Phylogenetic position of Polymorphus phippsi Kostylew, 1922 and Polymorphus magnus Skrjabin, 1913 (Palaeacanthocephala, Polymorphidae) ascertained on the basis of molecular data

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Acanthocephala, so-called thorny-headed worms, is a small phylum of obligatory endoparasites related to the Syndermata (Ahlrichs, 1997). To date only ca. 1300 species of acanthocephalans have been described (Khokhlova, 1986; Amin, 2013; Smales, 2015).

Also known as thorny-headed worms, acanthocephalans have a complex life cycle involving the intermediate host (an arthropod) and the definitive host (a vertebrate) (Khokhlova, 1986). It was shown that several species use paratenic hosts (Sharpilo, Salamatin, 2005). Classes and major orders of the phylum Acanthocephala were considered as monophyletic taxa with strong support by molecular and morphological data (Garcia-Varela et al., 2000; Amin, 2013; Monks, 2001). However, the composition of families and genera based on morphological characters is ambiguous (Amin, 2013). Data of molecular phylogeny are contrary to taxonomy based on phenotypic characters, especially in regard to family Polymorphidae (Garcia-Varela et al., 2013).

Polymorphidae is a species-rich family related to the class Palaeacanthocephala. Its type genus Polymorphus Lühe, 1911 demonstrated polyphyly (Garcia-Varela et al., 2013). An assemblage of new molecular data from other Polymorphus spp. can yield to changes in polymorphid phylogenetic tree topology. In this study we obtained new sequences of two widespread highly pathogenic for definitive hosts (water birds) Polymorphus spp. – P. phippsi Kostylew, 1922 and P. magnus Skrjabin, 1913, and added them to the phylogenetic reconstruction of the Polymorphidae.

MATERIALS AND METHODS

Larvae of P. phippsi 2-3 were collected from naturally infected amphipods Gammarus setosus (Luvenga, White Sea, Russia, 11.07.2019) and juvenile P. phippsi 1 were collected from Herring gull Larus argentatus (Dalnye Zelentsy, Barents Sea, Russia, 2001) and identified using Uspenskaya (1963) guide. A subadult specimen of P. magnus was isolated from Slaty-backed gull Larus schistisagus experimentally infected with cystacanths from naturally infected G. lacustris collected in the tundra lake situated close to the mouth of Chaun-Pucheveem river in area of Chaun research station (Chaun Bay coast, East-Siberian sea, Russia, July 2019). Species identification was carried out basing on Khokhlova (1986) guide and our experience.

The acanthocephalans were fixed in 96% ethanol, stained in Bömer’s hematoxylin (except for P. phippsi 1) and mounted in Canada balsam. Before staining, pieces of tissue were cut off and DNA was extracted from them using Chelex-100 procedure (De Lamballerie et al., 1992). Polymerase chain reactions (PCR) were performed with 5X ScreenMix (Evrogen, Moscow). Partial CDS of 28s rRNA were amplified using two overlapping PCR fragments of 2000–2500 bp. Primers for amplicon 1 were forward 5’-CAAGTACCGTGAGGGAAAGTTGCGC and reverse 5’-CTTCTCCAAC(T/G)TCAGTCTTCAA; amplicon 2 forward 5’-CTAAGGAGTGTGTAACAACTCACC and reverse 5’-CTTCGCAATGATAGGAAGAGCC (Garcia-Varela, Nadler, 2005). Partial CDS of cytochrome-oxydase I were amplified using forward primer 5’-AGTTCTAATCATAA(R)GATAT(Y)GG and reverse 5’-TAAACTTCAGGGTGACCAAAAAATCA (Folmer et al., 1994). PCR cycling parameters included denaturation at 94ºC for 3 min, followed by 35 cycles of denaturation at 94ºC for 1 min, annealing at 65ºC(28s)/60º(CO1) for 1 min, and elongation at 72ºC for 2 min, followed by final elongation at 72ºC for 4 min. PCR products were sequenced using Sanger’s technology in the resource center “Development of molecular and cellular technology” of St. Petersburg State University. Initial sequence analysis and reads assemblage was performed using ChromasPro 1.42.

We used 28S rDNA and CO1 mtDNA alignments from Garcia-Varela et al. (2013) as a template (table 1). Both amplicons of 28s rDNA were added to the corresponding alignment and then aligned in SeaView 4 (Gouy et al., 2010). The same procedure was performed for both CO1 sequences. CO1 alignment was trimmed according to the shortest sequence, 588 bp in length. Alignment of 28S rDNA was trimmed according to the common length of two overlapping amplicons from P. phippsi samples, so that its length was 2966 bp. We counted optimal substitution models and phylogenetic distances using MEGA 11.0.11 (Tamura et al., 2021) for each separate alignment. After that we created a concatenate matrix using SeaView 4.

 

Table 1. Source information

Species	Host	Locality	28s rDNA GenBank №	CO1 GenBank №
Andracantha gravida	Phalacrocorax auritus	Yucatán, México	EU267814	EU267822
Arhythmorhynchus frassoni 1	Uca spinicarpa	Yucatán, México	JX442176	EU189484
Arhythmorhynchus frassoni 2	Eudocimus albus	Sinaloa, México	JX442177	JX442188
Bolbosoma turbinella	Eschrichtius robustus	Monterey Bay, California, USA	JX442178	JX442189
Bolbosoma sp.	Callorhinus ursinus	St. Paul Island, Alaska, USA	JX442179	JX442190
Corynosoma australe	Phocarctos hookeri	New Zealand	JX442180	JX442191
Corynosoma enhydri	Enhydra lutris	Monterey Bay, California, USA	AY829107	DQ089719
Corynosoma magdaleni	Phoca hispida saimensis	Lake Saimaa, Finland	EU267815	EF467872
Corynosoma obtuscens	Callorhinus ursinus	St. Paul Island, Alaska, USA	JX442181	JX442192
Corynosoma strumosum	Phoca vitulina	Monterey Bay, California, USA	EU267816	EF467870
Corynosoma validum	Callorhinus ursinus	St. Paul Island, Alaska, USA	JX442182	JX442193
Ibirhynchus dimorpha	Eudocimus albus	Veracruz, México	GQ981437	GQ981438
Hexaglandula corynosoma	Nyctanassa violacea	Veracruz, México	EU267817	EF467869
Polymorphus brevis 1	Nycticorax nycticorax	Michoacán, México	AY829105	DQ089717
Polymorphus brevis 2	Nycticorax nycticorax	Michoacán, México	JX442183	JX442194
Polymorphus minutus	Gammarus pulex	Dijon, France	EU267819	EF467865
Polymorphus obtusus	Ahythya affinis	Baja California Sur, México	JX442184	JX442195
Polymorphus trochus	Fulica america	Sinaloa, México	JX442185	JX442196
Profilicollis altmani	Enhydra lutris	Monterey Bay, California, USA	AY829108	DQ089720
Profilicollis botulus 1	Somateria mollissima	Denmark	EU267818	EF467862
Profilicollis botulus 2	Anas platyrhynchos	USA	AY829109	DQ089721
Profilicollis bullocki	Emerita analoga	Caleta Lenga, Chile	JX442186	JX442197
Pseudocorynosoma constrictum	Anas clypeata	Estado de México, México	EU267812	EU267820
Pseudocorynosoma anatarium	Bucephala albeola	Durango, México	EU267813	EU267821
Pseudocorynosoma sp.	Oxyura jamaicensis	Durango, México	JX442187	JX442198
Southwellina hispida 1	ND	Hallai, USA	EU267810	EF467866
Southwellina hispida 2	Tigrisoma mexicanum	Veracruz, México	EU267811	EF467867
Centrorhynchus sp.	Falco peregrinus	California, USA	AY829104	DQ089716
Gorgorhynchoides bullocki	Eugerres plumieri	Quintana Roo, México	AY829103	DQ089715
Plagiorhynchus cylindraceus	Porcilio saber	Dijon, France	AY829102	DQ089724
Polymorphus phippsi 1 (This study)	Larus argentata	Dalnye Zelentsy, Barents sea, Russia	OK235421 (1st amplicon)	-
Polymorphus phippsi 2 (This study)	Gammarus setosus	Luvenga, White sea, Russia	-	OL676091
Polymorphus phippsi 3 (This study)	Gammarus setosus	Luvenga, White sea, Russia	ON667751 (2nd amplicon)	OL676687
Polymorphus magnus (This study)	Larus schistisagus	Chaun, Chukotka, Russia	-	OL689013

 

For P. phippsi 1 CO1 sequence and fragments of 28S rDNA for P. phippsi 3 and P. magnus was replaced with missing data, such as 28S rDNA ‒ for P. phippsi 3 and P. magnus (table 1).

Phylogenetic relationships were determined by maximum-likelihood analysis in CIPRES Science Gateway platform (Miller et al., 2010) with RaxML-HPC black box tool and by Bayesian analysis in MrBayes 3.2.7a (Ronquist et al., 2012). The following parameters were specified in Bayesian tree search: partitioned model (GTR+G and GTR+G+I for 28S rDNA and CO1, respectively) and setting outgroup identical to the taxset from Garcia-Varela et al. (2013).

RESULTS AND DISCUSSION

Interspecific genetic divergence between 28S rDNA sequences of P. phippsi 1 and P. phippsi 2 (0.028) was greater than that between the sequences from samples of one species (Arhytmorhynchus frassoni 0.016, Southwellina hispida 0.003, Polymorphus brevis 0.002) but smaller than the divergence between different congeneric species (average for Corynosoma spp. 0.06, for Profilicollis spp. 0.04). Divergence between P. phippsi 2 and P. phippsi 3 (0.008) was smaller than that between the sequences from samples of one species (Arhytmorhynchus frassoni 0.193 COI, Southwellina hispida 0.048, Polymorphus brevis 0.014). All the sequences from samples of P. phippsi were grouped together in a well-supported clade (fig. 1). We can conclude that the three samples were cophenetic. Overall topologies of our tree and the tree from the previous research (Garcia-Varela et al., 2013) were congruent. Bayesian and maximum-likelihood trees yielded the same topology (fig. 1). The clade containing P. phippsi sequences grouped with the clade containing Profilicollis bullocki and Pr. altmani, with Pr. botulus being basal to this clade. P. magnus grouped in one clade with P. obtusus, while P. minutus was basal to this clade.

 

Figure 1. Phylogenetic relationships inferred from combined 28S rDNA + COI data set. Numbers near internal nodes show ML/MP bootstrap clade frequencies (percents) and posterior probabilities clade frequencies (percents). Clade containing Polymorphus phippsi sequences is highlighted in violet and Polymorphus magnus – in blue.

 

The position of P. phippsi in our phylogenetic reconstructions supports the polyphyly of Polymorphus postulated by Garcia-Varela et al. (2013). The position of P. magnus corresponds to its accepted taxonomic status (Khokhlova, 1986). Thus, it is likely that assemblage of new molecular data can outline only more contradictions with accepted taxonomy of Polymorphidae. In this case the necessity of taxonomic characters revision, including additional data of comparative morphology, can appear.

ACKNOWLEDGEMENTS

Authors express their gratitude to K.V. Regel (Magadan, IBPN FEB RAS) for her help with sampling. Authors express gratitude to the administration of Kandalakschsky reserve and Murmansk Marine Biology Institute RAS for the possibility to collect and handle samples. The molecular studies were performed in the Saint-Petersburg State University resource center “Development of molecular and cellular technology”.

FUNDING

This work was supported by the Russian Science Foundation (grant no. 23-14-00329) and State Academic Programs no. 122031100260-0 “Biodiversity of parasites, their life cycles, biology and evolution” and no. 1021060307693-0 “Helminths in the Biocenoses of North-Eastern Asia: Biodiversity, Morphology, and Molecular Phylogenetics”.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

This work does not contain any studies involving human and animal subjects.

CONFLICT OF INTEREST

This work does not contain any studies involving human and animal subjects.

作者简介

A. Diumina

Zoological Istitute of Russian Academy of Sciences

编辑信件的主要联系方式.
Email: aleksandra.diumina@zin.ru
俄罗斯联邦, Universitetskaya emb., 1, Saint-Petersburg, 199034

K. Galaktionov

Zoological Istitute of Russian Academy of Sciences

Email: kirill.galaktionov@zin.ru
俄罗斯联邦, Universitetskaya emb., 1, Saint-Petersburg, 199034

G. Atrashkevich

Institute of the Biological Problems of the North Far East Branch of Russian Academy of Sciences

Email: gatr@ibpn.ru
俄罗斯联邦, Portovaya str., 18, Magadan, 685000

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