01_Sarabeev.indd UDC 595.133:597.556.333.7 MOLECULAR DATA CONFIRM THE SPECIES STATUS OF NEOECHINORHYNCHUS PERSONATUS AND N. YAMAGUTII (ACANTHOCEPHALA, NEOECHINORHYNCHIDAE) FROM THE ATLANTIC AND PACIFIC GREY MULLETS (TELEOSTEI, MUGILIDAE) V. Sarabeev1*, Ie. Tkach1, R. A. Sueiro2, J. Leiro2 1 Department of Biology, Zaporizhzhia National University, Zhukovskogo, 66, Zhaporizhzhia, 69063 Ukraine 2Departamento de Microbiología y Parasitología, Instituto de Investigación y Análisis Alimentarios, Universidad de Santiago de Compostela, Santiago de Compostela, 15782 Spain *Corresponding author E-mail addres: vosa@ext.uv.es, volodimir.sarabeev@gmail.com Molecular Data Confi rm the Species Status of Neoechinorhynchus personatus and N. yamagutii (Acanthocephala, Neoechinorhynchidae) from the Atlantic and Pacifi c Grey Mullets (Teleostei, Mugilidae). Sarabeev, V., Tkach, Ie., Sueiro, R. A., Leiro, J. — Neoechinorhynchus is known to be the most diverse acanthocephalan taxon with worldwide distribution; its species are characterized by uniformity of anatomical organization. Th e taxonomic status of Neoechinorhynchus agilis s. l. from grey mullets was recently reviewed that resulted in description of two new species, Neoechinorhynchus (Hebesoma) personatus and Neoechinorhynchus (Hebesoma) yamagutii. In the current study,18S rRNA partial gene sequences were obtained to molecularly characterize N. (N.) agilis, N. (H.) personatus and N.  (H.)  yamagutii from grey mullets, Chelon labrosus and Mugil cephalus, in the North-East Atlantic, the Japan, Azov and Mediterranean Seas. Th e universal (F-566 and R-1200) and specifi c eukaryotic set primers were used to amplify specimens from each species of Neoechinorhynchus. It has been found that three species can be clearly recognized using universal primers, which allow to obtain sequences of 590 to 664 bp in length. Th e genetic divergences estimated between three species sequenced here were relatively high, at ranged between 2.08 and 6.57 %. Phylogenetic analysis demonstrates that the studied species of Neoechinorhynchus from grey mullet fi sh share common ancestor, despite their diff erent geographic location, and are closely related. Th e terminal position of N. (N.) agilis and N. (H.) personatus on the evolution tree and the low genetic divergence found between them suggests the recent emergence of this group and that the colonization of the North-East Atlantic and the Mediterranean regions could represent a single event. Our phylogenetic analysis, which included several species of the subgenera Neoechinorhynchus and Hebesoma, showed that the latter is a polyphyletic taxon. K e y w o r d s : 18S rRNA partial gene sequences, Chelon labrosus, Mugil cephalus, Neoechinorhynchus agilis, Neoechinorhynchus personatus, Neoechinorhynchus yamagutii. Zoodiversity 54(1):1–10, 2020 DOI 10.15407/zoo2020.01.001 Fauna and Systematics 2 V. Sarabeev, Ie. Tkach, R. A. Sueiro, J. Leiro Introduction Th e taxonomic status of Neoechinorhynchus agilis s. l. (Rudolphi, 1819) from grey mullets (Mugilidae) across localities in the North-East Atlantic and the North-West Pacifi c areas was recently reviewed by Tkach et al. (2014). Th is taxonomic review performed on morphological features showed that there were three diff erent species of Neoechinorhynchus occurring in mullet hosts, two of those were recorded in the Atlantic and one in the Pacifi c waters (Neoechinorhynchus (Neoechinorhynchus) agilis (Rudolphi, 1819) and Neoechinorhynchus (Hebesoma) personatus Tkach, Sarabeev et Shvetsova, 2014, Neoechinorhynchus (Hebesoma) yamagutii Tkach, Sarabeev et Shvetsova, 2014, respectively). Morphological identifi cation of Neoechinorhynchus spp. from grey mullets is based on the combination of a set of specifi c characters describing the number of hypodermal nuclei, features of the hooks, lemnisci and genital organs. While the conventional microscopy applied to identifi cation of Neoechinorhynchus is complicated because of high species diversity and specifi c morphology of the group, which commonly requires experienced researcher, accurate taxonomic identifi cation is essential for biological and ecological studies. DNA barcoding techniques are the most technological and precise methods for the iden- tifi cation of a specimens bulk to the genus and species level (Morand, 2018). Biodiversity assessment method of metabarcoding uses a short section of DNA from a standardized region of the genome (Hebert et al., 2003). In the present study, we test the pair of the universal primers, F-566 and R-1200 to amplify variable regions of 18S rRNA gene (Hadziavdic et al., 2014) of three species of Neoechinorhynchus from grey mullets. We also designed a set of specifi c primers that were used to obtain longer sequences of the studied species and to perform com- parative phylogenetic analysis. Some regions of the 18S rRNA gene are highly conserved, others are variable or even highly variable (Hadziavdic et al., 2014); thus this gene can provide a basis for resolving relationships at the genus and species levels of studied here acanthocephalan parasites. Although Neoechinorhynchus is the most diverse acanthocephalan group with 121 known species, a few taxa of the genus were analysed using molecular data (Amin, 2013; Smales, 2013; Pinacho-Pinacho et al., 2014; Tkach et al., 2014; Melo et al., 2015; Gautam et al., 2018). Th ere is much fewer data available on 18S rRNA sequences for Neoechinorhynchus spp. Malyarchuk et al. (2014) investigated molecular phylogenetic relation- ships of six Neoechinorhynchus species from fi shes collected in freshwater localities of North-East Asia using DNA sequences of two genes, cytochrome CO1 of the mitochondrial DNA and 18S rRNA. Two additional almost complete 18S rRNA sequences were obtained by Near et al. (1998) and García-Varela & Nadler (2005) for Neoechinorhynchus (Neoechinorhynchus) crassus Van Cleave, 1919 and Neoechinorhynchus saginatus Van Cleave et Bangham, 1949, respectively, in their studies of acanthocephalan phylogeny. Finally, three sequences of the target regions of 18S rRNA for Neoechinorhynchus (Neoechinorhynchus) pseudemydis Cable et Hopp, 1954, N. (N.) cylindratus (Van Cleave, 1914) and N. (N.) dimorphospinus Amin et Sey, 1996 were found as deposited in GenBank (table 1). Here, 18S rRNA partial gene sequences of N. (N.) agilis, N. (H.) personatus and N. (H.) yamagutii were obtained from grey mullet hosts in localities across the North-East Atlantic and North-West Pacifi c regions, allow- ing us to molecularly characterize them, reveal interspecifi c relationships and test the systematic position within the genus. Materials and methods M a t e r i a l c o l l e c t i o n Grey mullets were collected from fi ve localities in the North-East Atlantic and the Japan, Azov and Medi- terranean Seas (table 1). In the current study, acanthocephalan parasites were sampled from two fi sh species, Chelon  labrosus (Risso) and Mugil  cephalus L. Fish intestine and pyloric caeca were examined for parasites within the day of capture, or refrigerated, and surveyed for infections with acanthocephalans under a stereo- microscope. The worms were isolated from the intestine, washed in 0.8 % saline water and after eversion of the proboscis fi xed and stored in 70 % or absolute ethanol prior to examination. Th e specimens of Neoechinorhyn- chus collected in this study were identifi ed as N. (N.) agilis, N. (H.) personatus and N. (H.) yamagutii based on morphological features and host-geographic information (Tkach et al., 2014). D N A e x t r a c t i o n , a m p l i f i c a t i o n , s e q u e n c i n g a n d a n a l y s i s Specimens of acanthocephalans were placed individually in tubes and sonicated in TE buff er for several seconds. Th en sodium dodecylsulfate and proteinase K were added to the mixture and digested 2 hours at 37 °C with subsequent phenol-chloroform deproteinization, isopropanol precipitation, and ethanol washing. DNA was analysed to estimate its purity and concentration with the NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, USA). PCR amplifi cation was performed as previously described by Leiro et al. (2000) and De Felipe et al. (2017). Th e universal eukaryotic primers (F- 566:5’-CAG CAG CCGCGGTAATTC C-3’ and R-1200:5’-CCC GTGTTGAGTCAA ATT AAG C-3’) proposed by Hadziavdic et al. (2014) were used to amplify V4 and V5 variable region of 18S rRNA gene as regions with high taxonomic information. Based on available sequences of 18S rRNA gene in GenBank (www.ncbi.nlm.nih.gov/genbank/) and new ones obtained in the current study for specimens of Neoechinorhynchus, the specifi c primer set (FNeo1086:5’–ATA GCCATG CAT GTG CAG TT-3’ and RNeo1086:5-GCC TTGCGACCATACTCC C-3’) was designed and optimized by 3Molecular data confi rm the species status of Neoechinorhynchus personatus and N. yamagutii… T ab le 1 . I so la te id en ti fi c at io n nu m be r (I D ), sp ec ie s a na ly se d, se a/ re gi on a nd lo ca lit y na m e, h os t s pe ci es , s eq ue nc e le ng th , G en B an k ac ce ss io n nu m be r an d so ur ce fo r sp ec im en s o f N eo ec hi no rh yn ch us st ud ie d in th is w or k Is ol at e ID Sp ec ie s Se a/ re gi on Lo ca lit y H os t Le ng th (b p) G en Ba nk a cc es si on n o. So ur ce V 1 N eo ec hi no rh yn ch us (H eb e- so m a) p er so na tu s T ka ch , Sa ra be ev e t S hv et so va , 2 01 4 M ed ite r- ra ne an V al en ci a co as ta l w a- te rs , S pa in M ug il ce ph al us L . 99 0 M N 14 90 66 Th e pr es en t s tu dy V 3 N . ( H .) pe rs on at us « « « 10 25 M N 14 90 67 « G 42 N . ( H .) pe rs on at us A zo v Se a Si va sh L ak e, U kr ai ne « 10 42 M N 14 90 68 « G 44 N . ( H .) pe rs on at us « « « 10 18 M N 14 90 69 « G 45 N . ( H .) pe rs on at us « « « 10 34 M N 14 90 70 « G 49 N . ( H .) pe rs on at us « « « 59 7 M N 14 90 71 « G 55 N . ( H .) pe rs on at us « « « 10 31 M N 14 90 72 « I1 N . ( H .) vi ol en tu s ( V an C le av e, 19 28 ) N or th -E as t A si a Pr im or ye re gi on , R us - si a Pe rc co tt us  g le ni i D yb ow sk i, 18 77 93 1 K F1 56 88 1. 1 M al ya rc hu k et a l., 2 01 4 A 2 N eo ec hi no rh yn ch us (H .) ya m ag ut ii T ka ch , S ar ab ee v et Sh ve ts ov a, 2 01 4 Ja pa n Se a A m ur B ay , R us si a M . c ep ha lu s 59 0 M N 14 92 19 Th e pr es en t s tu dy A 3 N . ( H .) ya m ag ut ii « « « 59 3 M N 14 92 20 « A 31 N eo ec hi no rh yn ch us (N eo ec hi - no rh yn ch us ) a gi lis (R ud ol ph i, 18 19 ) N or th -E as t A tla nt ic A ro us a R iv er , S pa in C he lo n  la br os us (R is so , 18 27 ) 66 4 M N 14 88 93 « A 12 N . ( N .) ag ili s « « « 61 6 M N 14 88 94 « A 5 N . ( N .) ag ili s « « « 65 1 M N 14 88 95 « A 1 N . ( N .) ag ili s « « « 65 1 M N 14 88 96 « 4 V. Sarabeev, Ie. Tkach, R. A. Sueiro, J. Leiro C 9 N . ( N .) ag ili s « C al de ba rc os co as ta l w a- te rs , S pa in « 10 34 M N 14 88 97 « C 12 N . ( N .) ag ili s « « « 99 4 M N 14 88 98 « B1 N . ( N .) be ri ng ia nu s M ik ha ilo - va e t A tr as hk ev ic h, 2 00 8 N or th -E as t A si a C he rn oe La ke , R us si a Pu ng iti us  p un gi tiu s (L in na eu s, 1 75 8) 89 3 K F1 56 87 5. 1 M al ya rc hu k et a l., 2 01 4 – N . ( N .) cr as su s V an C le av e, 19 19 N or th A m er ic a – C at os to m us  co m m er so ni i (L ac ep èd e, 1 80 3) 17 73 A F0 01 84 2. 1 N ea r e t a l., 1 99 8 – N . ( N .) cy lin dr at us (V an C le av e, 1 91 4) N or th A m er ic a Ba ck B ay M ic ro pt er us  sa lm oi de s (L ac ep èd e, 1 80 2) 15 01 M F9 74 92 5. 1 Bl ub au gh , G au th ie r, 20 17 w w w .n cb i.n lm .n ih .g ov / nu cc or e/ M F9 74 92 5. 1 – N . ( N .) di m or ph os pi nu s A m in et S ey , 1 99 6 So ut h C hi na S ea K ie nG ia ng G ul f, W ie t- na m Pl an ili za  su bv ir id is (V al en ci en ne s, 1 83 6) 16 73 M K 51 00 80 .1 A m in e t a l., 2 01 9 w w w . nc bi .n lm .n ih .g ov /n uc co re / M K 51 00 80 .1 – N . ( N .) ps eu de m yd is C ab le e t H op p, 1 95 4 So ut h- W es t A si a G an do m an La go on , Ir an C ap oe ta  a cu le at a (V al en ci en ne s, 1 84 4) 17 61 K U 36 39 73 .1 D ad ar , A de l, 20 15 w w w . nc bi .n lm .n ih .g ov /n uc co re / K U 36 39 73 .1 E4 a N . ( N .) sa lm on is C hi ng , 1 98 4 N or th -E as t A si a C hi st oe La ke , R us si a Sa lv el in us  m al m a (W al ba um , 1 79 2) 92 9 K F1 56 87 8. 1 M al ya rc hu k et a l., 2 01 4 C 1 N . ( N .) tu m id us V an C le av e et Ba ng ha m , 1 94 9 N or th -E as t A si a In di gi rk a R iv er , R us - si a C or eg on us n as us (P al la s, 17 76 ) 89 6 K F1 56 87 6. 1 M al ya rc hu k et a l., 2 01 4 – N .sa gi na tu s V an C le av e et Ba ng ha m , 1 94 9 N or th A m er ic a – Fr es hw at er fi sh 17 45 A Y 83 01 50 .1 G ar cí a- V ar el a an d N ad le r, 20 05 E1 N .si m an su la ri s R oi tm an , 1 96 1 N or th -E as t A si a En gt er i La ke , R us si a Sa lv el in us  a lp in us (L in na eu s, 17 58 ) 93 0 K F1 56 87 7. 1 M al ya rc hu k et a l., 2 01 4 – Fl or id os en tis m ug ili s ( M ac ha - do F ilh o, 1 95 1) N or th A m er ic a, M ex ic o – M ug il ce ph al us 17 60 A F0 64 81 1. 1 G ar cí a- V ar el a et a l., 2 00 0 C o n ti n u e d t a b le 1 5Molecular data confi rm the species status of Neoechinorhynchus personatus and N. yamagutii… use of the Primer 3Plus program (www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi), with default parameters. Th e PCR mixtures (25 μL) contained reaction buff er (10 mMTris-HCl, 50 mMKCl, 1.5 mM MgCl2), 0.2 mM of each deoxynucleoside triphosphate (dNTPs, Roche), 0.4 μM of each primer; 0.5 units of high fi del- ity Taq polymerase and 50 ng of genomic DNA. Th e reactions were run in an automatic thermocycler (Mas- tercycler, Eppendorf, Germany) as follows: initial denaturing at 94 °C for 5 min, followed by 35 cycles at 94 °C for 30 s, annealing at 57 and 55 °C (for F-566 and R-1200 and FNeo1086 and RNeo1086 primers, respectively) for 45 s, and 72 °C for 1 min; and fi nally, a 7 min extension phase at 72 °C. Th e PCR products were separated on a 4 % agarose gel in Tris acetate ethylenediaminetetraacetic acid (TAE) buff er containing Sybr Green at 1× concentration, to verify the presence of bands of the correct size under a variable-intensity UV transilluminator and auto image capture soft ware (Alpha Innotech, USA). Th e PCR product was purifi ed with Th ermo Scientifi c GeneJETPCR Purifi cation Kit and sequenced in complementary directions using Sanger sequencing service (GATC Biotech, Germany). Th e sequences were aligned using Clustal Omega online service (Madeira et al., 2019). All positions containing missing data were eliminated and phylogenetic analyses were performed. Trees were obtained using maximum likelihood (ML) with Tamura-Nei model, neighbour joining (NJ) and minimum evolution (ME) methods as applied in MEGA-X (Kumar et al., 2018). Clade support was assessed by bootstrap resampling with 100 replicates. Th e tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. Th e evolutionary distances were computed using the Kimura 2-parameter method (Kimura, 1980). Estimates of evolutionary divergence based on the number of base diff erences per site from averaging over all sequence pairs between and within groups (uncorrected p distances) were conducted in MEGA-X and expressed in percentages (table 2). For comparative analysis, the following DNA sequences of Neoechinorhyn- chus spp. were retrieved from GenBank: N. (N.) beringianus Mikhailova et Atrashkevich, 2008; N. (N.) tumidus Van Cleave and Bangham, 1949; N. simansularis Roitman, 1961; N. (N.) salmonis Ching, 1984; N. (H.) violentus (Van Cleave, 1928); N. saginatus; N. (N.) crassus; N. (N.) pseudemydis; N. (N.) cylindratus; N. (N.) dimorphospi- nus. Th e complete 18S sequence of Floridosentis mugilis (Machado-Filho, 1951) was used as outgroup based on its sister relationship to Neoechinorhynchus (Malyarchuk et al., 2014; Rosas-Valdez et al., 2012). Results G e n e t i c d i v e r g e n c e o f N e o e c h i n o r h y n c h u s f r o m g r e y m u l l e t s Seven specimens of N. (H.) personatus, 6 of N. (N.) agilis, and 2 of N. (H.) yamagutii were sequenced and used for phylogenetic analysis here (table 1). All species were ampli- fi ed and sequenced using both universal and specifi c set of primers (F-566 and R-1200; and FNeo1086 and RNeo1086, respectively), excepting for N. (H.) yamagutii for which sequenc- es were obtained only with universal primers. PCR products varied from 590 to 664 bp for T a b l e 2 . Estimates of evolutionary divergence between sequences encoding 18S rRNA gene of specimens of Neoechinorhynchus from grey mullets across localities in the North-East Atlantic, Mediterranean and Japan Sea No * 1 2 3 4 5 6 7 8 9 1 N. (H.) yamagutii A2 and A3 2 N. (H.) personatus V1 6.56 3 N. (H.) personatus V3 6.35 0.19 4 N. (H.) personatus G42 6.36 0.56 0.37 5 N. (H.) personatus G44 6.56 0.75 0.56 0.19 6 N. (H.) personatus G45, G49 and G55 6.16 0.37 0.19 0.19 0.37 7 N. (N.) agilis A31 6.57 2.85 2.66 2.66 2.85 2.47 8 N. (N.) agilis A1, A5 and A12 6.35 2.65 2.46 2.46 2.65 2.27 0.19 9 N. (N.) agilis C9 and C12 6.15 2.46 2.27 2.27 2.46 2.08 0.37 0.19 N o t e. Analyses were conducted using the Kimura 2-parameter model (Kimura, 1980) also, the evolutionary rates when the divergence times are known. Uncorrected p distances are shown as percentages. Th ere were a total of 541 bp in the fi nal dataset * Sequences of A2 and A3 of N. (H.) yamaguti, G45, G49 and G55 of N. (N.) personatus, A1, A5 and A12, and C9 and C12 of N. (N.) agilis were combined because their 18S partial sequences were identical. 6 V. Sarabeev, Ie. Tkach, R. A. Sueiro, J. Leiro the universal set of primers and from 990 to 1042 bp for specifi c ones. Th e aligned data set included 15 sequences with a length of 541 bp and occupying position from 551 to 1092 nu- cleotides along the 18S rRNA gene of F. mugilis. Th e genetic divergence estimated among specimens of the same species ranged from 0 to 0.75 % (table 2), while distances between taxa of Neoechinorhynchus were much higher (2.08–6.57 %). Th e 18S sequence divergence between N. (H.) yamagutii and both Atlantic species, N. (H.) personatus and N. (N.) agilis, were higher than that between the latter two (6.15–6.57 % vs. 2.08–2.85 %, respectively). A comparison of long sequences of N. (H.) personatus and N. (N.) agilis (positions from 96 to 550 nucleotides along the 18S rRNA gene of F. mugilis) supplied additional divergence in 3 bp between these species and revealed diff erence in 2 bp between specimens of C9 and C12 of N. (N.) agilis,and in 1 bp between G49 and other specimens of N. (H.) personatus. P h y l o g e n e t i c a n a l y s i s o f N e o e c h i n o r h y n c h u s f r o m g r e y m u l l e t s Th e phylogenetic analysis of specimens of Neoechinorhynchus from grey mullets re- vealed that 497 out of 541 characters were constant, 41 were parsimony-informative, and 3 variable characters were parsimony-uninformative. Th e ML analysis yielded a single tree with the highest log likelihood = −1242.96. Trees obtained with NJ and ME methods showed the same topology. Th e bootstrap support was high for all branches of the phy- logenetic tree (fi g. 1). All trees comprise of 3 major monophyletic clades, which corre- spond to N. (H.) yamagutii, N. (N.) agilis and N. (H.) personatus. Clades N. (N.) agilis and N. (H.) personatus are subsequently subdivided into 2 and 3 subclades, respectively, mainly refl ecting diff erent localities. P h y l o g e n e t i c r e l a t i o n s h i p s o f N e o e c h i n o r h y n c h u s f r o m g r e y m u l l e t s w i t h o t h e r m e m b e r s o f t h e g e n u s Th e longest sequence of each species obtained in the present study was combined with published sequences from GenBank, including 10 Neoechinorhynchus species and F. mugilis as outgroup taxa. Th e aligned data set included 14 sequences, with 442 characters, of which 86 were parsimony-informative. Th e ML tree with the highest log likelihood (–1619.55) is shown in fi gure 2. Trees obtained with NJ and ME methods showed a similar topology. Th is phylogenetic analysis revealed two main clades with strong bootstrap support. One includes Neoechinorhynchus spp. associated with freshwater and brackish water fi shes; another is 99/100/98 N. (H.) personatus G45, G49 and G55 N. (H.) personatus G44 N. (H.) personatus G42 N. (H.) personatus V3 N. (H.) personatus V1 N. (N.) agilis C9 and C12 N. (N.) agilis A1, A5 and A12 F. mugilis AF064811.1 N. (H.) yamagutii A2 and A3 100/100/100 62/67/69 100/100/100 59/66/70 62/63/63 99/100/100 0,020 Fig. 1 Phylogenetic tree of Neoechinorhynchus from grey mullets and across localities in the North-East Atlantic, Mediterranean and Japan Sea obtained with maximum likelihood (ML) method (− ln likelihood 1242.96) based on the 18S rRNA partial gene sequences. Floridosentis mugilis was used as an outgroup. Th e ML/neighborn joining/ minimum evolution bootstrap support is shown at each internal node. Identifi cation number of isolates is as in table 1. 7Molecular data confi rm the species status of Neoechinorhynchus personatus and N. yamagutii… composed by N. (H.) violentus and monophyletic subclade containing species from marine grey mullets. Th is subclade formed by the species N. (N.) dimorphospinus — N. (N.) agi- lis is phylogenetically distant from other species of the genus and strongly supported by bootstrap values (100 % for all methods of tree reconstruction). Th e bootstrap analysis also supports the sister relationship of N. (N.) dimorphospinus and N. (H.) yamagutii with the two terminal Atlantic species N. (N.) agilis and N. (H.) personatus. N. (N.) dimorphospinus and N. (H.) yamagutii are grouped together in one clade with moderate bootstrap support (49 %) in tree reconstructed by ME method (the result is not shown). Discussion Th e phylogenetic analysis inferred from partial sequences of the 18S rRNA gene supports the previous morphological observation of Tkach et al. (2014) indicating that N. (N.) agilis, N. (H.) personatus and N. (H.) yamagutii represent three independent spe- cies. Th e genetic divergences estimated between three species sequenced here were relative- ly high, at the range between 2.08 % and 6.57 %. Th ese levels of genetic divergence among species are similar or even higher to those exhibited within Neoechinorhynchus and ranging between 0.37 % and 5.55 % for the 18S rRNA gene (Malyarchuk et al., 2014). Our data, con- sistent with results of Malyarchuk et al. (2014), demonstrate the existence of genetic het- erogeneity in the species within Neoechinorhynchus. Th e closeness of species representing diff erent clades of the phylogenetic tree can be explained by their common origin in a cer- tain geographic region or in evolutionary related defi nitive hosts. Th e monophyletic clade formed with species of Neoechinorhynchus from grey mullets, N. (N.) dimorphospinus, N. (H.) yamagutii, N. (N.) agilis and N. (H.) personatus, indicates that all these species share a common ancestor. Th e terminal position of N. (N.) agilis and N. (H.) personatus and the low genetic divergence found between them suggests the recent emergence of this group and that the colonization of the North-East Atlantic and the Mediterranean regions could represent a single event. One possible explanation of the presence of two closely related species of Neoechinorhynchus in the Mediterranean is an evolutionary host-switching, as N. (N.) agilis and N. (H.) personatus tend to parasitize two diff erent host species, C. labrosus and M. cephalus, respectively (Tkach et al., 2014). Geographic barriers, such as land masses of Africa and India, prevent migration and consequent gene fl ow among acanthocephalans, N. simansularis KF156877.1 Floridosentis mugilis AF064811.1 94/98/100 78/63/0 100/100/100 64/5 8/54 22/59/55 52/55/59 58/55/60 23/44/40 21/43/16 11/0/0 20/0/16 0,020 Marine grey mullet fish Freshwater and brackish water fishes N. saginatus AY830150.1 N. (N.) salmonis KF156878.1 N. (N.) beringianus KF156875.1 N. (N.) tumidus KF156876.1 N. (N.) cylindratus MF974925.1 N. (N.) crassus AF001842.1 N. (N.) pseudemydis KU363973.1 N. (N.) violentus KF156881.1 N. (N.) dimo rp hospin us MK510080.1 N. (N.) agilis C9 N. (H.) personatus G42 N. (H.) yamagutii A3 Fig. 2 Phylogenetic tree of Neoechinorhynchus species obtained with maximum likelihood (ML) method (− ln likelihood 1619.55) based on the 18S rRNA partial gene sequences. Floridosentis mugilis was used as an out- group. Th e ML/neighborn joining/minimum evolution bootstrap support is shown at each internal node. 8 V. Sarabeev, Ie. Tkach, R. A. Sueiro, J. Leiro may explain a high divergence level between N. (H.) yamagutii and both Atlantic species, N. (N.) agilis and N. (H.) personatus. Th e basal position of N. (N.) dimorphospinus and N. (H.) yamagutii in relation to N. (N.) agilis and N. (H.) personatus indicates that the origin of the ancestor of the latter ones, most likely, related with colonization of the North- East Atlantic region by the Pacifi c species with subsequent vicariance event. Our results suppose that colonization of N. (H.) yamagutii of the Sea of Japan is associated with grey mullet fi sh rather than with freshwater and brackish water fi shes of North-East Asia, since the species of Neoechinorhynchus from these groups of hosts represent diff erent lineages. Although Hadziavdic et al. (2014) suggested that acanthocephalans could not be am- plifi ed using the primer pair F-566 and R-1200, the PCR product and sequences for all three species of Neoechinorhynchus studied here were successfully obtained by applying this set of universal primers. Th e amplicons of acanthocephalan individuals generated us- ing this universal set of primers averaged 623 nucleotides in length, whereas the specifi c primer pair supplied fragments with the average length in 1021 nucleotides covering the variable regions V4-V5 and V2-V5 of the 18S rRNA gene, respectively. In agreement with the results of Hadziavdic et al. (2014), our study demonstrates that the variable region V4- V5 yielded higher taxonomic information than the region V2-V3 providing 11 informative characters vs. 3 when comparing sequences of N. (N.) agilis and N. (H.) personatus. Th us, the universal primer pair F-566 and R-1200 can be eff ectively used to amplify acantho- cephalans for assessing their diversity in fi eld-based studies. Another important fi nding of the present study is that the subgenus Hebesoma is the polyphyletic group. While all three species of the subgenus Hebesoma are assigned to one clade, two species of the subgenus Neoechinorhynchus are nested within the same clade (see fi g. 2). In addition, the genetic divergence was higher between N. (H.) yamagutii and N. (H.) personatus than between the latter species and N. (N.) agilis. Th ese results match those inferred from CO1 gene by Malyarchuk et al. (2014) for the speciesof Neoechinorhyn- chus and Hebesoma from North-East Asia. Van Cleave (1928) erected the genus and the family Hebesomidae for Hebesoma violentum. In the diagnosis of the species he indicated that the eggs had globular polar enlargements of middle membrane, lemnisci were short, and the giant subcuticular nuclei were unnoticeable. Meyer (1932) transferred Hebesoma to Neoechinorhynchidae, leaving diagnoses of the genus, and of H. violentum without chang- es. Th e validity of Hebesoma was accepted by Petrochenko (1956) and Yamaguti (1963), they both provided more concise diagnosis of the genus. Salgado-Maldonado (1978) dis- missed the importance of the features listed in Hebesoma diagnosis, showing their presence in the descriptions of many Neoechinorhynchus species and proposed the synonymy of these genera. Amin (2002) argued against monophyly of Neoechinorhynchus. He proposed to combine the concepts of both genera and to lower the status of the Neoechinorhynchus (sensu stricto) and Hebesoma emphasizing on subgeneric importance of the egg structure. Neoechinorhynchus is characterized by uniformity of anatomical organization (Malyar- chuk et al., 2014), while the specifi c egg morphology observed in some species provides an important morphologic feature useful for species discrimination. At the same time, erec- tion of a species group to a higher level taxon based on the polar prolongations of the inner envelopes of an egg is not consistent with the molecular results arguing that Hebesoma is supposed to be an unnatural taxon. In conclusion, morphologically similar species N. (N.) agilis and N. (H.) personatus and N. (H.) yamagutii can be clearly recognized using universal primers (F-566 and R-1200) proposed by Hadziavdic et al. (2014) for 18S rRNA gene. Phylogenetic analysis demonstrates that the studied here species of Neoechinorhynchus from grey mullet fi sh share common ancestor, despite their diff erent geographic location, and are closely related. Hebesoma is shown to be a polyphyletic taxon. In future investigations, it might be possible to use additional genomic markers to assess the validity of the phylogenetic hypotheses inferred in the present study. 9Molecular data confi rm the species status of Neoechinorhynchus personatus and N. yamagutii… We are grateful to Raúl Míguez-Lozano from University of Valencia for providing specimens of N. (H.) personatus from Valencia coastal waters, Spain and Lyudmila Shvetsova from Pacifi c Research Fisher- ies Center for providing specimens of N. (H.) yamagutii from Amur Bay, Russia. Th is study is supported by MEDEA project of Erasmus Mundus Action 2, #2686 and partially by an Assemble + short-term fellowship #BA200119, Ministry of Education and Science of Ukraine (projects #1/17 and 1/19). Author contributions IeT, JL and VS conceived the ideas. IeT collected the data from the Azov Sea, VS from the North-East Atlantic. RAS designed methodology of DNA extraction and amplifi cation. JL designed primers set. VS obtained PCR product. 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