Introduction
Monogenea Van Beneden, 1858, is one of the three parasitic classes within the phylum Platyhelminthes Minot, 1876, the others being Trematoda and Cestoda. Monogeneans are mostly ectoparasites of fish, infecting all major taxa, from primitive Agnatha to Chondrichthyes to Osteichthyes, across freshwater, brackish, and marine environments. They may cause direct losses due to mortality, typically to younger fish and those in intensive culture or captivity (Thoney & Hargis, Reference Thoney and Hargis1991), by feeding on blood (Hayward et al., Reference Hayward, Bott, Naoki, Iwashita, Okihiro and Nowak2007) and/or the epithelial cells and mucus of fish (Buchmann & Bresciani, Reference Buchmann, Bresciani and Woo2006). Since their discovery in the 18th century by the German zoologist Müller (Reference Müller1776), nearly 7000 species have been described (Gibson et al., Reference Gibson, Bray, Hunt, Georgiev, Scholz, Harris, Bakke, Pojmanska, Niewiadomska, Kostadinova and Tkach2014).
Mizoram, located in northeast India, is recognised as a global biodiversity hotspot (Barman et al., Reference Barman, Singh, Singh, Saha, Singh, Laishram and Pandey2018). The region is rich in freshwater fish diversity, with at least 156 recorded species representing nine orders, 26 families, and 72 genera. Of these, approximately 78 species belong to the family Cyprinidae and are distributed across the diverse hilly terrain of the state (Lalhlimpuia et al., Reference Lalhlimpuia and Singh2017). Given that parasite species richness is generally positively correlated with host species richness, it is expected that Mizoram will support a diverse range of monogenean species. However, due to limited parasitological research conducted in the region, Dactylogyrus kolodynensis Trivedi, Prakash and Tripathi, Reference Trivedi, Prakash and Tripathi2022 is currently the only monogenean species documented from Mizoram.
Garra gotyla (Gray, 1830) (Cyprinidae, Labeoninae), commonly known as ‘sucker head’, is a benthopelagic species inhabiting the fast-flowing rivers, mountain streams, submerged rocks, and lakes of Asia (Froese & Pauly, Reference Froese and Pauly2023). It is one of the most important native ornamental fish species in India (Mandal et al., Reference Mandal, Mahapatra, Tripathi, Verma, Datta and Ngachan2007). Jha et al. (Reference Jha, Waidbacher, Sharma and Straif2005) reported that a substantial population in Nepal rely on G. gotyla as a protein source. Despite its prominence, diseases affecting G. gotyla in the region remain largely unexplored, with research primarily focusing on the impact of environmental pollutants (Munir et al., Reference Munir, Khan, Mian, Rafiq, Shahzadi, Naeem and Ahmad2021; Sharma & Langer, Reference Sharma and Langer2014). Only one species of Monogenea has been documented from the gills of G. gotyla, namely Lobotrema rajendrai, in north India over half a century ago (Srivastava & Kumar, Reference Srivastava and Kumar1983).
During a recent parasitological investigation in Mizoram, northeast India, two previously undescribed dactylogyrid species were found on the gills of G. gotyla. Subsequent morpho-molecular examination confirmed these specimens as novel species, herein described as Dactylogyrus labro sp. n. and Dogielius salpinx sp. n.
Dactylogyrus, Dogielius, and Dactylogyroides are three closely related dactylogyrid genera (subclass Polyonchoinea) whose member species are mostly found on cyprinid hosts (Gussev, Reference Gussev1963, Reference Gussev1976; Price & Yurkiewicz, Reference Price and Yurkiewicz1968). Of these, Dactylogyrus is the most species-rich, with over 1000 nominal species worldwide (WoRMS, 2025), including 58 species from India (Khwaja et al., Reference Khwaja, Prakash and Tripathi2023). Meanwhile, Dogielius is found in Africa, China (including the Amur region of Russia), the Indian subcontinent, and parts of Eurasia (Timofeeva et al., Reference Timofeeva, Gerasev and Gibson1997), with 43 nominal species globally, including five from India (WoRMS, 2025). On the other hand, Dactylogyroides Gusev, 1963 comprises 16 known species distributed across Africa, Ceylon, India, Peninsular Malaysia, Thailand, and China, including seven from India (WoRMS, 2025). The key morphological differences among these genera are summarised in Table 1.
Table 1. Diagnostic morphological characters distinguishing Dactylogyrus Diesing, 1850, Dogielius Bychowsky, 1936, and Dactylogyroides Gusev, 1963 (Dactylogyridae)
The taxonomic validity and phylogenetic relationships of these three genera have long been a subject of confusion. For example, Musilová et al. (Reference Musilová, Řehulková and Gelnar2009) stated that ‘Dactylogyrus and Dogielius are morphologically similar genera parasitising many species of the Cyprinidae…. One striking feature that separates these taxa is [that the] Dactylogyrus species lost the ventral anchor-bar complex, while Dogielius spp. lost the dorsal complex’. Meanwhile, Kritsky et al. (Reference Kritsky, Aquaro and Galli2010) noted that ‘the phylogenetic position of Dogielius and Thaparocleidus (both with freshwater species having ventral anchor/bar complexes) is currently unknown’. More recently, some studies, such as Raphahlelo et al. (Reference Raphahlelo, Přikrylová and Matla2020), assigned some monogenean species to Dactylogyrus which, based on illustrations and images, appear to correspond more closely with Dactylogyroides. In fact, one unpublished preprint even proposed synonymising both Dogielius and Dactylogyroides with Dactylogyrus (available at https://ecoevorxiv.org/repository/view/3947/).
Against this backdrop, this paper aims to describe and illustrate two new monogenean species found on G. gotyla in northeast India while also discussing the phylogenetic relationships among Dactylogyrus, Dogielius, and Dactylogyroides.
Materials and methods
Between March and May 2022, 50 specimens of G. gotyla were caught with electrofishing from three locations in the Serchhip District of Mizoram, northeast India: Mat River (23° 16′ 33.8″ N, 92° 49′ 40.1″ E), Tuichang River (23° 15′ 58.8″ N, 92° 57′ 07.7″ E) and Tuikum River (23° 24′ 26.0″ N, 92° 53′ 07.0″ E). Identification and nomenclature of fish followed the method of Nebeshwar and Vishwanath (Reference Nebeshwar and Vishwanath2013). Before parasitological investigations, individual fish were euthanized with an overdose of sodium bicarbonate-buffered tricaine methanesulfonate (MS-222 100 mg/L; Sigma Aldrich Co., USA).
Freshly excised gills were examined under a Leica EZ4HD binocular microscope, and any monogeneans spotted were carefully removed using fine dissection needles. These monogeneans were either mounted unstained in a small drop of Hoyer’s medium or glycerine or stained with Gomori’s trichrome before being mounted in dibutyl phthalate polystyrene xylene (DPX), following the protocols outlined by Kritsky et al. (Reference Kritsky, Thatcher and Boeger1986) and Tripathi et al. (Reference Tripathi, Matey and Agarwal2022). The slides were examined using a light microscope (DM4B, Leica Microsystems, Germany) equipped with phase-contrast and differential interference contrast optics and a Leica DFC7000 T digital camera. Composite line drawings were prepared using a drawing tube attachment fixed to a light microscope (BX 51, Olympus, Japan). The prevalence and mean intensity of infection were calculated according to Bush et al. (Reference Bush, Lafferty, Lotz and Shostak1997). Type specimens (one holotype and four paratypes of each species) were deposited in the Queensland Museum, Brisbane, Australia.
Individual monogeneans were identified morphologically and pooled by species (>5 individuals). Total genomic DNA was extracted from these pools using the Extracta DNA Prep (Quantabio, Beverly, USA). The variable domains D1-D2 of the 28S rRNA gene were amplified by employing the primer set C1 (5′-ACCCGCTGAATTTAAGCA-3′) and D2 (5′-TGGTCCGTGTTTCAAGAC-3′) (Hassouna et al., Reference Hassouna, Mithot and Bachellerie1984). The amplification profile was constructed following Šimková et al. (Reference Šimková, Matejusova and Cunningham2006). The PCR products were purified with the QIAquick PCR purification kit (Qiagen, USA) and sequenced in both forward and reverse directions using the same primers employed in the amplification. The resulting sequence chromatograms were edited with SnapGene version v5.3 (http://www.snapgene.com) and assembled using the DNA Baser Sequence Assembler (2013). The generated sequence contigs were submitted to GenBank and compared with related sequences in the NCBI Database (BLASTn, https://blast.ncbi.nlm.nih.gov/Blast.cgi) to achieve species-rank identification.
To ascertain the systematic position of the new species, we conducted phylogenetic analyses. All available 28S rRNA sequences for species of Dogielius, Dactylogyrus, and Dactylogyroides were retrieved from NCBI GenBank (search term 1: “Dactylogyrus AND 28S”; search term 2: “Dogielius AND 28S”; search performed on 19 May 2025) and converted into a local BLAST database, against which the newly generated partial 28S rRNA sequences were compared using the BLASTn algorithm. All sequences with sequence similarity of more than 80% across an alignment length of at least 320 bp were included in subsequent analyses, together with the partial 28S rRNA sequences of the new species.
Tetraonchus monenteron (Wagener, 1857) Diesing, 1858 (GenBank: AJ969953) and Paradiplectanum sillagonum (Tripathi, 1959) Domingues and Boeger, 2009 (GenBank: AY553626) were included as the outgroups to root the tree, following Roohi et al. (Reference Roohi, Asl, Pourkazemi and Shamsi2019) and Kmentová et al. (Reference Kmentová, Cruz-Laufer, Pariselle, Smeets, Artois and Vanhove2022), respectively.
Sequences were clustered at 100% identity using vsearch v. 2.23.0 (Rognes et al. Reference Rognes, Flouri, Nichols, Quince and Mahé2016), before multiple sequence alignment was performed using MAFFT v. 7.464 in L-INS-I mode (Katoh & Standley Reference Katoh and Standley2013). Alignments were subsequently trimmed using trimAl v. 1.4.1 in gappyout mode (Capella-Gutiérrez et al. Reference Capella-Gutiérrez, Silla-Martínez and Gabaldón2009). A maximum-likelihood tree was inferred using IQ-TREE v. 2.0.7 (Nguyen et al. Reference Nguyen, Schmidt, von Haeseler and Minh2015) with 1000 ultrafast bootstrap replicates, using the best-fitting model of sequence evolution as determined automatically by ModelFinder as implemented in IQ-TREE. Bayesian inference was performed on the same trimmed data matrix using MrBayes v3.2.6 (Ronquist et al. Reference Ronquist, Teslenko, van der Mark, Ayres, Darling, Höhna, Larget, Liu, Suchard and Huelsenbeck2012), with the model of evolution set to GTR with gamma-distributed rate variation across sites and a proportion of invariable sites (GTR+I+G; nst=6 rates=invgamma) and two independent MCMC (Markov chain Monte Carlo) runs with four chains each running for up to 10M generations (further relevant MCMC parameters: temp=0.2 mcmcdiagn=yes diagnfreq=1000 stoprule=yes stopval=0.01 samplefreq=100 relburnin=yes burninfrac=0.25), with the automatic stopping rule set to ASDSF≤0.01.
In addition to the large dataset, we compiled a smaller dataset comprising a random selection of publicly available sequences from the major clades. Multiple sequence alignment was performed with ClustalW (Thompson et al., Reference Thompson, Higgins and Gibson1994) in Molecular Evolutionary Genetic Analysis 11 (MEGA11) software (Tamura et al., Reference Tamura, Stecher and Kumar2021). Phylogenetic inferences (ML and BI) were then performed as described above. Genetic distances (uncorrected p-distance) were calculated in MEGA11 (Tamura et al., Reference Tamura, Stecher and Kumar2021) based on a reduced (small) dataset represented in the ML tree, with default settings (Rates among Sites: Uniform Rates; Gaps/Missing Data Treatment: Pairwise deletion).
The aligned sequences are provided in Supplementary Data S1 and S2, while the corresponding tree files are available as Supplementary Data S3 (ML tree, NEWICK format) and S4 (BI tree, NEXUS format). Trees based on the reduced dataset are provided in Supplementary Data S5–S6, and a list of sequences comprising the collapsed clades in Figure 5 is provided in Supplementary Data S7.
Results
Morphological characterization
Class Monogenea van Beneden, 1858
Order Dactylogyridea Bychowsky, 1937
Family Dactylogyridae Bychowsky, 1933
Genus Dactylogyrus Diesing, 1850
Dactylogyrus labro sp. n.
ZooBank registration: urn:lsid:zoobank.org:act:60304DFF-4D5F-4C42-9C72-62539F4E6C0C
Type host: Garra gotyla (Gray, 1830) (Cypriniformes, Cyprinidae)
Type locality: Mat River, Tuichang River, and Tuikum River in Serchhip district, Mizoram, India
Infection site: Gill lamellae
Infection parameters: See Table 2.
Table 2. Prevalence and mean intensity of infection of Dactylogyrus labro sp. n. and Dogielius salpinx sp. n. from Garra gotyla in the present study
Type material: Queensland Museum, Holotype, AHC37169; 4 Paratypes, AHC37170-AHC37173
Representative DNA sequence: 28S rRNA (850-bp) (GenBank: OR347559)
Etymology: From the Latin noun labrum (= flange), in reference to the presence of a projecting edge at the base of the copulatory tube of this species.
Description (Figures 1 and 2; metrical data in Table 3)
Figure 1. Line drawings of Dactylogyrus labro sp. n. from Garra gotyla (Gray, 1830). (A) dorsal anchor; (B) dorsal bar; (C) hook (pairs I– VII); (D) male copulatory organ; (E) vagina. Scale bar = 30 μm.
Figure 2. Phase-contrast micrographs of Dactylogyrus labro sp. n. from Garra gotyla (Gray, 1830). (A) whole mount; (B) anchor-bar complex and hooks; (C) male copulatory organ; (D) vagina. Scale bar = 20 μm.
Table 3. Measurement data (in μm) for Dactylogyrus labro sp. n. and Dogielius salpinx sp. n. [presented as range, followed by mean and number (n) of structures measured in parentheses]
With characters of the genus as defined by Diesing (1850). Single pairs of (dorsal) anchors, with distinct outward bulge on an otherwise evenly curved shaft. Single (dorsal) bar, rod-shaped with very short posteromedial expansion, and downward posterolateral expansions on both ends. Seven pairs of hooks, uniform in shape but diverse in size, each with delicate point, upright thumb, shank of two subunits (proximal subunit significantly expanded), and filamentous hook (FH) loop extending to the near union of shank subunits. Male copulatory complex comprises a male copulatory organ (MCO), accessory piece, and a thick ligament. Male copulatory organ is a loose coil of 1.1/2 clockwise rings and a swollen base with a lateral flange, narrowing to termination. Proximal part of the accessory piece articulates with the base of the copulatory tube through a thick ligament, and the distal part is twisted with a grooved sheath serving as guide for MCO and dorsal ribbon-like sclerotised strap. Vagina tubular, sclerotised, serpentine-coiled, with swollen proximal base and slightly expanded distal end with pore.
Remarks: Dactylogyrus labro sp. n. is distinguished from its congeners by the presence of a unique combination of morphological features, including an MCO with 1.1/2 complete clockwise coils with a swollen base bearing a lateral flange, an accessory piece comprising a grooved sheath to guide the MCO, accompanied by a ribbon-like sclerotised strap, and a serpentine, coiled vagina. To the best of our knowledge, the presence of a flange at the base of MCO has not been reported in any other Dactylogyrus species and, hence, may represent a diagnostic feature of D. labro sp. n.
Class Monogenea Van Beneden, 1858
Order Dactylogyridea Bychowsky, 1937
Family Dactylogyridae Bychowsky, 1933
Genus Dogielius Bychowsky, 1936
Dogielius salpinx sp. n.
ZooBank registration: urn:lsid:zoobank.org:act:8B8F9CB3-BE2F-440A-A31F-CB7BBDD0A677
Type host: Garra gotyla (Gray, 1830) (Cypriniformes, Cyprinidae)
Type locality: Mat River in Serchhip District of Mizoram, northeast India.
Additional localities: Tuichang River and Tuikum River in Serchhip District of Mizoram.
Infection site: Gill lamellae
Infection parameters: See Table 2.
Type material: Queensland Museum, Holotype, AHC 37164; 4 Paratypes, AHC37165-AHC37168.
Representative DNA sequence: 28S rRNA (873 bp) (GenBank: OR347563)
Etymology: From the Greek salpinx = a trumpet, in reference to the accessory piece of male copulatory organ.
Description (Figures 3 and 4; metrical data in Table 3)
Figure 3. Line drawings of Dogielius salpinx sp. n. from Garra gotyla (Gray, 1830). (A) ventral anchor; (B) ventral bar; (C) male copulatory organ; (D) hook (pairs I– VII); (E) vagina. Scale bar = 30 μm.
Figure 4. Phase-contrast micrographs of Dogielius salpinx sp. n. from Garra gotyla (Gray, 1830). (A) whole mount; (B) anchor-bar complex and hooks; (C) male copulatory organ; (D) vagina and egg. Scale bar = 20 μm.
With traits of the genus as defined by Bychowsky (1936). Single pair of (ventral) anchors, with a deep cleft between two well-developed roots; point recurved, bent at the end. Single (ventral) bar, wide U-shaped, robust, with medial part slightly twisted forward, extremities nearly bilobed. Seven pairs of hooks, similar in shape but dissimilar in size, each with a delicate point, erect thumb, and a shank of two subunits (proximal subunit expanded), along with a filamentous hook (FH) loop extending to the near union of shank subunits. Male copulatory complex comprises male copulatory organ (MCO) connected to accessory piece by laminar ligament attached to the base of the MCO. Male copulatory organ is straight and robust with inflated base. Accessory piece robust, comprising two subunits; anterior subunit serves as guide for the MCO, posterior subunit is somewhat trumpet-head-shaped, which articulates with the base of MCO via long and thick sclerotised thread. Vagina globular, seed-shaped, with a central slit-like opening, and highly sclerotised, and sinistral; vaginal tube not visible. Egg elongate-oval, shell surface thick, smooth; internal contents granular; no polar filaments or operculum observed.
Remarks: Dogielius salpinx sp. n. closely resembles D. kaelensis Narba, Matey, Tripathi 2022 from Garra annandalei Hora, 1921 and D. catlaius (Jain, 1962) Gusev, 1976 from Catla catla (Hamilton, 1822) (now Gibelion catla) in India, in the nearly identical shape and size of their anchors. The proposed new species, however, differs from D. kaelensis by possessing a larger ventral bar (66 μm vs 43 μm in D. kaelensis), an accessory piece of distinct morphology (trumpet-head-shaped vs. a ring-shaped middle part in D. kaelensis), and a differently shaped vagina (globular, seed-shaped vs. flower-shaped in D. kaelensis). The new species can also be differentiated from D. catlaius by the shape of the MCO, which is nearly straight in the new species (curved in D. catlaius), and an accessory piece that is trumpet-head-shaped in the new species (pivot-shaped with three processes in D. catlaius). This distinct haptoral and reproductive sclerite morphology distinguishes D. salpinx sp. n. as a novel species within the genus Dogielius.
Molecular characterization
Sequencing of the partial 28S rRNA gene of D. labro sp. n. resulted in an amplicon 850 base pairs long. Sequence comparison using a BLASTn search showed no exact matches in GenBank. The species with the closest nucleotide similarity to D. labro sp. n. was D. brevicirrus Paperna, 1973 from Labeo parvus (Boulenger, 1902) (KY629362; 99% query coverage, 88.62% maximum identity) in Senegal, West Africa, from which the new species differed by 13 gaps (1%) (Table 4). This similarity was confirmed in the phylogenetic tree, where D. labro sp. n. clustered with D. brevicirrus, D. senegalensis Paperna, 1969, and D. oligospirophallus Paperna, 1973 (three top BLASTn search matches) with 100% bootstrap support (Figure 5).
Table 4. Top three BLASTn search matches in GenBank for 28S rRNA gene sequences of Dactylogyrus labro sp. n. (850 bp) and Dogielius salpinx sp. n. (873 bp)
Figure 5. Rooted phylogenetic tree inferred from all available 28S rRNA sequences of Dactylogyrus spp., Dogielius spp., and Dactylogyroides spp. with sequences generated in the present study marked with solid circles. Node support is shown as posterior probability for Bayesian inference (BI) and bootstrap values (from 1000 replicates) for maximum likelihood (ML).
Sequencing of the partial 28S rRNA gene of D. salpinx sp. n. resulted in an amplicon 873 base pairs long. Sequence comparison using a BLASTn search revealed less than 100% similarity with any of the known sequences in GenBank. The species with the closest nucleotide similarity to D. salpinx sp. n. was D. brevicirrus from L. parvus (KY629362; 97% query coverage, 86.61% maximum identity) in Senegal, West Africa, from which the new species differed by 24 gaps (2%) (Table 4). The average genetic p-distance between D. labro sp. n. and other species of Dactylogyrus ranged between 11.44% and 21.86%, while that between D. salpinx sp. n. and D. catlaius was 11.78% (Table 5).
Table 5. A distance matrix viewer for uncorrected pairwise genetic p-distance based on partial 28S rDNA sequences included in the phylogenetic analysis based on a reduced (small) dataset. Newly sequenced taxa are in bold
Phylogenetic inference was performed based on partial 28S rRNA sequences obtained for D. labro sp. n. and D. salpinx sp. n. collected in India, combined with 219 published rRNA sequences, including 217 representatives of the genera Dactylogyrus, Dogielius, and Dactylogyroides, and two outgroup sequences. Maximum likelihood and Bayesian inference (ASDSF = 0.014, APRSF = 1.001, and ESS > 1000 for all parameters after 10M generations) of this dataset yielded congruent topologies, supporting the phylogenetic placement of the new species within a strongly supported clade comprising species infecting cyprinid fishes from Europe, Asia, and Africa (Fig. 5).
Dactylogyrus labro sp. n. and D. salpinx sp. n. were found most closely related to D. oligospirophallus and D. tripathii, respectively, with strong statistical support in ML and BI analyses. The two new species are grouped with species infecting African and Asian cyprinids (subclade A), with the majority of isolates from the subfamily Labeoninae. Notable exceptions were D. marocanus (ex. subfamily Torinae), D. pulcher (ex. subfamily Barbinae), and Dactylogyroides tripathii (ex. subfamily Smiliogastrinae).
Subclade B was dominated by Dactylogyrus spp. infecting cyprinids of the subfamily Smiliogastrinae, while subclade C comprised Dactylogyrus spp. infecting hosts of subfamilies Cyprininae and Barbinae. Subclade D comprised Dactylogyrus spp. isolates primarily from Cyprininae hosts (chiefly Cyprinus spp. and Carassius spp.), with few isolates reported from Xenocypridinae. Subclade B was consistently recovered as sister to subclade A in both ML and BI analyses based on the full dataset, albeit with only moderate statistical support; hence, this hypothesis must be considered tentative until further data become available.
Subclades C and D were not recovered in the analyses based on our reduced dataset (Supplementary Data S5–S6), indicating that limited taxon sampling may affect their inferred branching order. While the higher-level relationships in the genus Dactylogyrus are not the focus of the current study and will therefore not be discussed further, the branching order of the remaining clades appears robust and strongly supported in both ML and BI analyses of the full dataset (Figure 5) and is in agreement with a recent study (Nitta et al., Reference Nitta and Nagasawa2023). It is noteworthy, however, that sequences previously deposited as D. catlaius are consistently recovered at a basal position across all analyses.
Discussion
This study presents two new dactylogyrid species in the gills of G. gotyla in Mizoram: D. labro sp. n. and D. salpinx sp. n. Morpho-molecular comparisons with other closely related species confirmed the validity and placement of these two taxa within Dactylogyrus and Dogielius. Surprisingly, we did not find any evidence of L. rajendrai infection in any of the sampled host specimens. While epidemiological studies were not conducted, the high prevalence of two monogenean species suggests that these parasites may represent a significant risk to fish health in northeast India. As such, more experimental efforts are required to map the diversity of monogenean parasites and explore their impacts on the health of their host fishes in this region. Such research is especially necessary given that all monogeneans are potentially hazardous fish parasites.
According to our phylogenetic results, D. salpinx sp. n. and D. labro sp. n. were placed into a strongly supported clade (100% bootstrap support) primarily comprising Dactylogyrus spp. from labeonin cyprinids, alongside D. tripathii from smiliogastrin cyprinids (Figure 5, subclade A). Dogielius salpinx sp. n. was found to be a sister species to D. tripathii, indicating a closer relationship between Dogielius and Dactylogyroides than between Dogielius and Dactylogyrus. This relationship was supported by a lower genetic distance (16.80%) between Dogielius and Dactylogyroides compared to Dogielius and Dactylogyrus (14.15%–22.36%) (Table 5). Surprisingly, the sequence previously deposited for D. catlaius did not cluster with any of the main clades, not even subclade A, which includes D. salpinx sp. n. Rather, it diverged first (100% bootstrap support).
A major limitation in accessing the potential synonymy among the genera Dogielius, Dactylogyroides, and Dactylogyrus is the lack of high-quality, morpho-taxonomically validated reference sequences for Dogielius and Dactylogyroides in GenBank. For Dactylogyroides, only five 28S rRNA gene sequences are available, of which just one has been formally published (Dactylogyroides tripathii, JX993982; Chiary et al., Reference Chiary, Chaudhary and Singh2013). Moreover, only two of these sequences exceed 381 bp in length, thereby limiting their utility for robust phylogenetic inference.
The situation is even more complex for Dogielius. GenBank lists two distinct datasets: a single 28S rRNA sequence (KC687091; Dash et al., Reference Dash, Kar, Mishra and Sahoo2014) for Dactylogyrus catlaius Jain, 1961—a species reassigned to Dogielius by Gusev (1976)—and 22 sequences (MW479163–MW479184; Paul and Sahoo, Reference Paul and Sahoo2022), all labelled as Dogielius catlaius isolates despite being described as Dactylogyrus catlaius in the corresponding publication. All 23 sequences originate from a single laboratory and lack independent taxonomic validation.
Dash et al. (Reference Dash, Kar, Mishra and Sahoo2014) identified D. catlaius from Labeo rohita (Hamilton, 1822) (Cyprinidae), an atypical host, using only ‘a light microscopy image at 10x magnification’—an approach insufficient for definitive species-level morphological identification in monogeneans. Similarly, Paul and Sahoo (Reference Paul and Sahoo2022) reported D. catlaius from three unrelated cyprinid hosts: Labeo rohita, Catla catla, and Cirrhinus mrigala (Hamilton, 1822). Notably, neither study provided diagnostic-quality microscopic images or the line drawings of the specimens identified as D. catlaius, nor did they deposit voucher material in any recognised museum collection, further limiting the verifiability of their identifications.
In both studies, DNA was extracted from the host tissues (gills/mucus) rather than from isolated, morphologically confirmed parasites. This introduces a high risk of contamination from co-infecting taxa and increases the chances of misidentification, especially since L. rohita is known to host at least five monogenean genera: Dactylogyrus, Dogielius, Gyrodactylus von Nordmann, 1832, Mazocraes Hermann, 1872, and Paramazocraes Tripathi, 1959 (Pandey & Agarwal, Reference Pandey and Agarwal2008; Sailaja et al., Reference Sailaja, Shameem and Madhavi2016). Furthermore, the sequence submitted by Dash et al. (Reference Dash, Kar, Mishra and Sahoo2014) is 100% identical to a sequence labelled as D. labei (JX566720.1) from C. catla, indicating a potential misidentification and highlighting the need for critical re-evaluation of these sequences.
Collectively, these findings suggest that none of the sequences currently submitted to GenBank under the name Dogielius are taxonomically reliable. This likely explains the anomalous basal placement of D. catlaius in our phylogenetic analysis. If the sequence of Dash et al. (Reference Dash, Kar, Mishra and Sahoo2014) truly represented a Dogielius species, it would be expected to cluster as a sister taxon to Dogielius salpinx sp. n. The present study thus provides the first phylogenetically informative 28S rRNA sequence for Dogielius, generated from morphologically identified specimens using standard molecular protocols.
Our study supports the view that Dactylogyrus, in its traditional circumscription, should continue to be considered paraphyletic (see Aguiar et al., Reference Aguiar, Maia, Silva, Ceccarelli, Domingues and Adriano2017; Benovics et al., Reference Benovics, Desdevises, Vukić, Šanda and Šimková2018, Reference Benovics, Nejat, Abdoli and Šimková2021; Kmentová et al., Reference Kmentová, Cruz-Laufer, Pariselle, Smeets, Artois and Vanhove2022; Ondračková et al., Reference Ondračková, Seifertová, Tkachenko, Vetešník, Liu, Demchenko and Kvach2023; Hao et al., Reference Hao, Wei, Liu, Shi, Arken and Yue2023), with species from two other genera (Dogielius and Dactylogyroides) nested within it. While additional molecular sequences of the two latter genera are required for their generic status to be tested, this study provides the first confirmed molecular data on Dogielius spp., representing a step towards establishing a molecular taxonomic reference library for this genus. Although the pathogenic implications of D. salpinx sp. n. and D. labro sp. n. are currently unknown, their discovery highlights the unmarked diversity of fish parasites in Mizoram, India.
Conclusion
This study reports the discovery and description of two new monogenean species, Dactylogyrus labro sp. n. and Dogielius salpinx sp. n., parasitising the gills of Garra gotyla in Mizoram, northeast India. This region has high freshwater biodiversity yet limited parasitological investigations. Morphological examination, combined with partial 28S rRNA gene sequencing, confirmed the distinctiveness and novelty of both species. Phylogenetic analyses placed D. labro sp. n. and D. salpinx sp. n. within a well-supported clade of Dactylogyrus spp. infecting African and Asian Labeoninae hosts, consistent with established patterns of host–parasite co-evolution in monogeneans. Dogielius salpinx sp. n. was recovered as sister to D. tripathii, indicating a closer evolutionary relationship between Dogielius and Dactylogyroides. Notably, D. catlaius was consistently recovered in a basal position across all analyses, suggesting either unresolved generic boundaries within the Dactylogyrus–Dogielius complex or, more likely, that the available sequences currently attributed to D. catlaius are unreliable. While this study highlights ongoing confusion in the systematics of Dactylogyrus, Dogielius, and Dactylogyroides—compounded by the paucity of high-quality reference sequences for the latter two—it supports the prevailing view that Dactylogyrus is paraphyletic, with species of Dogielius and Dactylogyroides nested within it. These findings emphasise the urgent need for expanded molecular sampling and integrated taxonomic approaches to resolve the complex evolutionary relationships within this hyper-diverse genus.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0022149X25100813.
Data availability statement
The parasite materials that support this study are available at the Queensland Museum. Sequence data generated in this study are publicly available in GenBank.
Acknowledgments
SP gratefully acknowledges Mrs. Zonuntluangi (Joint Director, Department of Fisheries, Government of Mizoram), Dr. Kuldeep Kumar (ICAR-CIFA, Bhubaneswar, India), and Dr. K. D. Joshi (ICAR-NBFGR, Lucknow, India) for their valuable assistance with the collection and identification of fish specimens. We also sincerely thank the reviewers for their constructive feedback.
Author contribution
SP, AKT, SS: conceptualisation, funding acquisition, data collection and analysis, and figure preparation. AT, SS: writing the original draft of the manuscript. CH, AT: phylogenetic analysis and interpretation of molecular data. All authors approved the final draft of the manuscript for publication.
Financial support
The Department of Science and Technology, Government of India funded this research [No. DST/INSPIRE Fellowship/2019/IF190017]. AT acknowledges the use of laboratory facilities established under the research funding from the Science and Engineering Research Board (SERB), Government of India (SERB–EMR/2017/003232).
Competing interests
The authors declare no conflicts of interest.
Ethical standard
All procedures involving live fish were conducted in accordance with relevant guidelines and regulations, with permission from the Directorate of Fisheries, Government of Mizoram, India [Permit No. C.15015/1/2021-DTE(FY)].
Open access
