Genetic characterization of far eastern species of the genus Crepidostomum ( Trematoda : Allocreadiidae ) by means of 28 S ribosomal DNA sequences

Genetic divergence and phylogenetic relationships of four species of the genus Crepidostomum Braun, 1900 sensu Caira, Bogĕa (2005) were revealed using partial sequences of 28S ribosomal RNA gene. Genetic divergence between C. cf. farionis (Muller, 1784) and C. nemachilus Krotov, 1959 was 3.1%, which corresponds to the mean value of interspecific divergence between Crepidostomum species. These two species, therefore, can be recognized as bonafide species. However, we found no genetic differences between 28S rRNA gene sequences of C. nemachilus and C. cf. metoecus Braun, 1900 in spite of considerable morphological and ecological differences. Maximal values of genetic p-distances were revealed between C. cf. auriculatum Wedl, 1857 and C. cf. farionis. Phylogenetic relationships of Crepidostomum spp. for which sequence data are available, along with species in other related genera (Bunodera Railliet, 1896 and Allocreadium Loss, 1900) showed a paraphyly of the genus Crepidostomum. Considerable differentiation of C. cf. auriculatum from other Crepidostomum species was revealed, which may reflect the original description of this species in a separate genus Acrolichanus Ward, 1917. Our results are consistent with the conventional systematics that places the four genera (Crepidostomum, Bunodera, Megalogonia and Allocreadium) within the same family.


INTRODUCTION
Species of the Allocreadiidae are important components of the parasite fauna of the freshwater fishes and their systematics have not been clarified yet.The membership of these trematodes to either family Allocreadiidae Stossich, 1903 or family Bunoderidae Nicoll, 1914 has been discussed in the literature [1][2][3][4][5].However, molecular phylogenetic analyses that have included these trematodes have recovered them in a monophyletic Allocreadiidae [6][7][8].The genus Crepidostomum sensu Caira, Bogĕa, 2005 is one of the most taxonomically confused groups of trematodes of the family Allocreadiidae, which contains 40 nominal and 24 valid species.The life cycle of Crepidostomum spp.involves freshwater bivalves as first intermediate hosts, freshwater arthropods as second intermediate hosts, and freshwater fishes from a variety of families as definitive hosts.The most widely distributed species of this genus are C. auriculatum Wedl, 1857, C. farionis (Muller, 1784) and C. metoecus Braun, 1900.It is not surprising that these three species have been known by numerous synonyms.For example, a trematode of salmons was reported as C. ussuriensis by Layman (1930) [9] for south territory of Russian Far East from Salvelinus sp.Later this species was recognized as synonym of C. farionis by Skrjabin and Koval (1966).Another established species for Russian Far East-C.nemachilus, described by Krotov (1959) [10] from Barbatula toni, was recognized as synonymous taxon of C. ussuriensis [1].After that the taxonomical status of this species was not discussed in the Russian literature.Shi-mazu (1990) touched upon issue of taxonomic status of C. nemachilus and considered this species as synonymous with C. farionis [11].The problem of validity of C. nemachilus still needs to be resolved.
The taxonomy of Crepidostomum auriculatum, an intestinal flatworm of sturgeons, has a confusing history, and was reviewed by Choudhury (1997) [12].It was first described as Distoma auriculatum by Wedl from the sterlet Acipenser ruthenus from Austria (River Danube).This species was first reported in North America by Linton in 1897 under the same name [13].Pratt was the first who used the name "Crepidostomum auriculatum" for description of this species [12].The first detailed description of C. cf.auriculatum, based on whole mounts and histological sections, was given by Skwortzoff (1927Skwortzoff ( , 1928) ) from sterlet Acipenser ruthenus from Volga River [14,15].Taxonomical study of Hopkins (1933) resulted in the recognition of all North American forms from sturgeons as C. lintoni (Pratt in Linton, 1901) and recognition of C. auriculatum from sterlet as separate species [16].Later, these two species were recognized as synonymous by many researchers [1,3,[17][18][19].Crepisodostomum auriculatum was also considered by Hopkins (1934) as synonymous with Acrolichanus similis, described by Wisniewski in 1933 from Salmo trutta and Oncorhynchus mykiss [20].But further taxonomical studies showed that A. similis and C. auriculatum are separate species [3,12].According to Shul'man (1954) and Choudhury (1997), there are trends in a few differences between European and North American C. auriculatum specimens in spite of agreement in all essential details [12,19].Thus, placing C. auriculatum in either Crepi-dostomum or Acrolichanus is still a topical problem, as the identity of specimens of this species from different locations North America, Europe and Northern Asia, including Russian Far East.The aim of our study is to clarify the taxonomy of far eastern Crepidostomum species by genetic characterization using patrial sequences of the 28S rRNA gene.

MATERIALS AND METHODS
Trematodes C. cf.auriculatum, C. cf.farionis, C. cf.metoecus and C. nemachilus were obtained during parasitological field work in 2008-2010 from south of Russian Far East (water bodies of Primorsky Region and Amur River near Nikolaevsk-na-Amure city).Morphology of 1000 specimens (alive and mounted in Canada balsam) of trematodes was examined and four species were revealed.For molecular analysis adults of each species, obtained from the intestine of definitive host species, were preliminary checked under slight pressure between two glass slides and then fixed in 96% ethanol.Species identification was performed according to different authors [1][2][3]10].Additionally it was examined nine syntypes of C. nemachilus from muzeum of K. I. Skrjabin All-Russia Institute of Helminthology, Moscow, Russia (№ 7506 and 7507).List of species, incorporated into analysis, presented in Table 1.We used symbol "cf." for the names of three species (C.cf.auriculatum, C. cf.farionis, C. cf.metoecus) because of these specimens weren't compared with the trematodes from type location in spite of preliminary identification of these species by morphology.DNA extraction was performed from whole worms with silica technique as follows: 1) Trematode specimens were dried and homogenized in 3 M Guanidine thyocyanate.2) Homogenate was incubated for 10 minutes at 57˚C. 3) Water suspension of silica (silicium dioxide) was added to homogenate (5 µl of 50% suspension per 2 µg of expected amount of DNA), mixed and incubated about 5 minutes at 57˚C.4) Silica/Guanidine solution was centrifuged for 15 sec.at 10,000 rpm, supernatant was elucidated.5) Sediment was washed using cold washing buffer (20 mM TRIS-HCl, pH = 7.4, 1 mM EDTA, 50 mM NaCl, 50% ethanol) for four times by adding of 500 µl of the washing buffer, mixing and centrifuging as in previous step.6) Sediment was dried in air for one hour and then was resuspended in 100 -150 µl of nuclease free water, mixed and incubated for 10 minutes at 57˚C.7) Water suspension was centrifuged for 2 minutes at 12,000 rpm, supernatant was transferred to the clean tubes and used for PCR.
The 28S rRNA gene was amplified using the polymerase chain reaction (PCR) with the following primers: DIGl2 (5'-AAG CAT ATC ACT AAG CGG-3') and 1500R (5'-GCT ATC CTG AGG GAA ACT TCG-3') [21].Initial PCR reaction was carried out in a total volume of 20 µl.Each reaction contained 0.25 mM of each primer pair, combined with 1 µl of aqueous solution of DNA, 10x Taq buffer, 1.25 mM dNTP, 1.5 mM magnesium, and 1 unit of Taq polymerase.Amplification of a 1200-bp fragment of 28S rDNA was performed in a Ge-neAmp 9700 (Applied Biosystems) with 3 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 7 min extension hold at 72˚C.PCR contamination control was performed by including negative controls alongside positive controls, using both primers.PCR products were initially directly sequenced using ABI Big Dye Terminator v.3.1 Cycle Sequencing kit (as instructed by manufacturer) and internal sequencing primers: 300F, ECD2, 900F, 1200R [21].Reading of the cycle PCR products was performed with ABI 3130 genetic analyzer of Institute of Biology and Soil Sciences FEB RAS.Sequences of Crepidostomum spp. in this study were aligned with 28S sequence data of other species downloaded from NCBI GenBank database, representing following genera of the family Allocreadiidae: Crepidostomum, Megalogonia, Allocreadium, Bunodera.Also we used Callohelmis pichelinae as an outgroup [22] (Table 1).
DNA sequences were assembled with SeqScape v.2.6 software and aligned using MEGA 5.0 [23] alignment explorer with default options.Regions that could not be aligned unambiguously were excluded from the analyses.The number of variable, parsimony-informative sites, nucleotide composition and substitutions were obtained in MEGA 5.0.Frequencies of different substitution types and substitution rate were calculated using test of sub stitution pattern 4 by 4. Genetic divergence was estimated using genetic p-distance values, which were calculated by including all substitution types.Phylogenetic analysis of nucleotide sequences was undertaken, using all accessible methods, including maximum parsimony, maximum likelihood and Bayesian methods.All distance and parsimony trees were reconstructed with MEGA 5.0 and Bayesian inference was obtained using MrBayes v. 3.1.2software [24].The resulting networks were rooted with the outgroup taxa.Phylogenetic algorithms ML and BI performed using the GTR + G model (general-time reversible model with gamma distribution).This model showed the best fit to the data using Modeltest v. 3.07 software [25] using Akaike's information criterion [26].Bayesian inference was employed using the following nucleotide substitution parameters: lset nucmodel = 4 by 4, nst = 6, rates = gamma.Mcmc algorithm was performed using 4,000,000 generations and 2 independent runs.Burn-in (sump and sumt) values were 80000.A nonparametric bootstrap with 1000 replicates was used to evaluate the robustness of the clusters through nodal support [27] for maximum parsimony and maximum likelihood algorithms.Nodal support for Bayesian algorithm was estimated by posterior probabilities [28].Kishino-Hasegawa [29] and Shimodaira-Hasegawa [30] tests for phylogenetic trees were performed using PAUP 4b10 [31].

RESULTS
In the present study data on the genetic divergence and systematic of the far eastern species of the genus Creidostomum were obtained, using 28S rDNA partial sequences.Amplicons were about 1200 bp in length and the sequences, using for analysis after alignment were 1151 bp, including 70 variable and 66 parsimony-informative sites.There were no significant differences in nucleotide composition of 28S rDNA of the Crepidostomum species in comparison with the specimens from another genera and families of trematodes: A = 20.7%,T = 25.6%,C =21.7%, G = 32%.
The substitution pattern analysis revealed that significant differences of transition/transwersion rate ratio bias values (R) obtained by pairwise comparision of 28S rDNA sequences of different Crepidostomum species (Table 2 Genetic divergence between far eastern Crepidostomum species ranged from 0 (C.cf.metoecus/C.nema-chilus) to 3.8% (C.cf.auriculatum/C.cf.farionis) with a mean value 3.2% (Table 3).Divergence between 28S rDNA sequences between C. cf.farionis and C. nemachilus was 3.1% that close to the mean interspecific divergence between Creidostomum species.However, there are no divergence between 28S rDNA sequences of C. nemachilus and C. cf.metoecus in spite of considerable morphological differences.Genetic divergence between C. cf.auriculatum and B. luciopercae was 3.1% that corresponds with interspecific divergence level of Crepidostomum species.Maximal values of genetic divergence between far eastern Crepidostomum species covered with minimal values of intergeneric divergence (Table 3).
Phylogenetic analysis revealed six clusters with a high statistical support (Figure 1).First one was a basal and formed by the genus Allocreadium, and the second one included C. cf.auriculatum, a parasite of the sturgeons.Third cluster included the genus Bunodera, which was represented by two species: B. luciopercae (Muller, 1776) and B. acerinae Roitman and Sokolov, 1999, which were recognized early [32][33][34] where k 1 и k 2 -frequences of transitions between purines and pyrimidines, respectively.R becomes 0.5 when there is no bias towards either transitional or transversional substitution because, when the two kinds of substitution are equally probable, there are twice as many possible transversions as transitions.support, which was closely related to the (C.nemachilus + C. cf.metoecus) cluster.Genetic divergence between obtained clusters ranged from 3.3% (clusters IV/V and V/VI) to 6.0 (clusters II/VI).To test of significance of the tree topology, which showed paraphyly of the genus Crepidostomum, we compared this topology with tree constraining this genus as monophyletic using Kishino-Hasegawa and Shimodaira-Hasegawa tests (Table 4).
Obtained results showed significant differences between unconstrained phylogenetic tree, showed a paraphyly of Crepidostomum and phylogenetic tree with constraint monophyly of Crepidostomum for three phylogenetic algorithms used.

DISCUSSION
This is the first time that 28S sequence data have been used to elucidate the systematics of the far eastern Crepidostomum spp.and our data were useful for delineating some species boundaries and questioning at least one nominal taxon (C.nemachilus).All of the phylogenetic trees demonstrated the same topology and showed a paraphyly of the genus Crepidostomum (Figure 1).Firstly, C. cf.auriculatum, a parasite of the sturgeons, appears as more divergent species, that was reproduced on phylogenetic trees as a distinct cluster, which was not related to other Crepidostomum species.This cluster has a high bootstrap support as so as high mean value of genetic divergence (3.8%) in comparison with other Crepidostomum species from the Russian Far East (3.08%).Moreover, C. cf.auriculatum is characterized by lower value of transition/transversion ratio bias (R = 2.26 -2.74) in comparison with the other Crepidostomum species (Table 2), indicating slight bias from equal contents of transitions and transversions, i.e. prevalence of transversion substitutions type, which is typical for high level taxa.Results of KH and SH tests confirm significance of high differentiation of C. cf.auriculatum with other Crepidostomum species.In our opinion, presented results showed a currency of the question about systematic position of C. cf.auriculatum that was thought to be considered by Skwortzoff (1927) within the genus Acrolichanus [14].Analysis of 28S rDNA from 29 specimens of C. cf.auriculatum, collected from three different fishes (two specimens of Acipenser schrenckii and one of Huso dauricus) showed an absence of variation between obtained sequences.It can be interpreted by limited genetic affinity between C. cf.auriculatum and the host species and suggests that sturgeons were used as definitive host for C. cf.auriculatum not so far.Such genetic stability irrespective of the host species was shown for other trematode species which infect different species of animals [35][36][37].Moreover, C. cf.auriculatum differentiated from Bunodera luciopercae at interspecific divergence level (3.1%).Species B. luciopercae is a basal species on  Species C. cf.farionis and C. nemachilus, therefore, cannot be recognized as synonymous taxa using both morphological and genetic criteria.However, this conclusion needs to be examined using C. cf.farionis from type location, because of ambiguous taxonomy of C. cf.farionis from Russian Far East, which was firstly described as C. ussuriensis Layman, 1930 and reduced to synonym with C. cf.farionis without sufficient evidence.On the other hand, our results show high genetic identity of 28S rDNA sequences of C. nemachilus and C. cf.metoecus (one substitution per 1151 bp) in spite of their considerable morphological distinctions.This taxonomical problem need to be resolved by complex approach, including detailed description of life cycle and using more variable molecular markers for estimation of genetic divergence between these two species.

Figure 1 .
Figure 1.Phylogenetic tree of the family Allocreadiidae, obtained using maximum parsimony algorithm.Nodal numerals are the bootstrap statistics values (%) for MP/ML/BI algorithms, respectively.Roman numerals correspond to number of cluster, discussed in the text.

Table 1 .
List of the taxa incorporated in sequence analysis.
. The fourth cluster included C. cornutum and M. ictaluri.Last two clusters corresponded with different Crepidostomum species: C. nemachilus and C. cf.metoecus gathered in a single cluster and C. cf.farionis formed a distinct cluster with a high statistical

Table 3 .
Genetic divergence (%) between different species of the family Allocreadiidae using 28S rDNA partial sequences.

Table 4 .
[39]an et al. (2006) SH test of log-likelihood scores among unconstrained and constrained monophyletic trees of the genus Crepidostomum.*P < 0.05.Both tests showed significant differences between unconstrained tree, showed a paraphyly of Crepidostomum and phylogenetic tree with constraint monophyly of Crepidostomum for three phylogenetic algorithms used.Megalogonia ictaluri and Crepidostomum cornutum, ob-tained from USA (GenBank data).This result confirms the point ofCaira (1989)[3]about the membership of Megalogonia species to the genus Crepidostomum in spite of the point ofCurran et al. (2006), which accept the genus Megalogonia[39].Species C. cf.farionis and C. cf.metoecus have considerable external morphological differences between themselves, as so distribution and size of dorsal and dorsolateral oral lobes and tegumental papillae on anterior species and C. cf.auriculatum in the past.Sturgeons, therefore, possibly become definitive host for C. cf.auriculatum during recent host-switching processes.Secondly, our results showed close relationships of

farionis Crepidostomum cornutum EF032695 Megalogonia ictaluri EF032694 Bunodera luciopercae GU462124 Bunodera acerinae GU462122 C. cf. auriculatum C. cf. auriculatum C. cf. auriculatum C. cf. auriculatum C. cf. auriculatum C. cf. auriculatum Allocreadium lobatum EF032693 Allocreadium isoporum GU462126 Allocreadium isoporum GU462125 Callohelmis pichelinae FJ788495
[1]4,40].Also these two species differ by form of the genital bursa[1].C. nemachilus, included in our investigations, has the same form of the oral lobes and tegumental papillae as so as C. cf.farionis, and the form of genital bursa resembles with C. cf.metoecus.However, C. nemachilus is characterized by irregularity of testicles surface unlike both C. cf.farionis and C. cf.metoecus.C. nemachilus, therefore, can be recognized as a possible distinct species by general morphology, which will be presented in a separate article.In the present study, genetic divergence between C. cf.farionis and C. cf.metoecus was 3.1% as so as between C. cf.farionis and C. nemachilus that corresponds to mean value of interspecific values range of Crepidostomum species (3.2%).