Never ending diversity: two new species of the genus Allocreadium (Digenea: Allocreadiidae) including new keys to the genus

Abstract

Two new species of the genus Allocreadium were isolated from the intestines of the Lake minnow Rhynchocypris percnura caught in the backwater of the Komissarovka River in the South of the Russian Far East. The morphology of A. anastasii n. sp. corresponds to that of Allocreadium sp. from Lake Khar (Mongolia) and Allocreadium sp. Belous, 1952 from the Primorsky region of Russia except for the preacetabular anterior border of the vitelline follicles in A. anastasii n. sp. from the Komissarovka River vs. at anterior half of ventral sucker in Allocreadium sp. Genetic analysis revealed the identity of A. anastasii n. sp. to Allocreadium sp. 1 from the Nezhinka River and Lake Khar. Allocreadium macrolecithum n. sp. was differentiated from Palaearctic Allocreadium spp. by having the following features: respectively large vitelline follicles extending from posterior extremity to anterior margin of the ventral sucker; relatively short caeca reaching the border of middle and posterior thirds of hindbody; and small testes in the middle of hindbody. Interspecific genetic p-distances between Allocreadium spp. were 0.16–7.23% in 28S gene and 18.62–31.54% in Cox1 mtDNA gene. In the phylogenetic tree reconstructed with Maximum parsimony and Bayesian Inference methods, A. anastasii n. sp. and A. macrolecithum n. sp. were nested into different species groups of the genus Allocreadium – sister to A. khankaiense and A. bursense, respectively. Modified dichotomous keys were prepared for 31 Palaearctic species of Allocreadium including A. crassum, A. dogieli, A. papilligerum, A. bursense, A. anastasii n. sp., and A. macrolecithum n. sp.

Introduction

The genus Allocreadium Looss, 1900 represents cypriniform fish-associated trematodes comprising significant species richness in relation to other allocreadiids – 107 valid species (WoRMS 2024). At present, the systematics of Allocreadium is based on both morphological (mainly adult stages) and genetic data. Thus, for the last four years, four new species of Allocreadium were described from the Primorsky region of Russia, South Africa, and North Western Turkey (Vainutis 2020; Dos Santos et al. 2021; Vainutis et al. 2023; Aydogdu et al. 2023). It was revealed that 10 Allocreadium species representing different lineages maintain the general morphological characters of the type species A. isoporum (Aydogdu et al. 2023; Vainutis et al. 2023). Some species sharing great morphological similarity and infecting the same fish hosts in close geographic regions are still not genetically confirmed (e.g., over 30 species from India). Based on the WoRMS database, only 13% (14 species) of Allocreadium spp. have been confirmed with application of molecular genetic data: A. transversale (Rudolphi, 1802) Odhner, 1901; A. isoporum (Looss, 1894) Looss, 1900; A. lobatum Wallin, 1909; A. gotoi (Hasegawa & Ozaki, 1926) Shimazu, 1988; A. crassum (Wesenberg-Lund, 1934) Vainutis, Voronova, Urabe & Kazarin, 2023; A. schizothoracis Pande, 1938; A. dogieli Koval, 1950; A. neotenicum Peters, 1957; A. hemibarbi Roitman, 1963; A. papilligerum (Rees, 1968) Moravec, 1984; A. khankaiense Vainutis, 2020; A. apokryfi Dos Santos, Gilbert, Avenant-Oldewage & Dumbo, 2021; A. pseudoisoporum Vainutis, Voronova, Urabe & Kazarin, 2023; A. bursense Aydogdu, Vainutis, Voronova & Aydogdu, 2023 (Vainutis 2020; Dos Santos et al. 2021; Vainutis et al. 2023; Aydogdu et al. 2023; Petkevičiūtė et al. 2023; Sokolov et al. 2023; Solórzano-García et al. 2024).

This study was aimed at resolving the taxonomic status and phylogenetic relationships of two Allocreadium species found in the backwaters of the Komissarovka River (Primorsky region, Russian Far East). Description of the new species and recent confirmation of the species A. transversale, A. dogieli, A. papilligerum, and A. bursense, with genetic data (Aydogdu et al. 2023; Petkevičiūtė et al. 2023; Sokolov et al. 2023), resulted in modification of dichotomous keys including 31 Palearctic Allocreadium species to date.

Material and methods

Material collection and morphological analyses

Twelve adult specimens of Allocreadium spp. were isolated from the intestines of two Lake minnows Rhynchocypris percnura caught in two backwaters of the Komissarovka River, Khankaysky district, Primorsky region, Russia (44°57′56.4″N 131°44′37.3″E). Collected worms were identified to genus level according to morphological characteristics under a light microscope, rinsed in saline, killed in hot distilled water, and stored in 70% ethanol for morphological analysis and 96% ethanol for molecular genetic studies.

Six specimens of Allocreadium selected for the morphological study were stained in alum carmine, dehydrated in a graded ethanol series (75%, 85%, 95%, ~100% absolute ethanol), cleared in clove oil, and mounted in Canada balsam. Measurements of the entire body and hindbody were provided in millimetres (mm), and measurements of other structures were in micrometres (μm). The holotypes and paratypes of both species were deposited to the parasitological collection of the Water Bioresources and Aquaculture department of the Far Eastern State Technical Fisheries University (FESTFU), Vladivostok, Russia. Dichotomous keys (Vainutis et al. 2023) were modified by including six species of which two were described as new in this study and four from previous works (Aydogdu et al. 2023; Petkevičiūtė et al. 2023; Sokolov et al. 2023): A. bursense, A. transversale, A. dogieli, and A. papilligerum.

DNA extraction, amplification, and sequencing

Genomic DNA was extracted from four specimens using alkaline lysis method HotShot (Truett 2006). A fragment of the 28S rRNA gene was amplified using forward primer U178 (5′-GCA CCC GCT GAA YTT AAG-3′) and reverse primer L1642 (5′-CCA GCG CCA TCC ATT TTC A-3′) (Lockyer et al. 2003). A fragment of Cox1 mtDNA of A. anastasii was amplified using forward primer JB3 (5′-TTT TTT GGG CAT CCT GAG GTT TAT-3′) (Bowles et al. 1992) and reverse primer CO1-R-trema (5′-CAA CAA ATC ATG ATG CAA AAG G-3′) (Miura et al. 2005). For the analysis of genetic divergence, short fragments of three specimens of Allocreadium hemibarbi (ASP6 from Vainutis 2020) were amplified with forward primer JB3 and reverse primer JB4.5 (5′-TAA AGA AAG AAC ATA ATG AAA ATG-3′) (Bowles et al. 1992). The PCR mixture contained 2X DreamTaq Green PCR Master Mix (Thermo Scientific, USA), 0.5 μL forward and reverse primers and 5 μL templates in total volume of 20 μL. The amplification protocol for 28S rDNA was performed under the following conditions: 2 min denaturation hold at 94°C, 40 cycles of 30 s at 94°C, 30 s at 52°C, 2 min at 72°C, and a 7 min extension hold at 72°C. The amplification protocol for Cox1 mtDNA: 1 min denaturation hold at 94°C, 30 cycles of 15 s at 94°C, 30 s at 50°C, 2 min at 72°C, and a 7 min extension hold at 72°C. Each PCR reaction included a negative and positive control, using both primers to detect possible contamination. PCR products were directly sequenced using the Bright Dye Terminator Cycle Sequencing kit (Nimagen, The Netherlands) as instructed by the manufacturer. Internal sequencing primers implemented for the 28S gene fragment were as follows: 3S (5’-CGG TGG ATC ACT CGG CTC GTG-3’) (Bowles et al. 1995), 1200F (5’-CCC GAA AGA TGG TGA ACT ATG C-3’), 1200R (5’-GGG CAT CAC AGA CCT G-3’), 900F (5’-CCG TCT TGA AAC ACG GAC CAA G-3’) (Lockyer et al. 2003). The PCR products were read with an ABI Prism 3130 Genetic Analyzer (Applied Biosystems, USA) at the NSCMB FEB RAS.

Alignment and phylogenetic analyses

Four partial 28S rDNA and two partial Cox1 mtDNA sequences obtained in this study were assembled with MEGA X software and aligned using ClustalW (Kumar et al. 2018). New sequences of Cox1 gene of Allocreadium hemibarbi were deposited to GenBank with accession numbers OR945219–OR945221. Sequences used for the phylogenetic reconstruction are represented in Table 1. Genetic p-distances were estimated using the Tamura-Nei model with 1,000 bootstrap replicates in the MEGA X software; p-value was lower than 0.05. The species Acrolichanus auriculatus (NCBI Accession numbers MN524579, MN750364) was chosen as outgroup taxon. Following previous studies (Aydogdu et al. 2023; Vainutis et al. 2023), unidentified species from Ukraine and China were designated as Allocreadium sp. 2 and Allocreadium sp. 3, respectively. Phylogenetic analyses were carried out with Bayesian Inference (BI) algorithm (Huelsenbeck et al. 2001) with the GTR+I+G model selected in jModeltest v. 2.1.5 software as the best (Darriba et al. 2012). The MCMC algorithm was performed using two independent runs and 500,000 generations (the average standard deviation of split frequencies was less than 0.01); 25% of generations were discarded as burn-in in MrBayes v. 3.1.2 software (Huelsenbeck et al. 2001). An additional phylogenetic tree for the same sampling was reconstructed using the Maximum Parsimony (MP) method in the MEGA X software, with 1,000 bootstrap replicates. In this study, different species groups of Allocreadium on the phylogenetic trees were indicated with the Latin letters (from A to B) following the previous phylogenetic works (Aydogdu et al. 2023; Vainutis et al. 2023).

Table 1. Information on the species, host, geographic origin, and the genetic data (partial 28S rRNA and Cox1 mtDNA sequences) for the species used in the phylogenetic reconstructions

Results

Taxonomy

Phylum: Platyhelminthes Gegenbaur, 1859

Class: Trematoda Rudolphi, 1808

Subclass: Digenea Carus, 1863

Order: Plagiorchiida La Rue, 1957

Suborder: Xiphidiata Olson, Cribb, Tkach, Bray & Littlewood, 2003

Superfamily: Gorgoderoidea Looss, 1901

Family: Allocreadiidae (Looss, 1902) Stossich, 1903

Genus: Allocreadium Looss, 1900

Allocreadium anastasii n. sp. ( Figure 1 )

Figure 1. Allocreadium anastasii n. sp., registration No. T1: (a) holotype No. T1-1 from the backwater of the Komissarovka River, entire worm, ventral view, scale bar = 500 μm; (b) schematic drawing of the cirrus pouch, scale bar = 50 μm; (c) schematic drawing of posttesticular space with caeca and excretory vesicle, scale bar = 200 μm; abbreviations: t – testis, c – caeca, ev – excretory vesicle; (d) Microphotograph of A. anastasii n. sp. from the Nezhinka River, based on the material used for DNA extraction in the work of Vainutis (2020); (e) sketch drawing of A. anastasii n. sp. from the Nezhinka River.

urn:lsid:zoobank.org:act:EDC56D5A-C212-40D2-9DBB-E1D514C771D9

Material examined: Nine adult specimens were isolated from the intestine of one individual of Rhynchocypris percnura (Pallas, 1814).

Morphological description. Based on five specimens. Relatively small trematodes, body elongate, fusiform in contracted state, length 1.456–2.122 (1.774). Tegument smooth, unarmed. Widest part of body posterior to ventral sucker in utero-ovarian region in relaxed worms, 553–886 (703), or at level of anterior third of anterior testis when body contracted. Forebody narrowing at anterior end, short, 355–591 (426), 20.85–27.85% (23.92%) of body length; hindbody 0.868–1.278 (1.118), 59.62–65.65% (62.31%) of body length. Oral sucker subterminal, subrounded (n=3) or transversely oval (n=2), 199–254 ×214–250 (233×237), 11.26–15.73% (13.28%) of body length; perioral muscular papillae absent. Tegument protrusion absent at anterior margin of body. Small pigment eyespots present, in contracted worms at level of posterior margin of oral sucker, in relaxed worms at level of posterior margin of pharynx; eyespots 5.6–19×5–14(12×10) (n=4). Prepharynx not observed. Pharynx transversely oval 95–137×114–144 (114×134), 5.33–7.59% (6.5%) of body length. Oesophagus relatively long, 154–278 (199), 9.39–13.1% (11.14%) of body length, bifurcating dorsally to anterior third or middle of ventral sucker. Caeca reaching posterior third of posttesticular space, at 179–217 (201) from posterior end of body. Ventral sucker on border of anterior and middle thirds of body, subrounded (n=3) or transversely oval (n=2), larger than oral sucker, 230–288×221–280 (259×260), 13.29–15.97% (14.67%) of body length. Distance between oral and ventral suckers 121–356 (192). Suckers length ratio 1:1–1.18 (1:1.11); suckers width ratio 1:0.98–1.2 (1:1.1). Testes tandem or suboblique in middle third of hindbody, subrounded, transversely oval or irregular, tightly adjoining each other, anterior testis 179–489×195–404 (275×317), 10.01–27.11% (15.49%) of body length; posterior testis 185–632×220–523 (317×315), 12.7–35.03% (17.72%) of body length. Distance between anterior margin of anterior testis and posterior end of body 761–1023 (865); length of posttesticular space 364–471 (421). Cirrus pouch saccate, thin-walled, suboval, 118–168×73–85 (150×78), anterior to ventral sucker or its proximal part at anterior third of ventral sucker. Cirrus pouch containing seminal vesicle, short pars prostatica and short cirrus (119). Seminal vesicle tubular, curved crescently, locating in posterior half of cirrus pouch. Genital atrium containing male (mgp) and female (fgp) genital pores preacetabular, medium, 81×105 (n=1); mgp 23–27×21–29 (25×25); fgp 26–43×24–39 (33×29). Distance anterior to genital atrium 298–448 (343). Ovary subrounded or oval, 141–210×140–253 (183×223), 9.63–10.92% (10.35%) of body length, pretesticular, its anterior third dorsal to posterior third of ventral sucker; distance between ovary and anterior testis 147–190 (169). Seminal receptacle saccate, 85–193×73–219 (134×113), 5.83–9.09% (7.4%) of body length; posterior to ovary, biased posteriorly to anterior testis. Laurer’s canal not observed. Lateral fields of vitellarium extending ventrally and dorsally, overlapping caeca; its anterior border anterior to ventral sucker at middle of esophagus or at level of anterior margin of ventral sucker at 344–462 (379), posterior border at 60–88 (74) from posterior end of body. Vitelline follicles oval, subrounded or irregular, relatively small, 66–203×45–154 (109×86). Uterus between ventral sucker and posterior testis reaching posterior third of anterior testis; in contracted worms lower part of uterus at anterior third of posterior testis. Eggs 23–82, oval, 63–102×40–52 (82×46). Excretory vesicle tubular, reaching posterior margin of posterior testis or immediately posterior to it, length 279–492 (374); width at posterior part – 26–64 (50), anterior part – 39–89 (69). Excretory pore terminal.

Synonyms. Allocreadium sp. Belouss, 1952; Allocreadium sp. 1 (Vainutis, 2020); Allocreadium sp. (Sokolov et al. 2023).

Etymology. The species name anastasii was given after the remarkable scientist Anastasia Voronova (Leading researcher at Pacific branch of Russian Federal Research Institute of Fisheries and Oceanography (TINRO)) in gratitude for the guidance and support on my scientific path.

Type host and locality. Lake minnow Rhynchocypris percnura (Pallas, 1814) caught from a backwater of Komissarovka River, Khankaysky district, Primorsky region, Russia (44°57′56.4″N 131°44′37.3″E).

Other hosts. Phoxinus lagowskii оxycephalus (=Rhynchocypris oxycephala (Sauvage & Dabry de Thiersant, 1874)) in Belouss (1952); Oreoleuciscus potanini (Kessler, 1879) in Sokolov et al. (2023).

Other localities. Tributary of the Nezhinka River, Razdolnaya River basin, Nadezhdinsky district, Russia (43°25′57.1″N 131°46′21.8″E) (Vainutis, 2020); Lake Khar, Great Lakes’ Hollow, Mongolia (48° 19′ N; 93° 08′ E) (Sokolov et al. 2023).

Type material. Holotype (No. T1-1) and four paratypes (No. T1-2–5) were deposited to the helminthological collection of the Far Eastern State Technical Fisheries University, Vladivostok, Russia. Deposition date: 15 November 2023.

Molecular genetic data. Complete region ITS1-5.8S-ITS2 rRNA – MW480031–MW480032 (this study), partial 5.8S-ITS2 region OQ427388 (Sokolov et al. 2023); 28S rRNA gene – OR066230–OR066231 (this study), MK211209–MK211210 (Vainutis 2020), OQ427384 (Sokolov et al. 2023); Cox1 mtDNA gene – MK818870–MK818871 (Vainutis et al. 2021), OR945222 – OR945223 (this study).

Diagnosis. Allocreadium anastasii n. sp. has morphological characters of both Allocreadium sp. of Belouss (1952) from the Primorsky region and Allocreadium sp. of Sokolov et al. (2023) from Mongolia. Their common features are as follows: anterior half of pharynx dorsal to posterior margin of oral sucker; intestinal bifurcation dorsal to anterior half of ventral sucker; proximal part of cirrus pouch dorsal to anterior margin of ventral sucker; anterior margin of ovary dorsal to ventral sucker; lower part of uterus overlapping anterior third or half of anterior testis or anterior third of posterior testis ventrally; excretory vesicle reaching anterior quarter of posttesticular space, immediately posterior to posterior testis. Allocreadium anastasii n. sp. from the Primorsky region of Russia has several morphological features distinguishing it from Mongolia’s A. anastasii: anterior border of vitellarium preacetabular vs. at anterior half of ventral sucker; testes tandem or suboblique, subrounded, transversely oval or irregular and tightly adjoining each other vs. tandem, rounded and separated; excretory vesicle reaching posterior margin of posterior testis or immediately posterior to it vs. reaching anterior quarter of post-testicular space.

Among other Far Eastern Allocreadium species, A. gobii is the most similar to A. anastasii n. sp. by the following features: proximal part of cirrus pouch dorsal to ventral sucker reaching its midlevel; genital pore anterior to intestinal bifurcation; intestinal bifurcation dorsal to ventral sucker; anterior border of vitelline fields in forebody. But A. anastasii n. sp. differs from A. gobii by the following features: uterus posteriorly reaching anterior third of anterior testis vs. uterus in region between ventral sucker and anterior testis; anterior border of vitellarium at anterior half of ventral sucker (Mongolia) or anterior to it at level of genital pore (Primorsky region, Russia) vs. anterior border of vitellarium at posterior margin of pharynx.

Allocreadium anastasii n. sp. differs from relative species A. khankaiense by numerous morphological characters such as anterior border of vitellarium in forebody or at anterior third of ventral sucker vs. at level of anterior or posterior half of ventral sucker; proximal part of cirrus pouch reaching midlevel of ventral sucker vs. posterior margin of ventral sucker; uterus posteriorly reaching anterior third or posterior margin of anterior testis vs. strictly pretesticular or overlapping anterior margin of anterior testis.

Allocreadium macrolecithum n. sp. (Figure 2)

Figure 2. Allocreadium macrolecithum n. sp., registration No. T2: (a) holotype No. T2-1 from the backwater of the Komissarovka River, entire worm, ventral view, scale bar = 500 μm; (b) microphothograph of the entire worm, scale bar = 500 μm; (c) schematic drawing of the cirrus pouch, scale bar = 100 μm; (d) schematic drawing of posttesticular space with caeca and excretory vesicle; abbreviations: t – testis, c – caeca, ev – excretory vesicle, scale bar = 400 μm.

urn:lsid:zoobank.org:act:5F70A3B5-BDB7-4CDF-A7BC-C41AB7FB004E.

Material examined. Three adult specimens were isolated from the intestine of one individual of Rhynchocypris percnura.

Morphological description. Based on one specimen. Relatively small trematodes, body elongate-ellipsoid, length 2.451. Tegument smooth, unarmed. Widest part of body in utero-ovarian region, 917, posterior to ventral sucker. Forebody short, 485; hindbody 1.664, slightly narrowing to posterior end. Oral sucker subterminal, round, 309×326; perioral muscular papillae absent. Bipartite tegument protrusion present at anterior margin of body. Eyespots absent. Prepharynx not observed. Pharynx subrounded 171×163. Oesophagus short, 225, bifurcating at level of middle third of ventral sucker. Caeca reaching middle third of hindbody. Ventral sucker on border of anterior and second fourths of body, oval, larger than oral sucker, 334×413. Distance between oral and ventral suckers 157. Testes suboblique allocating in tandem in mid-line of body and in anterior half of hindbody, anterior testis subrounded, 196×175, length 7.99% of body length; posterior testis oval, 193×136, length 7.87% of body length. Distance between anterior and posterior testes 117; between ventral sucker and anterior testis 366; between posterior testis and posterior end of body 81. Cirrus pouch saccate, thin-walled, 245×141, dextro-dorsal to ventral sucker, its proximal part at middle third of ventral sucker. Cirrus pouch containing seminal vesicle, prostatic part and short cirrus. Seminal vesicle tubular, 116×39, with slightly widened posterior end, locating in posterior third of cirrus pouch, where at mid-length curving at right angle. Prostatic part 73×42. Genital pore preacetabular, dextro-submedian. Ovary subrounded, 223×304, pretesticular, in midsagittal line; distance between ovary and anterior testis 172. Seminal receptacle small, club-shaped, 138×89, postero-dorsal and antero-dorsal to ovary and anterior testis respectively. Laurer’s canal not observed. Vitellarium extending in lateral fields of body ventrally and dorsally, overlapping caeca; its anterior border at anterior third of ventral sucker, posterior border at posterior end of body. Vitelline follicles relatively large, 95–268×76–168, nuclei of vitellocytes oval or subrounded 23–26×19–23, containing nucleolus nearly four times smaller than nuclei in diameter. Follicles not reaching posterior end of body at 99. Uterus pretesticular, immediately posterior to ventral sucker. Eggs 21, relatively large, 78–96×59–69. Excretory vesicle tubular, reaching posterior margin of posterior testis. Excretory pore terminal.

Etymology. The species name macrolecithum is given due to enormously large vitelline follicles in respect to those of other Allocreadium species.

Type host and locality. Rhynchocypris percnura (Pallas, 1814) caught from a backwater of Komissarovka River, Khankaysky district, Primorsky region, Russia (44°57′56.4″N 131°44′37.3″E).

Type material. Holotype (No. T2-1) was deposited to the helminthological collection of the Far Eastern State Technical Fisheries University, Vladivostok, Russia. Deposition date: the 15th of November 2023.

Molecular genetic data. 28S rRNA gene – OR066228–OR066229.

Diagnosis. Allocreadium macrolecithum n. sp. is very similar to Allocreadium transversale in following morphological characters: ventral sucker is significantly larger than the oral sucker; testes small, oval, suboblique with a little distance between them; large vitelline follicles reaching the anterior margin of the ventral sucker (Bauer 1948; Koval 1957; Roitman 1963; Petkevičiūtė et al. 2023). Allocreadium macrolecithum differs from Allocreadium transversale by the following characters: bipartite tegument protrusion present at anterior end of body vs. protrusions absent; anterior border of vitelline fields at level of ventral sucker vs. in forebody; genital pore anterior to intestinal bifurcation vs. posterior. Vitelline follicles of A. macrolecithum are comparable with that of A. dogieli in Sokolov et al. (2023). The authors did not provide the measurements of follicles but noted the size of the follicles is almost equal to that of gonads. In A. macrolecithum, length of follicles (95–268) and width (76–168) are a little larger than those of A. dogieli gonads: anterior testis 74–124×62–106, posterior testis 62–149×62–92, and ovary 92–183×94–127.

Allocreadium anastasii n. sp. and A. macrolecithum n. sp. differ from each other by the following features: testes relatively large – anterior testis 10.01–27.11% and posterior testis 12.7–35.03% of body length vs. 7.99% and 7.87% of body length, respectively; anterior border of vitelline fields in forebody vs. at level of ventral sucker; vitelline follicles oval or subrounded, small, with maximum length 203 and width 154 vs. irregular, large in relation to body dimensions (268×168); uterus overlapping anterior half of anterior testis vs. pretesticular; excretory vesicle posterior to testes vs. reaching posterior testis.

Molecular genetic analysis

Genetic analysis of the 28S rRNA gene

Partial sequences of the 28S rRNA gene of two A. anastasii n. sp. (1734 bp) and two A. macrolecithum n. sp. (1686 bp) individuals were identical within each species. Pairwise genetic distances between 15 species of Allocreadium and unidentified Allocreadium sp. 2 and Allocreadium sp. 3, were 0.16 – 7.23% (Supplementary Table 1). Genetic distance between A. anastasii n. sp. and A. macrolecithum n. sp. was 4.51%. Allocreadium anastasii n. sp. was similar to A. khankaiense (0.89%) and differed from the other Allocreadium spp. on 2.13–6.37%. Allocreadium macrolecithum n. sp. was similar to the species A. papilligerum (1.21%), A. bursense (1.29%), and A. pseudoisoporum (1.45%), and distant from other Allocreadium spp. on 2.21–6.28%.

Both BI and MP phylogenetic trees based on the partial 28S rDNA sequences displayed the same branch topology (Figure 3). Allocreadiid species were divided into two clades of which the earliest branching taxon was Acrolichanus chosen as root and the second consisted of outgroup taxa (subclades I–III) and Allocreadium (subclade IV). Subclade I included two species of Bunodera. Subclade II comprised single species Crepidostomum chaenogobii. Subclade III was subdivided into two groups: first, two species of Crepidostomum, and second, two species of Stephanophiala. Subclade IV was formed with 15 species of Allocreadium and branched into eight separate groups. Group A belonged to A. pseudoisoporum, A. bursense, A. papilligerum, A. transversale, and A. macrolecithum n. sp. Group B included A. gotoi from Japan and Allocreadium sp. 2. Group C was presented with the single species A. hemibarbi. Group D included A. khankaiense and A. anastasii n. sp. from the Komissarovka and Nezhinka Rivers (Primorsky region, Russia). Group E included European species A. isoporum, A. crassum, and A. dogieli. Group F consisted of the single species Allocreadium sp. 3 from China, which is sister to the terminal node containing group G (A. neotenicum and A. lobatum) and group H (A. apokryfi).

Figure 3. Phylogenetic tree of five allocreadiid genera. Emphasis was made on the evaluation of phylogenetic relationships of 15 Allocreadium species (blue rectangle). The new species are in bold. Reconstruction made with the Bayesian Inference (BI) and Maximum Parsimony (MP) methods based on the 1269 bp of 28S rRNA gene fragment. Numbers on the branches are posterior probabilities of BI and % of MP.

Phylogenetic tree including Allocreadium schizothoracis was reconstructed using the shorter 28S fragment (Figure 4). Original alignment was trimmed to the shortest sequence based on the length of only sequence of A. schizothoracis available in GenBank (499 bp). In general, BI, MP, and ML reconstructions shared the same topology, but statistical support in nodes between species groups was highest in the BI tree and lower and unreliable in MP and ML. The genera Crepidostomum and Stephanophiala were chosen as outgroup taxa representing the first major clade and rooting the tree. African group H with Allocreadium apokryfi included A. schizothoracis with strongest support obtained by three methods (1.0/100/100).

Figure 4. Phylogenetic tree of three allocreadiid genera representing phylogenetic relationships of 16 Allocreadium species (blue rectangle). Four sequences of two new species are in bold. Reconstruction made with the BI, MP, and Maximum Likelihood (ML) methods based on the D2 domain of 28S rRNA gene fragment (499 bp). Numbers on the branches are posterior probabilities of BI and % of MP and ML.

Genetic analysis of the Cox1 mtDNA gene

Two identical sequences (767 bp length) were obtained for Allocreadium anastasii n. sp. The Cox1 fragment of A. macrolecithum n. sp. was not sequenced because of both pairs of primers (JB3 and CO1-R-trema, JB3 and JB4.5) were not specific to this species. Due to the short sequences of A. hemibarbi, the final length of analyzable fragment of the Cox1 gene was 381 bp for the whole sampling. Intraspecific genetic distances of two populations of A. anastasii (Nezhinka and Komissarovka Rivers) were 0.54–1.07%. The interspecific genetic distances distinguishing A. anastasii n. sp. from A. pseudoisoporum, A. bursense, A. gotoi, A. khankaiense, A. hemibarbi, and A. lobatum were in the range 18.36–30.22%. In general, the range of interspecific genetic distances of seven Allocreadium species was the following: 18.36–39.78% (Supplementary Table 2).

Phylogenetic trees based on the Cox1 fragment comprising ten allocreadiid species of two genera were reconstructed with BI and MP methods. The resulting tree had a different topology than that of the 28S tree. The consensus tree (Figure 5) was subdivided into two clades of which the earliest branching clade was formed by outgroup taxa (three species of Crepidostomum) chosen as the root-group. The second clade represented the ingroup of Allocreadium spp. and consisted of four subclades: A. pseudoisoporum occupied the first basal subclade; the second subclade included A. hemibarbi; the third subclade comprised A. gotoi and A. bursense, and was sister to the fourth subclade combining A. anastasii n. sp., A. khankaiense, and A. lobatum.

Figure 5. Phylogenetic tree reconstructed for seven Allocreadium species based on the 381 bp of Cox1 mtDNA gene fragment. The new species Allocreadium anastasii is in bold. Reconstructions were performed with the BI and MP methods. Numbers on the branches are posterior probabilities of BI and % of MP. (A) Variant of resolution of fourth subclade in BI tree; (B) variant of fourth subclade in MP tree.

Discussion

Current research supplemented several works devoted to the study of the type genus of the family Allocreadiidae mainly in the Russian Far East: description of three new species (Vainutis 2020; Vainutis et al. 2023; Aydogdu et al. 2023), genotyping of A. khankaiense and Allocreadium sp. 1 (=A. anastasii n. sp.) (Vainutis et al. 2021), and assumptions on co-evolution with cyprinid hosts (Bogatov and Vainutis 2022). The description of two new species increased the diversity of the genus Allocreadium in the Russian Far East, and particularly in the south of the Primorsky region. Morphological and genetic data obtained originally and from published materials (Vainutis 2020; Sokolov et al. 2023) clarified the taxonomic status of Allocreadium sp. 1 from the Primorsky region of Russia and Mongolia.

Considering the genetic distances estimated with 28S rRNA and Cox1 mtDNA genes and genetic data obtained in last several studies (Vainutis 2020; Dos Santos et al. 2021; Aydogdu et al. 2023; Petkevičiūtė et al. 2023; Sokolov et al. 2023; Vainutis et al. 2023), Allocreadium anastasii n. sp. and A. macrolecithum n. sp. are distinct species. Both species belong to already known species groups of Allocreadium – A. macrolecithum n. sp. to group A and A. anastasii n. sp. to Asian group D.

Among other species groups, group A is the earliest branching clade strongly supported with BI (Bogatov and Vainutis 2022; Aydoğdu et al. 2023; Sokolov et al. 2023; Vainutis et al. 2023), Maximum Likelihood (Vainutis et al. 2023; Petkevičiūtė et al. 2023), Neighbor-Joining (Aydoğdu et al. 2023), and Maximum Parsimony methods (Aydoğdu et al. 2023). It was revealed that group A, containing only A. pseudoisoporum in Vainutis et al. (2023) (Allocreadium sp. 2 in Bogatov and Vainutis (2022)), has wider species composition including A. pseudoisoporum, A. bursensis, A. papilligerum, and A. transversale (Aydogdu et al. 2023; Petkevičiūtė et al. 2023; Sokolov et al. 2023). Likewise, the group A is characterized by diverse diagnostic morphological features (Table 2) and geographic distribution – Eastern and Western Asia and Eastern and Northern Europe. Allocreadium macrolecithum n. sp. supplemented this group (Figure 3) forming a sister relationship with A. bursense and diverging from all representatives of group A on 1.21–2.21% based on the 28S rRNA gene. These values are characteristic for interspecific divergence of Allocreadium particularly (Aydogdu et al. 2023; Vainutis et al. 2023) and trematodes in general. The phylogenetic tree based on the Cox1 gene revealed alternative but unreliable topology. Allocreadium pseudoisoporum occupied basal subclade, but other representatives were rearranged (Figure 5). Of them, the fourth subclade had two variants depending on the methods: BI variant formed a separate branch for A. anastasii and showed a sister relationship of A. khankaiense and A. lobatum; the MP variant showed a sister relationship of A. anastasii and A. khankaiense and generated a separate branch for A. lobatum – in both cases, resolution had low statistical support. The selected fragment of the Cox1 gene is too short for trustworthy phylogenetic analysis; therefore, by reducing the number of informative sites, the impact of parallel mutations and reversions directly increases. Specifically, A. gotoi and A. bursense have more common conservative sites (312 of 381, 81.9%) than A. bursense and A. pseudoisoporum (301 of 381, 79%), although the close relationship between bursense and pseudoisoporum has been demonstrated (Aydogdu et al. 2023; Sokolov et al. 2023). Discrepancy occurred when comparing A. anastasii and A. khankaiense (325 of 381, 85.3%), and A. khankaiense and A. lobatum (315 of 381, 82.7%) – although A. anastasii and A. khankaiense are more similar, it did not affect their sister relationship in the BI tree. Thus, due to the higher rates of evolution of Cox1, the use of short fragments of this gene up to 700 bp, especially at the species level within the same genus, can lead to the generation of deliberately false phylogenies.

Table 2. Modified dichotomous keys to 31 Palearctic species of Allocreadium Looss, 1900, based on morphology of the adult worms

Allocreadium anastasii n. sp. is more widely distributed than its sister species, A. khankaiense; the former is found in the rivers Nezhinka (Razdolnaya River basin) and Komissarovka (Khanka Lake basin) (this study) of Southern Primorye of Russia and Lake Khar of Western Mongolia (Vainutis 2020; Sokolov et al. 2023), whereas A. khankaiense occurs in the rivers of Southern Primorye – Komissarovka, Pavlovka (Ussuri River basin) and Artyomovka (Muravyinaya Bay basin) (Vainutis 2020; Vainutis et al. 2021; Vainutis et al. 2023). The wider distribution of A. anastasii possibly affected its morphological variability that is an extension of the anterior border of vitellarium, spatial arrangement, shape, and interlocation of testes, and extension of excretory vesicle.

According to the values of genetic distances, Aydogdu et al. (2023) revealed that Indian species A. schizothoracis is closely related to group A, including five Allocreadium species. In this study, the short 28S sequence of A. schizothoracis does not provide opportunity to reconstruct a robust phylogeny with strong nodal support of ML and MP methods, except for the BI method, which revealed strong support and the same topology obtained based on the longer sequences. On the tree reconstructed with the BI method using the ITS2 rDNA region, Sokolov et al. (2023) revealed the sister position of A. schizothoracis in relation to the branch containing A. transversale and A. papilligerum. Considering this phylogeny, A. schizothoracis should be nested within the basal group or between groups A and B. In addition, newly added A. macrolecithum n. sp. is more related to A. schizothoracis (11.18%) than other species of group A (11.75%, 12.01%, 12.11%, 14.09%). It was shown earlier that among Allocreadium spp., A. apokryfi is most closely related to A. schizothoracis (6.05%) (Aydogdu et al. 2023). On the original tree (Figure 4) based on the second variable domain of the 28S rRNA gene (499 bp), A. schizothoracis was nested within the group H containing A. apokryfi. This group was strongly supported with BI, MP, and MP (1/100/100) methods, but bootstrap values of MP and ML in ancestral nodes of species groups was lower than 75%. The Indian species A. schizothoracis is sister to the South African A. apokryfi. This particularly confirms the initial assumption of Aydogdu et al. (2023) concerning the speciation of A. apokryfi and A. schizothoracis from the common South East Asian lineage. Since that, African group H should be considered Ethiopian-Oriental based on the names of the biogeographic realms which A. apokryfi and A. schizothoracis belong to – Ethiopia and Oriental realm, respectively.

Allocreadium apokryfi is not closely related to group A because of larger number of accumulated mutation steps. Among terminal groups G and H, Allocreadium apokryfi is the first confirmed of nine African species (Dos Santos et al. 2021). Genetic distances revealed relative equidistance of A. apokryfi in relation to other Allocreadium spp. – 5.03–7.23%. This could potentially explain the inconsistencies in resolution of apokryfi on the phylogenetic trees: (1) location between basal group A and A. gotoi (B) (Dos Santos et al. 2021; Sokolov et al. 2023), (2) sister in relation to group G (A. neotenicum and A. lobatum) (Figure 3; Aydogdu et al. 2023; Petkevičiūtė et al. 2023; Vainutis et al. 2023). First topology is controversial because of insufficient statistical support of the BI method: 0.8 (Dos Santos et al. 2021) and 0.62 (Sokolov et al. 2023).

Conclusion

The description of two new species, A. anastasii n. sp. and A. macrolecithum n. sp., revealed a higher diversity of Allocreadium spp. in Russian Far East, and particularly in the Southern Primorsky region of Russia than previously known. The list of Allocreadium species known from the basin of the Komissarovka River (Primorsky region, Russia) has been increased to four species: A. hemibarbi, A. khankaiense, A. anastasii n. sp., and A. macrolecithum n. sp. In total, ten species of Allocreadium inhabit inland waters of the Russian Far East. New species A. anastasii has the wider distribution in relation to its relative A. khankaiense: in the rivers Nezhinka (Razdolnaya River basin) (Vainutis 2020) and Komissarovka (Khanka Lake basin) (this study) of Southern Primorye of Russia and Lake Khar of Western Mongolia (Sokolov et al. 2023). Two new species, Allocreadium anastasii n. sp. and A. macrolecithum n. sp., share general morphological features characteristic for the most of 31 Palaearctic Allocreadium spp.: body elongate, anterior border of vitelline fields in acetabular region or in forebody (23 sp.), ventral sucker larger than oral (27 sp.), tandem testes (31 species), cirrus pouch pre- or dorso-acetabular, ovary pretesticular (31 species). On the phylogenetic tree reconstructed with the 28S rRNA gene using Bayesian Inference and Maximum Parsimony methods, the new species A. macrolecithum n. sp. and A. anastasii n. sp. belong to already known species groups of Allocreadium: basal group A and Asian group D, respectively.