Open Access

Phylogenetic position of Loricifera inferred from nearly complete 18S and 28S rRNA gene sequences

Zoological Letters20151:18

DOI: 10.1186/s40851-015-0017-0

Received: 9 March 2015

Accepted: 26 May 2015

Published: 30 June 2015

Abstract

Background

Loricifera is an enigmatic metazoan phylum; its morphology appeared to place it with Priapulida and Kinorhyncha in the group Scalidophora which, along with Nematoida (Nematoda and Nematomorpha), comprised the group Cycloneuralia. Scarce molecular data have suggested an alternative phylogenetic hypothesis, that the phylum Loricifera is a sister taxon to Nematomorpha, although the actual phylogenetic position of the phylum remains unclear.

Methods

Ecdysozoan phylogeny was reconstructed through maximum-likelihood (ML) and Bayesian inference (BI) analyses of nuclear 18S and 28S rRNA gene sequences from 60 species representing all eight ecdysozoan phyla, and including a newly collected loriciferan species.

Results

Ecdysozoa comprised two clades with high support values in both the ML and BI trees. One consisted of Priapulida and Kinorhyncha, and the other of Loricifera, Nematoida, and Panarthropoda (Tardigrada, Onychophora, and Arthropoda). The relationships between Loricifera, Nematoida, and Panarthropoda were not well resolved.

Conclusions

Loricifera appears to be closely related to Nematoida and Panarthropoda, rather than grouping with Priapulida and Kinorhyncha, as had been suggested by previous studies. Thus, both Scalidophora and Cycloneuralia are a polyphyletic or paraphyletic groups. In addition, Loricifera and Nematomorpha did not emerge as sister groups.

Keywords

Molecular phylogeny Ecdysozoa Scalidophora Cycloneuralia Nematoida Panarthropoda

Introduction

Since its first description as a new phylum [1], Loricifera has been one of the most enigmatic metazoan phyla. Although only 35 loriciferan species have been described worldwide, the actual species diversity is higher, as many new species await description [26]. All known loriciferan species are microscopic (80–800 μm) and occur in marine sediments, such as mud, sand, and shell gravel. The most extreme habitat for Loricifera is the hypersaline anoxic deep basin in the Mediterranean Sea, where members of this phylum are metabolically active [6, 7]. Our knowledge of loriciferan life cycles is also only fragmentary, given the recent findings of new life cycles and larval types [35, 8].

There are two alternative hypotheses on the position of Loricifera within Ecdysozoa, both based on morphological data. One is the ‘Scalidophora hypothesis’ [911], in which Loricifera, Kinorhyncha, and Priapulida together comprise a clade, Scalidophora. Morphological similarities between Scalidophora and Nematomorpha [1215] and between Scalidophora and Nematoida (Nematomorpha and Nematoda) [9, 11, 1621] have indicated that these five phyla in turn comprise a clade, Cycloneuralia [20, 21].

The alternative is the ‘Loricifera + Nematomorpha hypothesis’ [22]. While the first molecular phylogenetic study that included a loriciferan sequence (18S rRNA) failed to establish the phylogenetic position of Loricifera [23], Sørensen et al. [22] detected a sister group relationship between Loricifera and Nematomorpha based on 18S rRNA and histone-3 sequences, although with low nodal support (posterior probability = 0.83). The latter study also detected a sister group relationship between Priapulida and Kinorhyncha, but not monophyly for Cycloneuralia, which several previous molecular studies that lacked loriciferan sequences had indicated [2429].

The present study investigated the phylogenetic position of phylum Loricifera within Ecdysozoa using nearly complete 18S and 28S rRNA sequences. Also of interest was the phylogenetic status of the taxa Scalidophora and Cycloneuralia.

Materials and methods

Sampling and DNA sequencing

The loriciferan specimen used in this study was collected from Ise Bay, Japan, northwestern Pacific (34°9.77′N, 136°51.40′E, 161–174 m depth) during a cruise of the TR/V Seisui-maru (Mie University) on 21 November 2013. A sediment sample was collected with a biological dredge, subsequently frozen to prevent DNA degradation, and sent to the laboratory. In the laboratory, meiofaunal specimens were extracted by floatation [30] with Ludox® HS 40. The extracted sample was sorted under a stereomicroscope, and a single adult loriciferan specimen (Fig. 1a) was obtained and preserved in 99 % EtOH for DNA extraction.
Fig. 1

Rugiloricus sp., an undescribed loriciferan. Nomarski photomicrographs of the hologenophore of the specimen of Rugiloricus sp. used in this study. a, Entire animal before DNA extraction; b, Exoskeleton of the specimen after DNA extraction

Total genomic DNA was extracted [31] from the specimen with a DNeasy Tissue Kit (Qiagen, Tokyo). After DNA extraction, the exoskeleton was mounted in Fluoromount G® as a hologenophore (Fig. 1b). The loriciferan specimen was identified as Rugiloricus sp. based on the morphology of the hologenophore.

Nearly complete 18S rRNA (18S) and 28S rRNA (28S) genes sequences were amplified by PCR using previously published primer sets and conditions [31]. All nucleotide sequences were determined by direct sequencing with a BigDye Terminator Kit ver. 3.1 (Life Technologies, Co., USA) and a 3730 DNA Analyzer (Life Technologies, Co., USA). Sequence fragments were assembled by using MEGA 5 [32]. After assembly, 18S (1872 bp) and 28S (3450 bp) sequences were deposited in GenBank under accession numbers LC032019 and LC032020.

Phylogenetic analyses

18S and/or 28S sequences for 66 taxa were obtained from GenBank. We prepared the following five datasets for analyses (Table 1): “18S + 28S (50OTU)” including 18S and 28S sequences for all 50 taxa which both 18S and 28S are available (note that we treated the 18S sequence from Milnesium tardigradum and the 28S sequence from Milnesium sp. as a single OTU, because nearly complete 18S and 28S sequences were unavailable from a single tardigrade species); “18S (50OTU)” including 18S sequences for the same taxa of “18S +28S (50 OTU)”; “28S (50OTU)” including 28S sequences for the same taxa of “18S +28S (50 OTU)”; “18S (65 OTU)” including 18S sequences for more comprehensive taxon sampling especially in Tardigrada, Nematoda, Nematomorpha, Priapulida, and Kinorhyncha than the former three datasets; “18S (63 OTU)” including 18S sequences for same OTU to “18S (65 OTU)” except for Nanaloricus sp. due to its short sequence and Meiopriapulus fijiensis to avoid long branch attraction [22]. Sequences from each gene were pre-aligned separately with MAFFT software [33] using the FFT-NS-2 option and were subsequently divided into domains by eye. Domain sequences were realigned individually with MAFFT software using the L-INS-i option (Additional files 1, 2, 3 and 4). Alignment-ambiguous positions were removed with TrimAl software [34] in “strict setting”, and all positions bearing gaps were also removed. The trimmed domain sequences were recombined to form the final dataset for analysis (Additional files 5, 6, 7 and 8), which was 1426 bp long for 18S and 2189 bp long for 28S in “18S + 28S (50OTU)”, “18S (50OTU)”, and “28S (50OTU), 1277 bp long for 18S in 18S (65 OTU), and 1302 bp long for 18S in 18S (63 OTU). The chi-square test in Kakusan4 [35] indicated that the base composition of each dataset was significantly homogeneous.
Table 1

List of taxa included in each dataset

Taxa

Data set

Accession number

  

Species

18S + 28S (50OTU)

18S (50OTU)

28S (50OTU)

18S (65OTU)

18S (63OTU)

18S

28S

Loricifera

 

Rugiloricus sp.

LC032019

LC032020

  

Nanaloricus sp.

   

 

EU669461

 
  

Pliciloricus sp. 

   

AY746986

-

Arthropoda

Euchelicerata

Limulus polyphemus

U91490

AF212167

  

Calocheiridius cf. termitophilus

AY859559

AY859558

  

Siro rubens

U36998

AY859602

  

Eremobates sp.

AY859573

AY859572

  

Pandinus imperator

AY210831

AY210830

  

Mastigoproctus giganteus

AF005446

AY859587

  

Misumenops asperatus

AY210445

AY210461

 

Pycnogonida

Anoplodactylus portus

AY859551

AY859550

  

Callipallene sp.

AY210808

AY210807

 

Myriapoda

Polyxenidae sp.

AY859596

AY859595

  

Orthoporus sp.

AY210829

AY210828

  

Cherokia georgiana

AY859563

AY859562

  

Scutigera coleoptrata

AF173238

AY859601

  

Craterostigmus tasmanianus

AF000774

AY859569

 

Crustacea

Cyprididae sp.

AY210816

AY210815

  

Anaspides tasmaniae

L81948

AY859549

  

Squilla empusa

L81946

AY210842

  

Heteromysis sp.

AY859580

AY859578–79

  

Gaetice depressus

AY859577

AY859575–76

  

Panulirus argus

U19182

AY210833–35

  

Homarus americanus

AF235971

AY859581

  

Eulimnadia texana

AF144211

AY859574

  

Triops longicaudatus

AF144219

AY157606

 

Hexapoda

Podura aquatica

AF005452

AY210838

  

Sminthurus viridus

AY859604

AY859603

  

Dilta littoralis

AF005457

AY859570–71

  

Callibaetis ferrugineus

AF370791

AY859557

  

Mantis religiosa

AY859586

AY859585

  

Zootermopsis angusticollis

AY859615

AY859614

  

Gromphadorhina laevigata

AY210820

AY210819

  

Gomphocerinae sp.

AY859547

AY859546

  

Vespula pensylvanica

AY859613

AY859612

  

Merope tuber

AF286287

DQ202351

Onychophora

 

Peripatoides novaezealandiae

AF342794

AF342791–93

Tardigrada

 

Milnesium tardigradum

 

U49909

-

  

Milnesium sp.

 

  

-

AY210826

  

Echiniscus blumi

   

HM193375

 
  

Testechiniscus spitzbergensis

   

EU266967

 
  

Richtersius coronifer

   

AY582123

 

Nematoda

Spiurina

Ascaris lumbricoides

U94366

AY210806

 

Dorylaimia

Trichinella spiralis

U60231

AF342803

  

Xiphinema rivesi

AF036610

AY210845

 

Enoplia

Pontonema vulgare

   

AF047890

 
 

Desmodorida

Spirinia elongata

   

EF527426

 
 

Monhysterida

Theristus agilis

   

AY284695

Nematomorpha

 

Chordodes morgani

AF036639

AF342787

  

Gordius aquaticus

X80233

AY210817

  

Nectonema agile

   

AF421767

Priapulida

 

Priapulus caudatus

Z38009

AY210840

  

Halicryptus spinulosus

AF342790

AF342789

  

Tubiluchus corallicola

   

AF119086

 
  

Meiopriapulus fijiensis

   

 

JN211192

 

Kinorhyncha

 

Pycnophyes sp.

AY859598

AY859597

  

Dracoderes abei

   

AB738350

AB738351

  

Echinoderes dujardinii

   

LC007044

LC007065

  

Centroderes spinosus

   

KF372858

  

Campyloderes cf. vanhoeffeni

   

LC007037

 

Lophotrochozoa (Outgroup)

Nemertea

Amphiporus sp.

AF119077

AF342786

 

Mollusca

Placopecten magellanicus

X53899

AF342798

 

Platyhelminthes

Stylochus zebra

AF342801

AF342800

 

Echiura

Urechis caupo

AF342805

AF342804

Deuterostomes (Outgroup)

Hemichordata

Ptychodera fava

AF278681

AF212176

 

Chordata

Ciona intestinalis

AB013017

AF212177

Taxa included in each data set, with GenBank accession numbers for sequences

Before the analyses, the optimal substitution model was determined with Kakusan4 to be the general time-reversible model with the gamma distribution (GTR + Γ). Phylogenetic trees were constructed by maximum likelihood (ML) implemented in raxmlGUI 1.2 [36, 37], and Bayesian inference (BI) implemented in MrBayes 3.2.1 [38, 39]. Nodal support for the ML tree was assessed through analyses of 1000 bootstrap pseudoreplicates. For BI, Markov-chain Monte-Carlo searches were performed with four chains, each of which was run for 1,000,000 generations, with trees sampled every 100 generations. Stationarity was evaluated by monitoring likelihood values graphically. The initial 20 % of trees from each run were discarded as burn-in, and the remaining trees were used to construct majority-rule consensus trees and determine the Bayesian posterior probability for each clade [39].

Results and discussion

Overall topology in Ecdysozoa

None of the trees conflicted with the others in their overall topology; however, supporting values were lower in datasets with more OTU and shorter sequences (Table 2; Additional files 9, 10, 11 and 12). In our results, increasing the available sequence length with slightly limited taxa generated a better-resolved tree than using more taxa with markedly shortening the sequence length. Thus, we present and mainly discuss the result of 18S + 28S (50 OTU) dataset (Fig. 2). Both the ML and BI trees showed monophyly for the Ecdysozoa (nodal support ML/PP = 99/1.00) as well as for the phyla Priapulida (100/1.00), Nematoda (99/1.00), Nematomorpha (100/1.00), and Arthropoda (89/1.00). Although the monophyly of each phyla were not tested for Kinorhyncha, Loricifera, and Tardigrada in 18S + 28S (50 OTU) dataset, they were supported in 18S (65 OTU) and 18S (63 OTU) with the maximum supporting values (Table 2). Monophyly for Onychophora was not tested due to the inclusion of a single representative of the phylum in all datasets.
Table 2

Summary of the results of each dataset

Clade

supporting value (ML/BI)

18S + 28S (50 OTU)

28S (50 OTU)

18S (50 OTU)

18S (65 OTU)

18S (63 OTU)

Ecdysozoa

99/1.00

71/0.99

94/1.00

89/1.00

88/1.00

Priapulida + Kinorhyncha

100/1.00

96/1.00

89/1.00

-/0.93

76/0.99

Nematoida + Loricifera + Panarthropoda

96/1.00

72/0.99

-/0.90

−/−

-/0.95

Nematoida

71/0.91

50/-

-/0.91

−/−

−/−

Loricifera + Panarthropoda

63/-

75/0.95

−/−

−/−

−/−

Panarthropoda

54/-

54/-

−/−

−/−

−/−

Priapulida

100/1.00

100/1.00

96/1.00

−/−

98/1.00

Kinorhyncha

−/−

−/−

−/−

100/1.00

100/1.00

Nematoda

99/1.00

85/1.00

91/1.00

77/1.00

61/0.99

Nematomorpha

100/1.00

100/1.00

100/1.00

96/1.00

95/1.00

Loricifera

−/−

−/−

−/−

100/1.00

100/1.00

Tardigrada

−/−

−/−

−/−

100/1.00

100/1.00

Arthropoda

89/1.00

82/1.00

−/−

−/−

-/0.98

Nematoda + Tardigrada + Arthropoda

−/−

−/−

−/−

-/0.98

-/0.91

Tardigrada + Arthropoda

−/−

−/−

-/0.90

−/−

−/−

Nematoda + Tardigrada

−/−

−/−

−/−

−/−

-/0.93

Summary of the results of analyses based on each dataset. Reconstructed clades with supporting values (maximum-likelihood bootstrap/Bayesian posterior probability) in each dataset are listed. Supporting values lower than 50 % (bootstrap values) or 0.90 (posterior probability) are considered as nonsignificant and indicated by dashes. Dark highlighted clades are supported only in Bayesian tree of short-sequence datasets, 18S (50 OTU), 18S (65 OTU), and 18S (63 OTU), thus these clades are not regarded as actual clades

Fig. 2

Maximum-likelihood tree of 18S + 28S (50 OTU) dataset. The tree is based on 18S + 28S (50 OTU) dataset. Numbers near nodes are the maximum-likelihood bootstrap (BS) and Bayesian posterior probability (PP) values, respectively; values lower than 50 % (BS) or 0.90 (PP) are indicated by dashes. The scale bar indicates branch length in substitutions per site

Within the Ecdysozoa, two basal clades were detected with high nodal support: Priapulida + Kinorhyncha (Scalidophora, excluding Loricifera; nodal support 100/1.00) and Nematoda + Nematomorpha + Loricifera + Tardigrada + Onychophora + Arthropoda (99/1.00). The latter basal clade in turn comprised the clades Nematoda + Nematomorpha clade (= Nematoida), and Loricifera + Tardigrada + Onychophora + Arthropoda clade (= Loricifera + Panarthropoda) in both the ML and BI trees. Support for the Nematoida clade was only moderate (71/0.90), and that for Loricifera + Panarthropoda clade was low (63/0.66). Support for the monophyly of Tardigrada + Onychophora + Arthropoda (= Panarthropoda) was also low (54/0.76). Tardigrada, Onychophora, and Arthropoda formed an unresolved trichotomy.

Phylogenetic evaluation of loricifera, scalidophora, and cycloneuralia

The clade we detected consisting of Loricifera, Nematoida, and Panarthropoda received high nodal support (96/1.00), but the phylogenetic position of Loricifera within this clade remains unclear, as support for the node grouping Loricifera with Panarthropoda was quite low (63/0.66). However, the scalidophoran phyla Priapulida and Kinorhyncha together comprised a clade with high nodal support (100/1.00) to the exclusion of Loricifera, which instead grouped in a highly supported (96/1.00) clade with Nematoida and Panarthropoda. Our results thus do not support both the ‘Scalidophora hypothesis,’ in which Loricifera comprises a clade with Kinorhyncha and Priapulida, and the ‘Loricifera + Nematomorpha hypothesis’. Our trees also indicated non-monophyly for Cycloneuralia, as Loricifera and Nematoida showed closer relationships to Panarthropoda than to other cycloneuralian phyla (Priapulida and Kinorhyncha).

Evaluation of synapomorphies for scalidophora and cycloneuralia

Morphological synapomorphies have previously been proposed that uniting the scalidophoran phyla (Loricifera, Priapulida and Kinorhyncha) and the cycloneuralian phyla (Scalidophora plus Nematoda and Nematomorpha). Putative synapomorphies [11] among Loricifera, Priapulida, and Kinorhyncha include (1) an introvert that has short, spinose scalids that are staggered in arrangement and triradiate in cross-section, and that has (2) inner and outer retractor muscles; (3) a compound filter of protonephridia consisting of two or more terminal cells; (4) basally thickened cuspidate spines; and (5) sensory organs (flosculi) with external cuticular micropapillae and a central pore. The most important synapomorphy proposed for cycloneuralians is the collar-shaped circumoral brain consisting of a ring neuropil [20, 21]. Our results failed to support the monophyly of either Scalidophora or Cycloneuralia, and the putative synapomorphies supporting these groups thus need to be reevaluated.

With regard to the monophyly of Loricifera + Nematoida + Panarthropoda that we detected, three possible topologies among these groups (Fig. 3) in turn suggest two possible evolutionary scenarios for the three scalidophoran phyla (Priapulida, Kinorhyncha, Loricifera). If Loricifera is the sister taxon of Panarthropoda (Fig. 3a) or of Nematoida (Fig. 3b), the most parsimonious scenario is that ‘scalidophoran’ characters arose independently in Loricifera and in the common ancestor of Priapulida + Kinorhyncha and represent convergent characters. Alternatively, if Loricifera is basal in the Loricifera + Nematoida + Panarthropoda clade (Fig. 3c), the most parsimonious interpretation is that the common ancestor of Ecdysozoa possessed ‘scalidophoran’ characters, which the common ancestor of Nematoida and Panarthropoda subsequently lost.
Fig. 3

Hypotheses of evolutionary transitions in scalidophoran and cycloneuralian morphological characters. These hypotheses are based on the three possible relationships within the Loricifera + Nematoida + Panarthropoda clade. Sc and Cy above branches indicate morphological characters of the ‘Scalidophora’ and ‘Cycloneuralia,’ respectively; ‘ + ’ and ‘–’ indicate the presence and absence of characters; asterisks indicate well-supported nodes

In all three topologies (Fig. 3), the most parsimonious evolutionary scenario for ‘cycloneuralian’ characters is that they originated once in the common ancestor of Ecdysozoa and were lost once in the common ancestor of Panarthropoda. In other words, the ‘cycloneuralian’ characters are plesiomorphic in ecdysozoans.

Conclusions

We reconstructed the phylogeny of ecdysozoan phyla using nearly complete 18S and 28S rRNA gene sequences, and our results suggested a new hypothesis for the phylogenetic position of Loricifera. These results did not support the previously proposed ‘Scalidophora’ or the ‘Loricifera + Nematomorpha’ clades, but detected a ‘Loricifera + Nematoida + Panarthropoda’ clade with rather high nodal support. Cycloneuralia emerged as paraphyletic, with high nodal support. Relationships among phyla in the ‘Loricifera + Nematoida + Panarthropoda’ clade were not well resolved, and phylogenetic analysis using transcriptomic or genomic data will be necessary to reconstruct the relationships within this clade, and to elucidate evolutionary transitions within Ecdysozoa.

Availability of supporting data

The data sets supporting the results of this article are included within the article and its additional files.

Declarations

Acknowledgements

We thank Dr. Taeko Kimura (Mie University) and the captain and crew of TR/V Seisui-maru (Mie University) for assistance in collecting; Dr. Hiroshi Kajihara (Hokkaido University) for making available molecular laboratory facilities; and Dr. Matthew H. Dick (Hokkaido University) for comments and editing. This study was supported in part by a grant to H. Y. from the International Research Hub Project for Climate Change and Coral Reef/Island Dynamics from the University of the Ryukyus, and a Grant for the Cultivation of Young Scientists to H. Y. from the Research Institute of Marine Invertebrates, Japan.

Authors’ Affiliations

(1)
Department of Chemistry, Biology & Marine Science, Faculty of Science, University of the Ryukyus
(2)
Department of Zoology, Division of Biological Science, Graduate School of Science, Kyoto University
(3)
Seto Marine Biological Laboratory, Field Science Education and Research Center, Kyoto University

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© Yamasaki. 2015

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