Research Article |
Corresponding author: Matthew T. Wayland ( mw283@cam.ac.uk ) Academic editor: Boyko Georgiev
© 2015 Matthew T. Wayland, Jouni K. Vainio, David I. Gibson, Elisabeth A. Herniou, D. Timothy J. Littlewood, Risto Väinölä.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Wayland MT, Vainio JK, Gibson DI, Herniou EA, Littlewood TDJ, Väinölä R (2015) The systematics of Echinorhynchus Zoega in Müller, 1776 (Acanthocephala, Echinorhynchidae) elucidated by nuclear and mitochondrial sequence data from eight European taxa. ZooKeys 484: 25-52. https://doi.org/10.3897/zookeys.484.9132
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The acanthocephalan genus Echinorhynchus Zoega in Müller, 1776 (sensu
Acanthocephala , Echinorhynchus bothniensis , Echinorhynchus brayi , Echinorhynchus cinctulus , Echinorhynchus gadi , Echinorhynchus salmonis , Echinorhynchus truttae , Acanthocephalus lucii , phylogeny, molecular phylogeny, taxonomy, parasite, systematics, zoogeography
The acanthocephalan genus Echinorhynchus Zoega in Müller, 1776 (sensu
Given the species diversity and genetic divergence within Echinorhynchus, it would be useful to split the genus if monophyletic groups with supporting morphological characters can be identified.
Although molecular systematics have revealed that species of Echinorhynchus show a degree of genetic divergence that would indicate a generic division, such a division would not produce taxa concordant with Petrochenko’s system (
A further problem for Petrochenko’s classification is the taxonomic status of P. clavula, his type-species for Pseudoechinorhynchus. When Petrochenko published his classification, two morphologically distinct species were conflated under the specific binomen Echinorhynchus clavula Dujardin, 1845. Dujardin’s original description did not include drawings and lacked sufficient detail for the taxon to be reliably identified by other workers. Subsequently,
The incompatibility between E. clavula Dujardin and E. clavula Dujardin sensu
Further attempts at revising Echinorhynchus should be underpinned by evidence of the phylogenetic relationships of its constituent taxa. To this end we have used sequences from two genes with different patterns of inheritance and different rates of evolutionary change (28S rRNA and cytochrome c oxidase subunit I) to reconstruct a phylogeny for nine populations of Echinorhynchus, representing eight distinct biological taxa (Table
Species | Host | Locality | Date collected | Genus sensu |
Environment | GenBank # (28S rDNA / COI) |
Voucher specimens |
---|---|---|---|---|---|---|---|
Acanthocephalus lucii (outgroup) | Perca fluviatilis (L.) (Percidae) | Lake, Bleasby, Nottinghamshire, UK | 4/06/1997 | Acanthocephalus | Freshwater | KM656148 / KP261016 | BM(NH) 2002.2.4.284–292 |
E. bothniensis | Osmerus eperlanus (L.) (Osmeridae) | Lake Keitele, central Finland | 10/10/1996 | Metechinorhynchus | Freshwater | KM656146 / KP261018 | BM(NH) 2002.2.4.102–122 |
E. 'bothniensis' |
Platichthys flesus (L.) (Pleuronectidae) Mysis segerstralei Audzijonyte & Väinölä (Mysidae) |
Lake Pulmankijärvi, northern Finland | 11/06/1990 | Echinorhynchus | Freshwater | KM656143 / KP261019 | NA |
E. brayi | Pachycara crassiceps (Roule) (Zoarcidae) | Porcupine Seabight, 49°49.9'N, 13°08.2'W, depth 2,444 m | 13/08/1997 | Metechinorhynchus | Marine, deep-sea | KM656151 / KP261015 | BM(NH) 1997.12.8.3 (holotype); BM(NH) 1997.12.8.4–28 |
E. cinctulus (= E. borealis) |
Lota lota (L.) (Lotidae) | Kuopio, Finland | 15/10/1996 | Pseudoechinorhynchus | Freshwater | KM656142 / KP261014 | BM(NH) 2002.2.4.123–131 |
E. gadi sp. I | Gadus morhua L. (Gadidae) | Baltic Sea, off Tvärminne, Hanko | 21/10/1992 | Echinorhynchus | Marine | KM656144 / KP261022 | BM(NH) 2002.2.4.90–101 |
E. gadi sp. I | G. morhua | Mys Kartesh, Gulf of Kandalaksha, White Sea | 31/08/1994–2/09/1994 | Echinorhynchus | Marine | KM656150 / KP261021 | NA |
E. gadi sp. III | G. morhua | Mys Kartesh, Gulf of Kandalaksha, White Sea | 31/08/1994–2/09/1994 | Echinorhynchus | Marine | KM656149 / KP261020 | NA |
E. salmonis | Coregonus lavaretus (L.) (Salmonidae) | Bothnian Bay, Baltic Sea | 27/08/1996 | Metechinorhynchus | Freshwater | KM656145 / KP261017 | BM(NH) 2002.2.4.132–226 |
E. truttae | Salmo trutta L. (Salmonidae) | Loch Walton Burn, River Carron catchment, central Scotland (National Grid Reference NS 668 865) | 24/06/1996 | Metechinorhynchus | Freshwater | KM656147 / KP261013 | BM(NH) 2002.2.4.264–275 |
Collection data for the samples are provided in Table
E. bothniensis Zdzitowiecki & Valtonen, 1987 is known from fresh- and brackish-water environments of Northern Fennoscandia. Based on molecular differences, it may be further subdivided into two allopatric taxa (
E. brayi Wayland, Sommerville & Gibson, 1999 was described from Pachycara crassiceps (Roule) (Zoarcidae) collected from the Porcupine Seabight at a depth of 2,444 metres (
Cement gland arrangement in male Echinorhynchus spp. Notation for cement gland pattern from
Species | A | B | C | D | E |
---|---|---|---|---|---|
E. bothniensis | 0 | 1 (5.3%) | 4 (21.1%) | 10 (52.6%) | 4 (21.1%) |
E. 'bothniensis' | 0 | 0 | 0 | 4 (44.4%) | 5 (55.6%) |
E. brayi | 1 (8%) | 7 (54%) | 3 (23%) | 2 (15%) | 0 |
E. cinctulus | 218 (100%) | 0 | 0 | 0 | 0 |
E. gadi | 0 | 0 | 0 | 3 (8%) | 34 (92%) |
E. truttae | 0 | 1 (3%) | 16 (53%) | 13 (43%) | 0 |
E. salmonis | 6 (37.5%) | 10 (62.5%) | 0 | 0 | 0 |
As explained in the Introduction, E. cinctulus Porta, 1905 is the correct name for the type-species of Petrochenko’s genus Pseudoechinorhynchus that has commonly been referred to as E. borealis Linstow. This species is found in fresh and oligohaline waters of the Palaearctic (
E. gadi Zoega in Müller, 1776, the type-species of Echinorhynchus, is the most frequently reported acanthocephalan from fish of the North Atlantic and North Pacific Oceans (
E. salmonis Müller, 1784 is the type-species of
E. truttae Schrank, 1788 is another common parasite of salmonid fishes in northern Europe. In the original description of E. bothniensis,
In order to root the phylogenetic trees, sequence data were also determined from Acanthocephalus lucii (Müller, 1776), another member of the subfamily Echinorhynchinae. Acanthocephalus and Echinorhynchus appear to be closely related genera discriminated on the basis of only one morphological character, the position of the nerve ganglion or “brain”, which is situated at the base of the proboscis receptacle in Acanthocephalus but mid-way along the receptacle in Echinorhynchus (see
All acanthocephalans were washed in saline and then fixed in 90–100% alcohol immediately after collection, or alternatively frozen in liquid nitrogen and stored at -80 °C. Single specimens of each sample were used for the sequencing of each gene, but different individuals were analyzed for the different genes (in different laboratories). The anterior ends of the worms were removed before DNA extraction to avoid contamination of the samples with any host tissue attached to the proboscis. For the 28S analysis, individual acanthocephalans were washed in TE, ground in 150 µl TE (pH 8.0), 0.5% SDS, and digested overnight with the addition of 6 µl proteinase K (10 mg ml-1) at 37 °C. DNA was phenol-chloroform extracted and precipitated for 15 minutes at -20 °C with 0.1 vol. sodium acetate, at pH 5.0, and 2.5 vols 100% ethanol. DNA pellets were washed in 70% ethanol, dried, resuspended in TE (pH 8.0) and stored at -20 °C. Spectrophotometry was used to estimate the concentration of nucleic acids. Alternatively, for the COI data set, the CTAB extraction protocol of
For most taxa, a c.1,600 base-pair segment of the 28S rRNA gene spanning variable regions D1 to D6 was amplified using the primers LSU5 (5´-TAGGTCGACCCGCTGAAYTTAAGCA-3) and LSUD6-3 (5´-GGAACCCTTCTCCACTTCAGTC-3´) (
For analysis of a part of the mitochondrial COI gene, the universal “barcoding” primers of
The 28S rDNA and COI sequences were analyzed independently and also concatenated into a single dataset. Three methods of phylogenetic reconstruction were applied to each dataset: Bayesian inference (BI), maximum likelihood (ML) and maximum parsimony (MP). A. lucii was used as an outgroup in all analyses. For the phylogenetic reconstruction methods involving modelling of sequence evolution (BI and ML), the data-sets were partitioned to accommodate heterogeneity in patterns and rates of substitutions between genes and/or codon positions. The COI data-set was divided into three partitions, one for each codon position. The concatenated 28S rDNA and COI data-set was separated into four partitions, one for the 28S rDNA sequence and three for each of the codon positions in the COI sequence. The 28S rDNA data-set was not partitioned.
Mr Bayes version 3.2.2 (
ML analysis was carried out using the genetic algorithm implemented in MetaPIGA 3.1 (
MP analysis was performed using PAUP version 4.0b10 (
Phylograms and other graphics were created using R (
All sequence data have been submitted to GenBank; accession numbers are provided in Table
The aligned partial 28S rDNA sequence data consisted of 1,607 nucleotide sites for all taxa except E. cinctulus, for which only the first 750 base pairs of the segment could be sequenced (Suppl. material
Observed sequence divergence (%) between pairs of echinorhynchid species for the 28S rDNA (below the diagonal) and COI sequence data (above the diagonal).
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | |
---|---|---|---|---|---|---|---|---|---|---|
1. A. lucii | — | 36.1 | 33.3 | 34.5 | 32.8 | 34.2 | 34.4 | 34.0 | 34.0 | 34.7 |
2. E. salmonis | 18.5 | — | 29.7 | 27.7 | 28.7 | 29.7 | 29.7 | 29.4 | 28.7 | 28.9 |
3. E. cinctulus | 31.1 | 23.1 | — | 21.7 | 22.2 | 21.5 | 21.7 | 22.9 | 22.9 | 23.1 |
4. E. brayi | 19.1 | 15.5 | 6.6 | — | 16.8 | 17.4 | 17.3 | 19.0 | 17.1 | 18.0 |
5. E. truttae | 19.3 | 15.3 | 7.5 | 0.8 | — | 8.2 | 8.4 | 9.1 | 8.9 | 8.9 |
6. E. gadi sp. I (Baltic Sea) | 19.2 | 15.4 | 7.1 | 0.5 | 0.3 | — | 0.2 | 7.2 | 6.5 | 6.3 |
7. E. gadi sp. I (White Sea) | 19.2 | 15.4 | 7.1 | 0.5 | 0.3 | 0.0 |
— | 7.4 | 6.5 | 6.3 |
8. E. gadi sp. III | 19.2 | 15.4 | 7.1 | 0.5 | 0.3 | 0.0 |
0.0 |
— | 3.3 | 3.1 |
9. E. bothniensis | 19.2 | 15.4 | 7.1 | 0.5 | 0.3 | 0.0 |
0.0 |
0.0 |
— | 1.5 |
10. E. 'bothniensis' | 19.2 | 15.4 | 7.1 | 0.5 | 0.3 | 0.0 |
0.0 |
0.0 |
0.0 |
— |
In the 585 base-pair alignment of the COI sequences, 249 (42.6%) of the nucleotide sites were variable within Echinorhynchus, of which 62 (24.9%) were at a first codon position, 23 (9.2%) at a second codon position and 164 (65.9%) at a third codon position (Suppl. material
Since identical sequences were obtained from members of the E. gadi complex, E. bothniensis and E. ‘bothniensis’, the 28S rDNA data-set could only be used to resolve the deeper branches in the phylogeny. BI identified a hierarchy of three clades, each with a maximal posterior probability (Fig.
A fully resolved tree was recovered from the mitochondrial COI data-set (Fig.
MP analysis of the COI data-set produced a single most parsimonious tree, 542 steps long (CI = 0.795, RI = 0.615), which differed from the BI and ML phylograms at a single point, regarding the basal placement of E. cinctulus instead of E. salmonis (Fig.
Phylogenetic relationships of Echinorhynchus spp. inferred from maximum parsimony analysis of COI data-set. Trees are rooted on the outgroup A. lucii. A Phylogram estimated using maximum parsimony analysis of COI sequence data. Numbers at nodes indicate bootstrap support (n = 10,000) B Consensus cladogram from maximum parsimony analysis of COI sequence data excluding third codon positions. Numbers at nodes indicate bootstrap support (n = 10,000).
BI, ML and MP analysis of the combined data-sets all yielded the same phylogram, which was topologically identical to the BI/ML tree for the COI data-set and displayed similar support for most clades (Fig.
The following discussion is based on the fully resolved phylogeny recovered from the total molecular data. It is important to note that, whereas the deeper branches in the phylogeny are supported by sequence data from both genes, the interrelationships of the five most closely related species were resolved using the COI data-set alone.
No support for
Aquatic environment (freshwater/marine) mapped on to the fully resolved phylogeny inferred from the concatenated 28S and COI sequences. Bold letter indicates genus according to
Cement gland arrangement displays continuous variation, from the pattern of three regular pairs through to the strictly moniliform pattern, with each Echinorhynchus species displaying a range of variation along this continuum (Table
Further and more conclusive evidence that the ancestral cement gland arrangement is three regular pairs is available from both outgroup comparison and ontogeny. Firstly, outgroup comparison is based on the assumption that the character state found in related groups is the plesiomorphic condition (
E. cinctulus and E. salmonis exhibit a relatively strong genetic divergence from each other and from the other taxa of the ingroup (Table
The acanthors of E. cinctulus display a unique pattern of hooks and spines which has not been observed in other species of Echinorhynchus, although relatively few taxa have been studied (
Another taxonomic finding of the current study is paraphyly of the E. gadi group with respect to the monophyletic E. bothniensis group (Fig.
One significant problem in the systematics of Echinorhynchus, which could not be addressed with the current data, is the monophyly of the genus. Further phylogenetic analyses incorporating a range of echinorhynchid acanthocephalans will be needed to resolve this issue. The relatively slowly evolving 28S rRNA gene, along with nuclear protein coding genes, should prove to be particularly useful in this respect.
Since our phylogeny represents only a small proportion of the species in the genus, it is impossible to make any definitive claims about the zoogeography of this group of worms. However, the limited observations do suggest hypotheses that could be tested with additional data.
Echinorhynchus spp. are distributed from the Arctic (e.g.
From this suggested freshwater origin and radiation, Echinorhynchus spp. have invaded the sea, most likely several times (Fig.
Evidence of a re-invasion of freshwater by marine stock can also be found in the fully resolved phylogeny (Fig.
This preliminary investigation of the phylogenetic relationships within Echinorhynchus (sensu lato) underscores the argument for rejecting
We would like to thank Professor Tellervo Valtonen (University of Jyväskylä, Finland) for collecting the specimens of E. bothniensis, E. ‘bothniensis’, E. cinctulus and E. salmonis, and Dr Rod Bray (Natural History Museum, London) for collecting specimens of E. brayi.
Aligned and concatenated partial sequences of COI and 28S rDNA
Data type: Nexus file
Explanation note: Aligned and concatenated partial sequences of COI and 28S rDNA in nexus format. Aligned partial sequences of COI and 28S rDNA from each acanthocephalan population have been concatenated. Gaps are indicated by ‘-’. The first 585 characters in each block correspond to COI and the remainder to 28S rDNA. The file contains data for all nine Echinorhynchus samples and the outgroup taxon, Acanthocephalus lucii. This nexus file was used in all phylogenetic analyses.
Maximum likelihood model parameters
Data type: Adobe PDF file
Explanation note: Model parameters used in the maximum likelihood approach to phylogenetic reconstruction.
Nucleotide substitutions
Data type: Comma-separated-value file of measurements
Explanation note: Substitutions of nucleotides (transitions/transversions) for 28S rDNA (below the diagonal) and COI sequence data (above the diagonal).
Patterns of COI sequence variation
Data type: Adobe PDF file
Explanation note: Patterns of COI sequence variation. Graphs and discussion of patterns of nucleotide substitions in the COI data-set.