores.

The European fauna of Allocreadium consists of nine nominal species: A. baueri, A. carparum Odening, 1959, A. dogieli Kowal, 1950, A. isoporum, A. markewitschi Kowal, 1949, A. neotenicum Peters, 1957, A. papilligerum (Rees, 1968), A. striatum Dinulescu, 1942 and A. transversale. The validity of some of the species is questionable in view of the fact that much reliance has been placed on morphological characters for their segregation. Molecular markers were available only for A. isoporum and A. neotenicum. Four Allocreadium species are registered in Lithuania and neighboring Poland, namely A. dogieli, A. isoporum, A. markewitschi and A. transversale [10,59,60]. Allocreadium crassum, as well as A. isoporum, significantly differs from A. dogieli in size and arrangement of vitelline follicles and gonads; the vitelline follicles of A. dogieli are very large (their diameter is almost equal to the diameter of the testes) vs. small vitelline follicles in the first two species; A. dogieli is characterized by small gonads arranged in a triangle; the testes of A. crassum and A. isoporum are significantly larger and tandem.

Allocreadium crassum and A. isoporum differ from A. markewitchi mainly in the arrangement of the vitellaria. The vitellaria of A. markewitchi extend anteriorly up to the pharynx level; the vitellaria of A. crassum and A. isoporum do not reach anteriorly the ventral sucker.

Allocreadium transversale is characterized by the ventral sucker being conspicuously larger than the oral, and large vitelline follicles extending anteriorly to the anterior margin of the ventral sucker [17], while A. crassum and A. isoporum are characterized by the presence of approximately equal suckers and small vitelline follicles not reaching anteriorly the ventral sucker. In our molecular phylogenies, the sequences of A. transversale form separate, well-supported branches within the Allocreadium clade (Figure 1 and Figure 2).

4. Discussion

Research into European helminth fauna has a long history and this region is as well-known as any [61]. However, DNA markers are still not available for the vast majority of species, and morphology remains the basis on which the vast majority of trematode species have been recognised. This study showed once again that even well-known trematode species, especially those species that had historically been erected based solely on morphological characters, may in fact be complexes of related species, and that hidden diversity can be revealed only through molecular approaches.

The phylogenetic analysis of sequences of the ITS2 region and 28S rRNA ribosomal gene, obtained in this study and our previous studies, revealed that specimens consistent with the diagnosis and description for Allocreadium isoporum and recovered from the intestines of different cyprinid fish, A. alburnus, B. barbatula, R. rutilus and S. erythrophthalmus, from different localities in Lithuania and Russia represented two genetically distinct species-level lineages. Some new sequences corresponded with those of A. isoporum obtained from Alburnus alburnus and Barbatula barbatula in our previous studies [4,6], while the others formed a separate clade together with sequences of Cercariaeum crassum from Pisidium amnicum [33]. The levels of the sequence divergence observed between the two clades strongly support the two distinct species status of these isolates. Moreover, the adults of these two genetically different clades appear to be associated with distinct patterns of first intermediate host identity and cercarial morphology. Therefore, we suggest that the specimens of Allocreadium conspecific with Cercariaeum crassum Wesenberg-Lund, 1934, should now be referred to as Allocreadium crassum (Wesenberg-Lund, 1934).

It is difficult to find morphometric distinctions that are parallel with the molecular results; these distinctions can be slight and only clearly seen when specimens are examined in substantial numbers. Morphological studies are complicated by the fact that the identity of the host is not an infallible indicator of parasite identity. Specimens of both lineages can exist in the same host species, common roach, and therefore cannot be even preliminary differentiated without molecular markers. In the present study, there is evidence for speciation independent of definitive hosts specificity. The species differ in their larval stages and specificity to the first intermediate host. Thus, discriminating available molecular markers, different cercariae morphology and differing first intermediate host specificity is a sufficient basis for species separation.

A key component of the understanding of the biodiversity of trematodes is an elucidation of their life cycles and all host species involved in the parasite’s circulation in an ecosystem. It is generally agreed that elucidating life cycles is critical to gaining a complete understanding of digenean trematodes, but despite the advantages offered by modern methods, relatively few of the large number of species assigned to the genus Allocreadium have had their larval stages described. In our previous studies, it was established that the enigmatic European species C. crassum from the sphaeriid bivalve P. amnicum shows a close relationship with type-species A. isoporum in the molecular tree inferred from 28S rDNA sequences [33]. No match was found in the rDNA internal transcriber spacer 2 (ITS2) or 28S sequences of C. crassum or any adult allocreadiids sequenced up to that time. It was surprising that C. crassum, a fairly common species in Central and North European freshwaters, was not associated with any adult, despite the fact that fish parasites are comparatively well studied in this area. In the light of the new data, it becomes clear that C. crassum, associated with the single sphaeriid species P. amnicum, is the larval stage of Allocreadium crassum, the sibling species of A. isoporum.

Our knowledge of the involvement of bivalves in the trematode life cycles remains relatively scarce. Especially little is known about the role of small Pisidium spp. in the circulation of these parasites in ecosystems. Small clams are often overlooked in malacological samples and, moreover, the diagnosis of sphaeriids using shell morphologies is greatly hampered by the high variability of species in different environments (see [62]).

The digenean cercariae exhibit morphological diversity and can behave in many distinct ways that ultimately lead to the infection of the vertebrate host [63]. (They infect their hosts in many different ways.) Most of the allocreadiid cercariae emerge from the first intermediate host, the sphaeriid bivalve, and infect the second intermediate host, the aquatic arthropod (encyst in insect larvae or crustacean) [3]. The final host, the fish, becomes infected by feeding on infected arthropods. In some cycles, a tendency to eliminate the second or final host may be inferred. Cercariaeum crassum can develop directly from a germ ball with or without the cercarial stage and can remain in the first intermediate host; the emergence of cercariae from clams has never been observed [58]. This supports the notion of the possibility of direct infection of the definitive host when clams are eaten. The common roach, R. rutilus, and rudd, S. erythrophthalmus, are both omnivorous fish and eat a wide variety of vegetation and invertebrates. The importance of benthos in the roach diet, including mollusks, increases with the size of the fish [64]. Adult S. erythrophthalmus feed mainly on aquatic vegetation as well as insects, snails, crustaceans, diatoms and occasionally fish eggs [64,65,66,67,68]. Such diets are consistent with the possibility of direct ingestion of cercariaeum along with infected clams.

Host ecological factors such as diet and feeding habitats can play a role in the speciation of passively transmitted intestinal parasites [69]. Allocreadium isoporum sensu lato is a widely distributed parasite recorded in a number of species of cyprinids and some other Palearctic fish [8,58]. The most important definitive hosts are chub, Leuciscus cephalus and other leucisines [9,11]. In Lithuania, this trematode has been found in Rutilus rutilus, Leuciscus leuciscus, L. idus, Squalius cephalus, Aspius aspius, Alburnus alburnus, Blicca bjoerkna and other cyprinid fish hosts [60], but it is evident that the degree of host specificity of A. isoporum has been underestimated. When the species is encountered in a wide host range and/or in a wide geographic region, the existence of cryptic species may be suspected [70,71]. The host species recorded for A. isoporum sensu lato are different ecologically, have different diets and occupy different habitats. The basis of pattern of host-specificity is far from clear, but must be partly based on the mode of transmission. The transmission of A. isoporum sensu stricto is possibly via metacercariae encysted in arthropods which are ingested by fish.

There has been some ecological evidence of separate closely related species under the name A. isoporum in fish species across the range. During the study of trematodes in northern Finland, A. isoporum was found only in Carassius carassius and Alburnus alburnus, but not other cyprinids such as Leuciscus leuciscus and Rutilus rutilus, the latter of which was examined in large numbers [72]. The authors pointed out that “the apparent absence of this worm from certain cyprinids in Finnish waters is difficult to explain, unless, as suggested by Ergens [73] and others, A. isoporum is composed of a number of morphotypes, often given the status of subspecies, and that these forms have different host specificities”.

Another species of the genus Allocreadium whose DNA was first sequenced in this study, A. transversale, was described by Rudolphi in 1802 from the intestine of the weatherfish, Misgurnus fosilis. Szidat [74] redescribed this species from a single specimen found in the spined loach, Cobitis taenia, in East Prussia. During the course of studies on helminth fauna of C. taenia from Lake Balsys (Vilnius, Lithuania), adult Allocreadium trematodes obtained from the intestine were identified as A. transversale. Koval and Izyumova [75] are of the opinion that A. transversale is a specific parasite of weatherfish, while allocreadiids from spined loach belong to another species, A. baueri Spassky et Roytman, 1960. Allocreadium baueri was originally described from Phoxinus phoxinus and P. czekanowskii from the basin of the river Yenisei, though later it was synonymized with A. transversale by Roytman [76]. However, Koval [17] revealed morphological differences between the two species and restored the validity of A. baueri. Nevertheless, A. transversale was recorded from three cobitid fishes, Cobitis taenia, C. elongatoides and Misgurnus fosilis, in Poland [59]. Recently, A. transversale has been recorded from C. elongatoides, C. elongata and C. strumicae in Bulgaria [77]. However, there is still a shortage of information about these species that may generate inconsistencies in species delimitations and host-parasite associations. The molecular markers obtained in this study will be useful to clarify questions on species diversity. In our 28S phylogenetic tree (Figure 1), sequences of A. transversale form a separate strongly supported subclade with Allocreadium pseudoisoporum from Carassius gibelio.

New information allows us to better understand patterns of host specificity exhibited by species of Allocreadium and to elucidate their life cycles. Our results show that molecular methods can be expected to reveal hidden diversity even in long-established and widely studied European trematode species, while contributing to a better understanding of their life cycles and circulation in ecosystems.

In their study of cryptic species recognition in tropical Indo-west Pacific fishes using an integrative paradigm, Bray et al. [78] considered morphology, molecular data, host distribution and geographic distribution and concluded that “this combination creates a system that is both conceptually satisfying while generating a workable classification, although not one without complexity”. We believe that in addition to abovementioned datasets, consideration of life cycle data of trematodes is essential for assessing species diversity and delimitation. Our data on the life histories of related allocreadiid trematodes (Bunodera acerinae + B. luciopercae and Allocreadium isoporum + A. crassum) clearly show that although adults are morphologically similar (morphologically indistinguishable and biologically cryptic in that they infect the same species of fishes at the same or different localities), their larval stages can differ in morphology and host specificity.

We would like to draw special attention to the fact that when speaking of morphologically identical trematode species, data on the morphology of adult forms is usually used. However, the obtained results clearly show that sibling species can reliably differ in the morphology of the cercariae and intermediate host specificity. Therefore, for a reliable assessment of biodiversity, it is very important to know all the stages of development of species and to clarify life cycles.