Introduction
There is a great diversity of monogenetic parasite species in vertebrates, including sea and freshwater fish (Thatcher, Reference Thatcher2006). Up to 300 monogenean species belonging to the Gyrodactylidae and Dactylogyridae have been described in Brazil, even though only a small percentage of species is known (Eiras et al., Reference Eiras, Takemoto and Pavanelli2010).
The genus Urocleidoides is one of the monogenetic genera found in freshwater fish in Brazil. Mizelle & Price (Reference Mizelle and Price1964) originally characterized this genus by the presence of a sinistral vaginal sclerite, overlapping gonads, clockwise cirral rings, simple anchors, hooks with enlarged shanks and the reduction of the first and fifth pair in infected fish (Kritsky et al., Reference Kritsky, Thatcher and Boeger1986). Based on the aforementioned features, the genus was reviewed by Kritsky et al. (Reference Kritsky, Thatcher and Boeger1986), and only 8 out of 30 species listed by Kritsky & Thatcher (Reference Kritsky and Thatcher1983) were considered to belong to the genus.
Sixteen species of Urocleidoides are known in Brazil (Takemoto et al., Reference Takemoto, Luque, Bellay, Longhini, Graça, Pavanelli, Takemoto and Eiras2013; Moreira et al., Reference Moreira, Scholz and Luque2015), of which five are hosted by Hoplias aff. malabaricus (Bloch, Reference Bloch and Bloch1794), namely Urocleidoides eremitus (Kritsky et al., Reference Kritsky, Thatcher and Boeger1986), U. malabaricusi, U. cuiabai, U. brasiliensis and U. naris (Rosim et al., Reference Rosim, Mendonza–Franco and Luque2011).
Morphological identification of Urocleidoides species is straightforward with well-established taxonomies. However, doubts exist with regard to the monophyletic origin of the genus because several species listed as Urocleidoides by Cohen et al. (Reference Cohen, Justo and Kohn2013) do not have the vaginal sclerite, which is an important criterion for the inclusion of the species within this genus (Kritsky et al., Reference Kritsky, Thatcher and Boeger1986). Genetic data may be a great help in testing the hypothesis of the monophyletic origin for the species of Urocleidoides.
Although monogeneans are the most studied group of fish parasites in Brazil (Karling et al., Reference Karling, Ueda, Takemoto and Pavanelli2014), few studies have employed genetic data to investigate the phylogeny of these parasites in taxonomic studies (Gasques et al., Reference Gasques, Souza, Graça, Pavanelli, Takemoto and Eiras2013). Recently, Mendoza-Palmero et al. (Reference Mendoza-Palmero, Blasco-Costa and Scholz2015) conducted a phylogenetic study with 28S rRNA sequences and reported that there was no taxonomical support for the two subfamilies of the monogeneans in the neotropical region, and allocated them to other groups. The aforementioned work highlights the importance of such tools for taxonomists who aim at achieving greater precision in their studies.
Nucleotide sequence analysis of the barcode region of the mitochondrial cytochrome c oxidase gene (COI) has been very important in taxonomical studies. Sequence alignment of barcoded DNA of the gene, approximately 650 base pairs (bp) long, has been done to use the gene with a total size of 1590 bp in Benedenia seriola (Monogenea: Monopisthocotylea), a parasite whose mitochondrial genome has already been sequenced (Perkins et al., Reference Perkins, Donnellan, Bertozzi and Whittington2010).
The degree of conservation of the COI gene makes it a good marker for species differentiation (Solé-Cava, Reference Solé-Cava2008). Since it is a mitochondrial gene, it is haploid without any chance of genetic recombination, and may provide important information in the phylogenetic studies of closely related species (Perkins et al., Reference Perkins, Martinsen and Falk2011).
Our study investigated the COI sequences of U. malabaricusi and U. cuiabai from the trahira fish, H. aff. malabaricus, of the flood plain of the Upper River Paraná, to collect the genetic information to be used in further phylogenetic studies on the genus.
Materials and methods
Collection and examination of monogeneans
Sixteen specimens of the fish H. aff. malabaricus (trahiras) were caught in the River Paraná (Long Term Ecological Research – LTER) in April 2012, using nets of different mesh sizes, at locations near Porto Rico PR Brazil (22°43′S; 53°10′W) and the research centre Núcleo de Pesquisas em Limnologia, Ictiologia e Aquicultura (Nupélia) of the Universidade Estadual de Maringá.
Processing of the specimens comprised thawing, removal of gills and analysis by stereoscopic microscopy to separate the parasites. Parasites were later identified by morphological analysis using an optical microscope and slides with water, according to Rosim et al. (Reference Rosim, Mendonza–Franco and Luque2011). Each specimen was identified and stored individually in a microtube containing 50 μl of 96% ethanol. Urocleidoides cuiabai and U. malabaricusi were not found in all the fish analysed.
Molecular analysis
DNA was extracted following the simplified protocol by Beltran et al. (Reference Beltran, Galinier, Allienne and Boissier2008). Sequencing of the partial COI gene was conducted using the amplification product of COI sequences obtained with primers COI_Mono_5′: 5′-TAATWGGTGGKTTTGGTAA-3′, COI_Mono_3′: 5′-AATGCATMGGAAAAAAACA-3′ and COI_Mono_int3′: 5′-ACATAATGAAARTGAGC-3′ according to the protocol of Plaisance et al. (Reference Plaisance, Rousset, Morand and Littlewood2008). Polymerase chain reaction (PCR) amplicons were sequenced using the primer COI_Mono_int3′ in MegaBACE 1000 (Amersham Biosciences, Little Chalfont, Bucks, UK).
The sequences were edited with BioEdit (Hall, Reference Hall1999) and aligned using Clustal W (Thompson et al., Reference Thompson, Higgins and Gibson1994). The determination of p-distance, the test for the best substitution model (ModelTest) and phylogeny reconstruction built by the maximum likelihood algorithm were carried out with MEGA 6 (Tamura et al., Reference Tamura, Stecher, Peterson, Filipski and Kumar2013).
Results
The continuous alignment of 420 bp from the nine sequences could be performed after manual editing. The mean nucleotide frequency reached 30% for A, 20% for T, 20% for C and 30% for G. The best sequences derived from U. cuiabai (Uc) and U. malabaricusi (Um) sequencing were taken into account for analysis. They were deposited in GenBank with the following accession numbers: Uc1 (KT625591), Uc2 (KT625592), Uc3 (KT625593), Uc4 (KT625594), Uc5 (KT625595), Um1 (KT625587), Um2 (KT625588), Um3 (KT625589) and Um4 (KT625590).
The alignment of sequences used as the basis for the preparation of the divergence index while taking p-distances into consideration is provided in table 1.
Divergence matrix with p-distances between specimens of Urocleidoides cuiabai (from Uc1 to Uc5) and Urocleidoides malabaricusi (from Um1 to Um4).
The mean p-distance between U. cuiabai and U. malabaricusi was 23.7% ( ± 0.4%), i.e. 12.5 times higher than the intra-species mean of U. cuiabai (1.9%) ( ± 0.73%). The mean intra-specific p-distance rate of U. malabaricusi was 14.4% ( ± 7.0%), i.e. 7.4 times higher than the rate found in U. cuiabai.
The reconstruction of phylogeny was performed with the best substitution model indicated by ModelTest, which was the Hasegawa–Kishino–Yano test (Hasegawa et al., Reference Hasegawa, Kishino and Yano1985) with gamma distribution by maximum likelihood algorithm with 10,000 bootstrap re-samplings. Figure 1 demonstrates that the grouping of specimens of the species U. cuiabai is more concise, with lower bootstrap rates than U. malabaricusi ramifications.
Phylogenetic relationships of Urocleidoides cuiabai (Uc) and Urocleidoides malabaricusi (Um), with the maximum likelihood algorithm by the Hasegawa–Kishino–Yano method, with gamma distribution and 10,000 bootstrap re-sampling. The barcode sequence of Tetrancistrum nebulosi (KJ0001340) was used as the outgroup.
Discussion
Two sequences, namely the internal transcribed spacers (ITS1 and ITS2) of rDNA and the mitochondrial cytochrome c oxidase I (COI), have been widely used in the molecular analysis of parasites of the phylum Platyhelminthes. The mutation rates were about 1% for ITS among the researched congener species in the phylum and were about 10% for the COI gene (Vilas et al., Reference Vilas, Criscione and Blouin2005). Locke et al. (Reference Locke, Mclaughlin, Dayanandan and Marcogliese2010) reported similar results in parasites of the genus Diplostomum of the subclass Digenea.
Although molecular analysis for the establishment of phylogenetic relationships of some monogenetic species, such as Gyrodactylus, was successful, others did not have similar success because of the high variability of the regions under analysis. This was the case for Lamellodiscus in the molecular analysis using COI and ITS. Poisot et al. (Reference Poisot, Verneau and Desdevises2011) registered high degrees of variability within the group, with a low co-relationship between morphological and molecular characteristics.
Vilas et al. (Reference Vilas, Criscione and Blouin2005) investigated variability rates in Platyhelminthes and reported distances of approximately 10% divergence for congener species (for gene COI). The authors recommended that rates above 5% divergence should be investigated. Ratnasingham & Hebert (Reference Ratnasingham and Hebert2007) suggested that species should be investigated when the COI nucleotide sequence divergence of a group is higher than 2%. In our study, the mean divergence rate among the four U. malabaricusi specimens is much higher than suggested.
Additionally, the inter-species p-distance rates were higher than those obtained for other Monopisthocotylea species (Poisot et al., Reference Poisot, Verneau and Desdevises2011). However, it must be noted that our study contained a small number of specimens; an increase in sampling size will probably decrease the inter-species p-distance rates. Despite the aforementioned statement, a diversity greater than expected in U. malabaricusi may be verified when the mean rate of intra-species p-distance (14.4 ± 7.0%) is compared with that of other monogenetic species, which varied between 0.32% in Euryhaliotrema grandis and 8.6% in Protopolystoma símplices (Poisot et al., Reference Poisot, Verneau and Desdevises2011).
Furthermore, the topology in the phylogeny of U. malabaricusi reinforces the hypothesis of cryptic species, due to the high bootstrap rates occurring in these branches. Conversely, U. cuiabai shows much lower rates in a more concise clade. The situation is repeated when the tree is elaborated with amino acid sequences from the analysed sequences, indicating a complex with three different species in U. malabaricusi.
Because the number of cryptic species is directly related to the number of individual sequences of parasites analysed (Poulin, Reference Poulin2011), the COI sequences are adequate for characterization of cryptic species (Criscione et al., Reference Criscione, Poulin and Blouin2005; Vilas et al., Reference Vilas, Criscione and Blouin2005; Nadler & De León, Reference Nadler and De León2011). Additionally, because only four U. malabaricusi specimens were investigated in this study, other cryptic species may occur in the complex.
Analysis of COI sequences seems to be promising for the differentiation of the species in this genus. The region's sequence analysis is thus an identification tool for new species by reducing the role of phenotypic plasticity as interference in the process. Molecular data will make the elaboration of clearer phylogenetic reconstructions among the species of Urocleidoides possible and enable clarification of their origin.
Finally, further investigations on the genus should be conducted, with more representative samples and associated with a specific region of the nuclear genome, for the better understanding of cryptic species.
Acknowledgements
The authors would like to thank the Research Nucleus in Limnology, Ichthyology and Aquiculture (Nupélia/UEM), UNIPAR (Universidade Paranaense) and the Postgraduate Course in Comparative Biology of the Universidade Estadual de Maringá (UEM).
Financial support
The current work was funded by Nupélia/UEM through the project: The Flood Plain of the River Paraná: Structure and Environmental Processes (LTER/CNPq) and by UNIPAR. R.J.G. received a research fellowship from the Brazilian Council for Scientific and Technological Development (CNPq).
Conflict of interest
None.
Ethical standards
The project 21944/2012 was approved by the Ethical Committee for Animal Research of UNIPAR on 22 September 2011.
