The complete mitochondrial genome sequence of Oncicola luehei (Acanthocephala: Archiacanthocephala) and its phylogenetic position within Syndermata

Parasitology International 61 (2012) 307–316

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The complete mitochondrial genome sequence of Oncicola luehei (Acanthocephala: Archiacanthocephala) and its phylogenetic position within Syndermata Mohiuddin Gazi a, Tahera Sultana b, Gi-Sik Min b, Yung Chul Park c, Martín García-Varela d, Steven A. Nadler e, Joong-Ki Park a,⁎ a

Graduate Program in Cell Biology and Genetics and Department of Parasitology, College of Medicine, Chungbuk National University, Cheongju 361-763, Republic of Korea Department of Biological Sciences, Inha University, Incheon 402-751, Republic of Korea Department of Forest Environment Protection, College of Forest and Environmental Science, Kangwon National University, Chuncheon 200-701, Republic of Korea d Departamento de Zoología, Instituto de Biología, Universidad Nacional Autónoma de México, Avenida Universidad 3000, Ciudad Universitaria, C.P. 04510, Distrito Federal, Mexico e Department of Nematology, University of California, Davis, CA 95616, USA b c

a r t i c l e

i n f o

Article history: Received 30 October 2011 Received in revised form 2 December 2011 Accepted 7 December 2011 Available online 17 December 2011 Keywords: Mitochondrial genome Molecular phylogeny Oncicola luehei Archiacanthocephala Acanthocephala Syndermata

a b s t r a c t In the present study, we determined the complete mitochondrial genome sequence of Oncicola luehei (14,281 bp), the first archiacanthocephalan representative and the second complete sequence from the phylum Acanthocephala. The complete genome contains 36 genes including 12 protein coding genes, 22 transfer RNA (tRNA) genes and 2 ribosomal RNA genes (rrnL and rrnS) as reported for other syndermatan species. All genes are encoded on the same strand. The overall nucleotide composition of O. luehei mtDNA is 37.7% T, 29.6% G, 22.5% A, and 10.2% C. The overall A + T content (60.2%) is much lower, compared to other syndermatan species reported so far, due to the high frequency (18.3%) of valine encoded by GTN in its protein-coding genes. Results from phylogenetic analyses of amino acid sequences for 10 protein-coding genes from 41 representatives of major metazoan groups including O. luehei supported monophyly of the phylum Acanthocephala and of the clade Syndermata (Acanthocephala+ Rotifera), and the paraphyly of the clade Eurotatoria (classes Bdelloidea + Monogononta from phylum Rotifera). Considering the position of the acanthocephalan species within Syndermata, it is inferred that obligatory parasitism characteristic of acanthocephalans was acquired after the common ancestor of acanthocephalans diverged from its sister group, Bdelloidea. Additional comparison of complete mtDNA sequences from unsampled acanthocephalan lineages, especially classes Polyacanthocephala and Eoacanthocephala, is required to test if mtDNA provides reliable information for the evolutionary relationships and pattern of life history diversification found in the syndermatan groups. © 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction The Acanthocephalan (thorny headed worms) is a phylum of endoparasites with a worldwide distribution and approximately 1200 described species. These parasites use vertebrates (fishes, amphibians, reptiles, birds, and mammals) as definitive hosts, arthropods (insects and crustaceans) as intermediate hosts, and in some cases, fishes, reptiles, and amphibians are used as paratenic (transport) hosts [1,2]. The phylum is currently represented by 4 classes, i.e., Archiacanthocephala,

Abbreviations: atp6 and atp8, genes for ATP synthase subunits 6 and 8; BI, Bayesian inference; bp, base pair; BP, bootstrap percentage; BPP, Bayesian posterior probability; cob, gene for cytochrome oxidase b; cox1–cox3, genes for cytochrome oxidase c subunit 1–3; dNTP, deoxyribonucleotide triphosphate; kb, kilo base; ML, maximum likelihood; mtDNA, mitochondrial DNA; nad1–6 and nad4L, genes for NADH dehydrogenase subunits 1–6 and 4L; NCR, non-coding region; nt, nucleotide; PCR, polymerase chain reaction; pp, posterior probability; rrnS and rrnL, genes for small and large mitochondrial ribosomal RNA subunits; tRNA, transfer RNA. ⁎ Corresponding author. E-mail address: [email protected] (J.-K. Park). 1383-5769/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.parint.2011.12.001

Palaeacanthocephala, Eoacanthocephala, and Polyacanthocephala [3–6]. This classification is based mainly on morphological features such as the location of the lacunar system (network of cavities in the epidermis), the persistence of ligament sacs in females, the number and shape of cement glands in males, the number and size of proboscis hooks, and host taxonomy and ecology [3,7–9]. Molecular and morphological phylogenetic hypotheses for acanthocephalans show substantial congruence and support the monophyly of these classes [6,10–12]. Rotifers, which are microscopic organisms that inhabit freshwater and marine habitats, are now established as close relatives of acanthocephalans. The phylum Rotifera is currently divided into 3 classes, Bdelloidea, Monogononta and Seisonidea. Bdelloid rotifers lack males and reproduce strictly by parthenogenesis, representing one of the few likely instances of ancient asexuality among animals [13]. Monogononts represent the largest group of rotifers, and are characterized by a heterogonic life history involving an alternation between generations of parthenogenesis and sporadic sexual reproduction. Seisonidea is a marine group represented by only 3 described species that reproduce by amphimixis and has symbiotic lifestyles including

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some association as ecto-commensals of marine leptostracan crustaceans of the genus Nebalia [14–16]. Currently the phyla Rotifera + Acanthocephala are recognized as a clade named Syndermata [17]; this clade has been supported by some morphological [18–20] and several molecular phylogenetic analyses (SSU rDNA [5,21]; SSU + LSU rDNAs and mitochondrial cox1 [6]). Although syndermatan monophyly has been continuously supported by morphological and molecular investigations, the internal phylogeny within Syndermata (e.g., Eurotatoria monophyly/paraphyly, relationships among major syndermatan classes including those of acanthocephalans) is still matters of debate [6,15,22–24] but with certain recent combined analysis of molecular and morphological data supporting the inclusion of Acanthocephala as a rotiferan subgroup [25]. Full resolution of this question requires analysis of additional molecular data, including mtDNA genomes. With a very few exceptions, metazoan mtDNA are typically circular DNA molecules, ranging in size from 14 kb to 42 kb, encoding 37 genes that consist of 13 protein coding genes (but atp8 is lacking in most flatworm and nematode species), 2 ribosomal RNA genes (rrnS and rrnL), and 22 transfer RNA (tRNA) genes. Although there is a growing body of recent reports for extensive gene rearrangement even among closely related species, the gene content of mitochondrial genomes is generally relatively conserved across most metazoans. Based on the view that gene order is rather stable and that gene rearrangement (i.e., gene order changes) resulting from convergent evolution appears relatively uncommon (in certain taxonomic groups), comparison of mitochondrial gene order pattern has been proposed as a reliable tool for resolving deep node phylogenetic relationships [26–28]. More than 2400 complete mitochondrial genome (mt genome) sequences are available from different metazoan groups (www.ncbi.nlm.nih.gov./genomes/ OGANELLES/mztax_short.html). Despite this wealth of mitochondrial genome information from diverse metazoans, there are some phyla for which mtDNA is still underrepresented or unavailable to date [29,30]. This lack of genome information for as yet unsampled animal groups has rendered development of phylogenetic hypotheses based on mitochondrial sequences far less comprehensive. Mitochondrial genomes from underrepresented animal groups can provide a wealth of genetic information for understanding both animal and mitochondrial genome evolution. The Acanthocephala is one phylum represented by only a single mitochondrial genome. The mitochondrial genome sequence of Leptorhynchoides thecatus is the only species reported for Acanthocephala [31]. Within Syndermata there have been two reports for rotifers (a monogonont Brachionus plicatilis [32] and a bdelloid Rotaria rotatoria [24]), thus there are three complete genomes available representing Syndermata. Considering the remarkable diversity in their ecology (parasitic life cycles versus free-living, sexual versus asexual reproduction) and number of described species (approximately 1200 acanthocephalan [8] and 2000 rotifer species [33]), the relative lack of genome information necessitates further mitogenomic investigation. In the present study, we report the complete mitochondrial genome

sequence of Oncicola luehei, the first archiacanthocephalan species for which the entire genome sequence has been determined, and the second representative of the phylum Acanthocephala. O. luehei is an endoparasite of the intestine of small mammals, such as coatis and opossums, that are distributed in regions of North, Central and South America. In molecular phylogenies based on nuclear ribosomal DNA and the mitochondrial cox1 gene, Oncicola spp. are members of a clade with other Archiacanthocephala; this class is the sister group to a clade consisting of Palaeacanthocephala, Polyacanthocephala and Eoacanthocephala [6]. Therefore, the Oncicola mtDNA sequence, which is considered representative for the archiacanthocephalan lineage, was used along with 40 published metazoan species to assess phylogenetic relationships within the syndermatan clade and among major metazoan phyla. 2. Materials and methods 2.1. Sampling and molecular techniques Specimens of the species O. luehei Travassos, 1917 were isolated from the intestine of its final host animal Didelphis virginiana (opossum) captured from Veracruz, México. After isolation from the host intestine, the specimens were thoroughly washed, kept in 70% ethanol and stored at −20 °C until genomic DNA extraction. Total genomic DNA was extracted using the Masterpure DNA extraction kit (Epicentre Biotechnologies Co.) according to the manufacturer's protocol. Initially, three partial gene fragments for cox1, cob, and 16S (rrnL) were amplified using universal primer sets (Cox1F/Cox1R) or primer sets (Syn-CytbF/ Syn-CytbR, and Syn-16S-F/Syn-16S-R) designed from conserved regions of the published acanthocephalan and two rotifer species (Table 1). PCR reactions for these partial fragments were carried out in a 50 μl reaction volume consisting of 10 units of Taq polymerase (Roche), 2.5 mM dNTP mixture, 2.5 mM MgCl2, and 20 pmol of each primer with the following amplification conditions: 1 cycle of the initial denaturation at 95 °C for 1 min, followed by 35 cycles of denaturation at 95 °C for 1 min, primer annealing at 45 °C for 30 s and elongation at 72 °C for 1 min, and the final extension at 72 °C for 10 min. The nucleotide sequences obtained from these partial gene fragments were then used to design O. luehei specific primers for long PCR amplification (Table 1). These overlapping long PCR products (approximately 1.8 kb, 5.5 kb, and 10 kb in size, respectively; Fig. 1), covering the entire mitochondrial genome, were amplified using the long PCR primer sets and the Expand Long Template PCR System (Roche) with the following amplification conditions: 1 cycle of initial denaturation (2 min at 94 °C), 30 cycles of denaturation–primer annealing–elongation (15 s at 94 °C, 30 s at 50–60 °C, and 10 min at 68 °C), and 1 cycle of the final extension (10 min at 68 °C). The amplified long PCR products were gel-isolated, and extracted using the TOPO Gel Purification reagents supplied with the TOPO XL cloning kit (Invitrogen Co.). After gel purification, each of the long PCR products was ligated using the TOPO XL cloning kit and then transformed into competent Escherichia coli. Cycle sequencing

Table 1 PCR primers used in the study of Oncicola luehei. Primer

DNA sequence (5′-3′)

Estimated size of PCR products

Primer source

LCO1490 HCO2198 Syn-16S-F Syn-16S-R Syn-Cytb-F Syn-Cytb-R Onci-Cox1-F Onci-16S-R Onci-Cob-F Onci-Cox1-R Onci-16S-F Onci-CO2-R

GGTCAACAAATCATAAAGATATTGG TAAACTTCAGGGTGACCAAAAAATCA GACYGTRCTWAGGTAGCRTRATC AWRDRATRATCCAACATCGAGGTA CTTTTTTAGGGTATGTTTTACC TCWACARYAYAWCCTCC GTGGGTCTATAGAAGTGGAGCATCTGTGG CTAATGATTACGCTACCTTAGCACAGTC GATGGCTTTGGCAGTGACTATTGTTG TACCAAACCCTCCTATTATCACCGGTATTGC GACTGTGCTAAGGTAGCGTAATCATTAG TACTCCCAATACCACTGATGGC

~ 680 bp

[63]

~ 600 bp

This study

~ 600 bp

This study

~ 1.8 kb

This study

~ 5.5 kb

This study

~ 10 kb

This study

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309

Onci-Cox1-F/Onci-16S-R (~1.8 kb) Onci-Cob-F/Onci-Cox1-R (~5.5 kb)

Oncicola luehei mtDNA 14,281 bp

Onci-16S-F/Onci-CO2-R (~10 kb)

Fig. 1. Circular representation of the complete mitochondrial genome for Oncicola luehei. All genes are encoded in the same direction and 22 tRNA genes are designated by a single-letter abbreviation. The two leucine and two serine tRNA genes are labeled, according to their anticodon sequence, as L1 (trnL-uag), L2 (trnL-uaa), S1 (trnS-ucu), and S2 (trnS-uga), respectively.

reactions for each of the long PCR products were performed in both directions by the ‘primer walking’ method using a Big Dye Terminator Cycle-Sequencing Kit (Applied Biosystems). A complete strand of the entire mtDNA sequence was then assembled by double-checking the sequences of overlapping regions of the long PCR fragments and using partial sequences obtained from separate gene fragments.

groups, the ClustalX multiple alignment program cannot guarantee uniform results due to length variation, sequence divergence within certain regions, and rate variation among lineages. To avoid problems associated with errors inferring positional homology we selected the most conserved sequence regions for phylogenetic analysis from each of 10 aligned genes using the web-based Gblocks program with default options [38].

2.2. Gene annotation and phylogenetic analyses 2.4. Phylogenetic analyses Nucleotide sequences were initially analyzed using MEGA version 4.1 [34] and Geneious version 5.1 (computer software for nucleotide sequence analysis; [Biomatters Co.]). Twelve mitochondrial proteincoding genes and two ribosomal RNA genes were identified by finding gene boundaries based on comparison with other syndermatan mitochondrial DNA sequences. The 22 tRNA genes were identified using the tRNA scan-SE program [35], DOGMA [36] and by manually inspecting potential secondary structures and anticodon sequences. Most of the tRNAs were identified by comparing specific anticodon sequences and secondary structures with those found in L. thecatus, the published mitochondrial genome sequence from the Acanthocephala. 2.3. Alignment For the phylogenetic analysis of 41 metazoan mtDNA genomes, 10 of 12 protein-coding genes were used. The nad4L and atp6 genes were excluded due to their extreme variability in gene length across metazoans. The metazoan groups represented in the analysis included 15 lophotrochozoans, 10 platyzoans, 8 ecdysozoans, 5 deuterostomes, and 1 acoel species. The mitochondrial genome sequence data from two cnidarians (Acropora tenuis and Aurelia aurita) were included in the analysis as outgroups. The complete list of species, taxonomy, and GenBank accession numbers is given in Table 2. Nucleotide sequences for each of 10 protein-coding genes of O. luehei were first translated into amino acids using the invertebrate mitochondrial genetic code. The resulting amino acid sequences were then aligned for each gene using ClustalX with default options [37]. For this wide taxonomic range of metazoan

Phylogenetic analyses for the concatenated dataset of conserved amino acid sequences were performed using two different treebuilding methods; maximum likelihood (ML) and Bayesian Inference (BI). For ML analysis we estimated the best fit model for our amino acid sequence dataset by using Akaike Information Criterion (AIC) and ProTest version 2.0 [39]. We performed ML analysis in Treefinder October version [40] using the MtART + I + G matrix [41], the best-fit model for amino acid substitution selected from ProTest implementation. Bootstrap ML analysis was performed using the nonparametric bootstrap with 500 replicate datasets using Treefinder for ML inference. Bayesian inference for the amino acid dataset was performed using MrBayes version 3.1.2 [42] with the MtRev matrix of amino acid substitution. Bayesian analysis was run for 10 6 generations, and sampled every 1000 generations with four Markov Chain Monte Carlo (MCMC) chains using Bioportal, a web portal for phylogenomic analysis (www. bioportal.uio.no). Bayesian posterior probability (BPP) values were determined after discarding the initial 200 saved trees (the first 2 × 105 generations) as burn-in. 3. Results and discussion 3.1. Gene content and organization The complete mitochondrial genome of O. luehei is a circular, double-stranded DNA molecule (14,281 bp in size), and it is considerably larger than L. thecatus;13,888 bp [31]. O. luehei contains 36 genes

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Table 2 The species, taxonomy, and GenBank accession numbers for metazoan species used in phylogenetic analyses in this study. Species Lophotrochozoa Nautilus macromphalus Katharina tunicata Clymenella torquata Loxocorone allax Loxosomella aloxiata Lumbricus terrestris Urechis caupo Sipunculus nudus Terebratalia transversa Terebratulina retusa Phoronis psammophila Bugula neritina Flustrellidra hispida Spadella cephaloptera Paraspadella gotoi Platyzoa Oncicola luehei Leptorhynchoides thecatus Rotaria rotatoria Brachionus plicatilis Benedenia seriolae Fasciola hepatica Microcotyle sebastis Gyrodactylus salaris Gyrodactylus thymalli Hymenolepis diminuta Ecdysozoa Limulus polyphemus Lithobius forficatus Drosophila melanogaster Panulirus japonicus Epiperipatus biolleyi Priapulus caudatus Trichinella spiralis Caenorhabditis elegans Deuterostomia Branchiostoma lanceolatum Strongylocentrotus purpuratus Acipenser dabryanus Xenopus laevis Balanoglossus carnosus Acoelomorpha Symsagittifera roscoffensis Radiata (outgroups) Acropora tenuis Aurelia aurita

Taxonomic group

GenBank accession no.

Mollusca Mollusca Annelida Entoprocta Entoprocta Annelida Echiura Sipunculida Brachiopoda Brachiopoda Phoronida Bryozoa Bryozoa Chaetognatha Chaetognatha

NC_007980 NC_001636 NC_006321 NC_010431 NC_010432 NC_001673 NC_006379 NC_011826 NC_003086 NC_000941 AY_368231 NC_010197 NC_008192 NC_006386 NC_006083

Acanthocephala, Archiacanthocephala Acanthocephala, Palaeacanthocephala Rotifera, Bdelloidea Rotifera, Monogononta

JN710452 NC_006892

Platyhelminthes Platyhelminthes Platyhelminthes Platyhelminthes Platyhelminthes Platyhelminthes

NC_013568 NC_010472 Part-I, NC_010484 Part-II NC_014291 NC_002546 NC_009055 NC_008815 NC_009682 NC_002767

Arthropoda Arthropoda Arthropoda Arthropoda Onychophora Priapulida Nematoda Nematoda

NC_003057 NC_002629 NC_001709 NC_004251 NC_009082 NC_008557 NC_002681 NC_001328

Table 3 The mitochondrial genome organization of Oncicola luehei. Gene

cox1 trnG trnQ trnY lrRNA trnL1 nad6 trnD trnS2 atp6 nad3 trnW trnK NCR1 trnV trnE trnT nad4L nad4 trnH nad5 trnL2 trnP cob nad1 trnI trnS1 NCR2 trnM srRNA trnF cox2 trnC cox3 trnA trnR trnN nad2

Position

Size

Codons

Start

Finish

No. of nt No. of aa Initiation Termination

1 1535 1589 1658 1713 2663 2717 3151 3219 3275 3861 4195 4391 4452 4704 4814 4949 5003 5260 6520 6575 8217 8277 8331 9439 10419 10508 10577 10908 11047 11600 11653 12326 12380 13149 13198 13322 13378

1534 1588 1650 1712 2662 2719 3150 3203 3274 3859 4196 4255 4451 4703 4762 4874 5001 5251 6519 6574 8216 8276 8330 9430 10350 10478 10576 10907 10966 11599 11652 12327 12379 13148 13203 13255 13377 14280

1534 54 62 55 950 57 434 53 56 585 336 61 61 252 59 61 53 249 1260 55 1642 60 54 1100 912 60 69 331 59 553 53 675 54 769 55 58 56 903

511

GTG

T

144

ATT

TA

195 112

GTG TTG

TAG TAA

83 420

GTG GTA

TAG TAG

547

GTG

T

366 304

ATG GTG

TA TAA

225

GTG

TAA

256

GTG

T

301

ATT

TAA

Intergenic sequence 0 0 7 0 0 −3 0 15 0 1 −2 135 0 0 51 74 1 8 0 0 0 0 0 8 68 29 0 0 80 0 0 −2 0 0 −6 66 0 1

*Stop codons were not included, nt = nucleotide, aa = amino acid. Cephalochordata Echinodermata Chordata Chordata Hemichordata

NC_001912 NC_001453 NC_005451 NC_001573 NC_001887

Acoela

NC_014578

Cnidaria Cnidaria

NC_003522 NC_008446

including 12 protein coding genes (lacking atp8), 22 transfer RNA (tRNA) genes and 2 ribosomal RNA genes (lrRNA and srRNA), with all genes encoded by the same strand (Fig. 1). The absence of the atp8 gene has also been reported in some other species of syndermata and many nematode and flatworm species [24,31,32,43,44]. A list of gene order, gene length, and intergenic spacer regions is given in Table 3. The nucleotide composition of the entire O. luehei mtDNA sequence is 37.7% T, 29.6% G, 22.5% A, and 10.2% C (see Table 4 for details of nucleotide composition). The overall A + T content (60.2%) is much lower, compared to those found in some other syndermatan species reported so far (an acanthocephalan L. thecatus [71.3%], a bdelloid rotifer R. rotatoria [73.1%], but rather similar to the monogonont B. plicatilis [63.9% and 62.9% for mtDNA-I and mtDNA-II, respectively]). The difference in the A + T content among these groups is related to some extent to lineage-specific non-random codon usage of nucleotides in protein-coding genes, and this bias will be discussed in more detail in the following section.

3.2. Protein coding genes and codon usage Like all characterized species of Syndermata, a total of 12 proteincoding genes (atp8 is missing) were identified in the O. luehei mtDNA: The nad5 (1642 bp) and cox1 (1534 bp) are relatively large in size, whereas nad4L (249 bp) is among the smallest. Among 12 proteincoding genes, seven genes (cox1, cox2, cox3, nad1, nad4L, nad5, and atp6) were inferred to use GTG as start codon: in contrast, the remaining five are initiated with ATG (cob), ATT (nad2, nad6), TTG (nad3), and GTA (nad4), respectively. Out of 12 protein-coding genes, seven were inferred to terminate with the complete stop codon (TAA or Table 4 Nucleotide composition of the mitochondrial genome of Oncicola luehei. Nucleotide

Length (bp)

A (%)

C (%)

T (%)

G (%)

A+T (%)

G+C (%)

Entire sequence Protein coding sequence Codon position* 1st 2nd 3rd Ribosomal RNA genes sequence Transfer RNA genes sequence Non-coding regions (NCRs) NCR 1 NCR 2

14,281 10,371

22.5 20.1

10.2 10.1

37.7 38.7

29.6 31.1

60.2 58.8

39.8 41.2

3457 3457 3457 1503 1265 1126 252 331

24.1 15.1 21.2 29.1 28.2 30.4 30.2 30.5

9.4 12.9 7.9 10.4 10.4 9.3 12.3 6.9

30.0 51.1 35.1 35.2 37.2 32.2 27.0 36.3

36.5 20.9 35.8 25.2 24.0 28.1 30.6 26.3

54.1 66.2 56.3 64.4 65.5 62.6 57.1 66.8

45.9 33.8 43.7 35.6 34.5 37.4 42.9 33.2

*Termination codons were excluded.

M. Gazi et al. / Parasitology International 61 (2012) 307–316

Frequency (%)

TAG): TAA for nad1, nad2, nad3 and cox2 and TAG for nad4, nad4L and atp6, respectively. The remaining five genes are terminated with an incomplete stop codon (T or TA): T for cox1, cox3 and nad5, and TA for nad6 and cob, respectively. The truncated incomplete stop codon (T) has also been found in cox1, nad1, nad4, nad5 and cob genes for L. thecatus. Termination codon TAA used for nad3 and cox2 is inferred to overlap with trnW and trnC by two nucleotides. The start/termination codon usage among syndermatan members differs greatly depending on genes and/or species, and there is no noticeable synapomorphic patterns (e.g., sharing the same codon usage between species pairs from the same taxonomic groups, i.e., within rotifer and/or acanthocephalan species). Details of initiation and termination codons of 12 protein-coding genes are shown in Table 3. The O. luehei mtDNA also shows nucleotide compositional bias toward T and A (60.2% A + T content), but the degree of bias is less prominent, compared to those found in some other published syndermatan species (L. thecatus [71.3%], R. rotatoria [73.1%]). The proteincoding genes of O. luehei mtDNA are composed of amino acids that are encoded by T-rich codons (more than 2 Ts in a triplet), as previously documented for some other invertebrates including the published syndermatan species (nematodes [45], mollusk [46], dipteran insect [47], acanthocephalan [31], rotifer [24]). In R. rotatoria mtDNA proteincoding genes, leucine2 (Leu2) and phenylalanine (Phe) are among the most frequently found amino acids with the frequency of TTA (11.0%) and TTG (2.4%) for Leu2 and TTT (9.5%) and TTC (1.5%) for Phe, respectively. In L. thecatus mtDNA, the leucine2 (encoded by TTR; 12.5%), valine (encoded by GTN; 10.1%), and phenylalanine (encoded by TTY; 9.9%) occur most abundantly, accounting for 32.5% of total amino acid components (Fig. 2). Like other syndermatan mtDNAs, leucine2 (encoded by TTR) is also very abundant in O. luehei mtDNA, accounting for 10.6% of the total amino acid composition. However, a more notable feature in the amino acid composition of O. luehei mtDNA is that valine (encoded by GTN) is most abundant (18.3% of total amino acid composition; Table 5), showing an obvious contrast with other syndermatan species in which valine is the second or third most abundant amino acid (accounting for 8.3% [B. plicatilis] to 10.1% [L. thecatus] of their total amino acid composition; Fig. 2). This notably higher occurrence of GTN encoding valine is responsible for the relatively higher percentage of G both in the protein-coding gene sequence (31.1%) and the entire mtDNA genome sequence (29.6%; Table 4), compared to those of other syndermatan species, which range from 16.2% G (B. plicatilis mtDNA-I) to 17.4% G (L. thecatus). Unequal use of synonymous codons

311

Table 5 Genetic code and codon usage for the 12 mitochondrial protein coding genes of Oncicola luehei. Codon

AA

NO.

%

Codon

AA

NO.

%

TTT TTC TTA TTG CTT CTC CTA CTG ATT ATC ATA ATG GTT GTC GTA GTG TCT TCC TCA TCG CCT CCC CCA CCG ACT ACC ACA ACG GCT GCC GCA GCG

Phe Phe Leu Leu Leu Leu Leu Leu Ile Ile Met Met Val Val Val Val Ser Ser Ser Ser Pro Pro Pro Pro Thr Thr Thr Thr Ala Ala Ala Ala

187 21 164 198 41 8 39 40 118 27 116 175 180 25 166 260 108 9 28 16 40 13 13 12 49 14 12 6 44 28 30 25

5.40 0.61 4.74 5.72 1.19 0.23 1.13 1.16 3.40 0.78 3.38 5.06 5.20 0.73 4.80 7.51 3.12 0.26 0.81 0.46 1.16 0.38 0.38 0.35 1.42 0.40 0.35 0.18 1.27 0.80 0.87 0.72

TAT TAC TAA TAG CAT CAC CAA CAG AAT AAC AAA AAG GAT GAC GAA GAG TGT TGC TGA TGG CGT CGC CGA CGG AGT AGC AGA AGG GGT GGC GGA GGG

Tyr Tyr * * His His Gln Gln Asn Asn Lys Lys Asp Asp Glu Glu Cys Cys Trp Trp Arg Arg Arg Arg Ser Ser Ser Ser Gly Gly Gly Gly

130 31 4 3 45 10 8 18 44 9 30 40 48 20 18 70 29 7 26 83 14 2 7 15 46 15 31 102 91 33 44 179

3.76 0.90 0.12 0.09 1.30 0.29 0.23 0.52 1.28 0.26 0.87 1.16 1.39 0.58 0.52 2.02 0.84 0.20 0.75 2.40 0.40 0.05 0.20 0.44 1.33 0.44 0.90 2.95 2.63 0.96 1.27 5.17

*Stop (termination) codon.

in two-fold and/or four-fold codon families, another factor that causes nucleotide compositional bias, is also prominent in the O. luehei mtDNA (Table 5), as reported in many other metazoan mitochondrial protein-coding genes (echinoderm [48], nematode [49], rotifer [24]). Differential preference of thymine (T) or adenine (A), and avoiding cytosine (C) at the third codon positions is apparent: among four synonymous codons that encode valine, three codons (GTG [7.51%], GTT [5.20%], and GTA [4.80%]) are preferentially used, whereas GTC occupies only 0.73% of total amino acid composition. For phenylalanine, the frequency of TTT is very high (5.40%), but the relative frequency of TTC is drastically decreased (0.61%). In other four-fold codon families, such as leucine1 and serine2, there is a strong avoidance of cytosine (C) at the third codon position (Leu1: CTT [1.19%], CTC [0.23%], CTA [1.13%], CTG [1.16%]; Ser2: TCT [3.12%], TCC [0.26%], TCA [0.81%], TCG [0.46%]).

3.3. Ribosomal RNA and transfer RNA genes

Amino acids

Fig. 2. Comparison of amino acid composition of 12 protein-coding genes for four syndermatan species.

The two ribosomal RNA genes (rrnS and rrnL) of O. luehei mtDNA were identified by sequence comparison with those of L. thecatus, and the entire flanking regions between the gene boundaries of their respective adjoining genes were designated as rrnS and rrnL. The rrnL is 950 bp in length and found between trnY and trnL1, the same position as in L. thecatus. The rrnS is 553 bp in length, and positioned between trnM and trnF. Twenty two tRNA genes, ranging in size from 53 bp to 69 bp, are predicted to fold into cloverleaf-like secondary structure, similar to those found in the acanthocephalan L. thecatus [31] in that the trnS1 and trnS2 lack a dihydrouridine (DHU) arm, whereas the remaining 20 tRNAs lack a pseudouridine (TΨC) arm, features were common to L. thecatus mtDNA (see Fig. 3 for more details of the secondary structures of tRNAs).

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O. luehei mtDNA and two cnidarian outgroups. Both maximum likelihood (ML) and Bayesian inference (BI) were used for phylogeny estimation. Out of 12 protein-coding genes universally found in metazoan mtDNA (atp8 is lacking in most of platyhelminthes, nematodes [except for Trichinella spiralis], and syndermatan species), nad4L and atp6 were excluded from the analyses because there were no conserved sequence blocks selected following Gblocks implementation; these two genes are the least conserved both in their sequence and length across the examined metazoan species. The resulting trees from both analytic methods recovered the split comprising protostomes versus deuterostomes, each forming a monophyletic clade with high nodal support (95% BP, 1.00 BPP for the Deuterostomia clade and 100% BP, 1.00 BPP for Proteostomia clade in ML and BI, respectively; Figs. 4 and 5). The phylogenetic tree topology from ML analysis differed from the Bayesian tree. In some respects the most notable difference is that Acoela (represented by Symsagittifera roscoffensis) was unresolved in ML analysis, whereas in Bayesian analysis it was placed sister to platyhelminth

3.4. Non coding region The total length of the non-coding region (NCR) for O. luehei is 1126 bp that is composed of 16 intergenic spacer sequences, ranging from 1 to 331 bp. Among these, two regions (NCR1 and NCR2) are most prominent in their length. The NCR1, located between trnK and trnV, is 252 bp and NCR2, located between trnS1 and trnM, is 331 bp. The third largest intergenic spacer region (135 bp) was also found between trnW and trnK. The A + T contents of NCR1 and NCR2 for O. luehei are 57.1% and 66.8%, respectively (Table 4). 3.5. Systematic position of Syndermata (Acanthocephala + Rotifera) within major metazoan groups To evaluate phylogenetic relationships among major syndermatan groups, we reconstructed mitochondrial gene phylogeny for 10 protein-coding genes from 41 metazoan representatives including Ala n in e (A)

U A C C U U A

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Fig. 3. The predicted secondary structures of 22 tRNAs of the Oncicola luehei mtDNA determined in this study.

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Hymenolepis diminuta

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Fasciola hepatica Benedenia seriolae 100 Gyrodactylus salaris Gyrodactylus thymalli

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Phoronida Bryozoa Annelida Sipunculida Echiura Annelida

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Leptorhynchoides thecatus 77 Oncicola luehei 96 70 Rotaria rotatoria Brachionus plicatilis 100 Spadella cephaloptera Paraspadella gotoi Loxosomella aloxiata 100 Loxocorone allax Phoronis psammophila 100 100 Flustrellidra hispida Bugula neritina Lumbricus terrestris 97 Sipunculus nudus 100 100 Urechis caupo Clymenella torquata 94 Terebratalia transversa 100 Terebratulina retusa Nautilus macromphalus Katharina tunicata 85 Lithobius forficatus 89 Limulus polyphemus Epiperiputus biolleyi 100 Priapulus caudatus Panulirus japonicus 96 Drosophila melanogaster Acipenser dabryanus 100 Xenopus laevis 75 Branchiostoma lanceolatum 95 Balanoglossus carnosus Strongylocentrotus purpuratus Symsagittifera roscoffensis Acropora tenuis 100 Aurelia aurita 100

S y n d e rm a ta

Microcotyle sebastis 82

P la ty z o a

100

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Fig. 4. A single maximum likelihood tree inferred using Treefinder program for amino acid sequence dataset of 10 protein-coding genes for 39 metazoan mitochondrial genomes (excluding two nematode sequences). The bootstrap values, when 70% or greater, are indicated near internal nodes or using vertical brackets to the right of taxon names.

taxa, with high nodal support (0.96 BPP; Fig. 5). A recent phylogenetic analysis including the mitochondrial genome sequence of S. roscoffensis suggested that along with Nemertodermatida, Acoela represents one of the earliest bilaterian lineages [50]. Nemertodermatida was not included in this analysis because only the partial genome of Nemertoderma westbladi (5.2 kb) is available [51]. Another difference between the ML and Bayesian methods was found in the level of support for relationships of the chaetognath species. Chaetognaths were sister to the Platyzoan clade in both ML and BI analyses, but with strong support in BI (1.00 BPP) versus weak support by ML (70% BP). Other topological differences between the ML and Bayesian results generally were found for clades having lower BP in ML analysis. Additional taxon sampling of Acoela and Nemertodermata may be important for increasing the reliability of inferred relationships in such cases. Within the Protostomia clade, monophyly of Ecdysozoa was robustly supported with high nodal support (100% BP and 1.00 BPP in ML and BI analyses, respectively), but lophotrochozoan monophyly was not supported in either analysis. Lophotrochozoa excluding Platyzoa + Chaetognatha was sister to ecdysozoans, but this relationship received very low supporting values (59% BP and 0.78 BPP in ML and BI, respectively) and is inconsistent with some earlier molecular analyses (EST genome sequences [52], mitochondrial genome sequences [24]). Within the Lophotrochozoan clade, platyzoan monophyly

(including Platyhelminthes, Acanthocephala and Rotifera in this study) was consistently supported (82% BP in ML and 1.00 BPP in BI, respectively). However, monophyletic grouping of platyzoan members was collapsed when nematode species (Caenorhabditis elegans and T. spiralis) were included in the analyses (Supplementary data 1 and 2): The nematodes were sister to platyhelminths in the ML tree (but received ≤50% BP), or sister to platyhelminthes + Acoela in the Bayesian analysis (1.00 BPP). Nematode sequences have long been regarded as one of the most fast-evolving among animal groups and thus they show a propensity to cluster with other long-branching taxa, platyhelminthes in the present study, causing artificial grouping due to longbranch attraction (LBA) as documented previously [31,50,53–55]. To avoid this artificial factor in the tree reconstruction, nematode species were excluded from subsequent analyses. The platyzoa was then recovered as monophyletic in the resulting phylogeny by both maximum likelihood and Bayesian methods (Figs. 4 and 5). In addition, the ML tree depicted monophyly for both Platyhelminthes and Syndermata, each group receiving very high nodal support (100% and 96% BP, respectively). Acoela was sister to the platyhelminth species with 0.96 BPP, and this clade was sister to Syndermata with 1.00 BPP in Bayesian phylogeny. The sister-group relationship between Acoela and Platyhelminthes recovered in the Bayesian analyses of amino acid sequences (Fig. 5 and Supplementary data 2) may be due to LBA artifacts.

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1.00

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Priapulus caudatus Epiperipatus biolleyi Xenopus laevis Acipenser dabryanus Branchiostoma lanceolatum Strongylocentrotus purpuratus Balanoglossus carnosus Acropora tenuis Aurelia aurita

Bryozoa Brachiopoda

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Spadella cephaloptera Paraspadella gotoi Loxosomella aloxiata Loxocorone allax Phoronis psammophila Flustrellidra hispida Bugula neritina Terebratalia transversa Terebratulina retusa Nautilus macromphalus Katharina tunicata Lumbricus terrestris 1.00 Sipunculus nudus 1.00 Urechis caupo Clymenella torquata Lithobius forficatus Limulus polyphemus Panulirus japonicus Drosophila melanogaster

Protostom ia

1.00

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Lophotrochozoa Acoelomorpha

Gyrodactylus salaris Gyrodactylus thymalli 1.00 Benedenia seriolae 0.86 Hymenolepis diminuta 1.00 Fasciola hepatica Microcotyle sebastis Symsagittifera roscoffensis Leptorhynchoides thecatus 1.00 Oncicola luehei Rotaria rotatoria Brachionus plicatilis 1.00

1.00

0.91

Cnidarian outgroups

0.1

Fig. 5. Bayesian phylogenetic tree for amino acid sequence dataset of 10 protein-coding genes for 39 metazoan mitochondrial genomes (excluding two nematode sequences). The numerical values near internal nodes or at vertical brackets to the right of taxon names represent Bayesian posterior probability values after discarding the initial 200 trees (the first 2 × 105 generations) as burn-in.

Comparative analyses of multiple nuclear protein-coding genes indicates that the grouping of Acoela and Platyhelminthes is subject to LBA, which is overcome when a substitution model that is less sensitive to LBA artifacts is employed [56]. Monophyly of syndermatan species was recovered in all phylogenetic analyses with very high supporting values, regardless of including or excluding nematode species and for both inference methods (96% BP in ML and 1.00 BPP in BI [without nematodes] and 100% BP in ML and 1.00 BPP in BI [with nematodes]). Within the Syndermatan clade, the two acanthocephalans O. luehei (Archiacanthocephala) and L. thecatus (Palaeacanthocephala) were sister taxa with R. rotatoria (a bdelloid rotifer), and B. plicatilis (a monogonont rotifer), each forming successive sister groups to the acanthocephalans. Acanthocephalan monophyly was very robustly supported (100% BP in ML and 1.00 BPP in BI, respectively) in all phylogenetic analyses. Interestingly, eurotatorian paraphyly was consistently recovered in all analyses of this study, as asserted by some previous molecular analyses (nuclear 18S rDNA +

mitochondrial 16S rDNA [22]; EST genome [23]; mitochondrial genome [24]). As more mitochondrial genome information has become available from as yet unstudied groups, there is more evidence of extensive gene rearrangement even among some closely related metazoan groups (e.g., enoplean nematodes [57], mollusks [58,59], tunicates [60], crustacean arthopodes [61]). Such exceptions challenge the longheld view that gene order data is particularly useful for inferring the relationships among ancient lineages for metazoa. In contrast, the gene content of mitochondrial genome remains relatively unchanged in most metazoans. Based on the view that gene order is rather stable, comparison of mitochondrial gene order pattern has often been used as a supplementary tool for assessing phylogenetic affinity in metazoans [26–28,44,62]. Comparison of the gene order for O. luehei with L. thecatus reveals that these two acanthocephalans have almost identical gene arrangements, the only difference being the two reciprocal translocations of tRNAs between trnK and trnV and between trnS1 and

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N nad2 c o x1 G Q Y rrnL L1 nad6 D S 2 atp6 nad3 W K V E T nad4Lnad4 H nad5 L2 P c o b nad1 I S 1 M rrnS F c o x2 C c o x3 A R

Oncicola lue he i (Archiacanthocep hala: Acanthocep hala) N nad2 c o x1 G Q Y rrnL L1 nad6 D atp6 nad3 W V K E T nad4Lnad4 H nad5 L2 P c o b nad1 I M S 1 rrnS F c o x2 C c o x3 A R

Le ptorhynchoide s the catus (P a la e a ca nthoce pha la : Aca nthoce pha la ) N nad2 c o x1 G L2 W I Y rrnL L1 nad6 A Q D atp6 E nad1 c o b nad4L nad4 H nad5 F M rrnS c o x2 K c o x3 nad3 T P R S 2 V S 1

R otaria rotatoria (Bde lloide a : Rotife ra ) E S 1 H rrnS T V G nad2 W nad1 Q atp6 D rrnL Y P M c o b L1

Brachionus plicatilis mtDNA-I (Monogononta : Rotife ra )

F S 2 c o x1 R

I nad6 C nad4Lnad4 nad5 N c o x2 c o x3 A nad3 K L2

Brachionus plicatilis mtDNA-II

Fig. 6. Linearized comparison of the mitochondrial gene arrangement of four syndermatan species. Gene and genome size are not to scale. All genes are transcribed in the same direction (from left to right). The tRNAs are labeled by single-letter abbreviations. Three gene clusters shared between a bdelloidean rotifer and two acanthocephalan species are represented by shadowed areas.

trnM that are directly adjoined to each other, respectively (Fig. 6). In comparing the gene arrangement among available syndermatan species, three gene clusters (trnN–nad2–cox1–trnG, trnY–rrnL–trnL1–nad6, and nad4L–nad4–trnH–nad5; highlighted in Fig. 6) that are shared between the bdelloidean rotifer R. rotatoria and acanthocephalan species (O. luehei and L. thecatus), are not shared with any other metazoan mtDNAs so far reported including the monogonont rotifer B. plicatilis. Thus, the sister relationship of the bdelloidean rotifer R. rotatoria with acanthocephalans inferred from phylogenetic analysis of mtDNA amino acid sequence data is also corroborated by the similar gene arrangement pattern shared between these two groups. Considering that Acanthocephala is composed of obligatory endoparasitic species, the relationship of the monophyletic acanthocephalan species within the Syndermatan clade suggests that obligate parasitism within this group evolved in the common ancestor of the acanthocephalans after this lineage diverged from its common ancestor with the Bdelloidea. Additional comparison of complete mtDNA genomes from unstudied acanthocephalan lineages, such as the classes Polyacanthocephala and Eoacanthocephala is required to fully assess the phylogenetic relationships and evolutionary patterns of different life histories represented in the syndermatan groups. 4. Conclusion In the present study, we determined the complete mitochondrial genome sequence of O. luehei (14,281 bp), the first archiacanthocephalan representative (and the second complete sequence from the phylum Acanthocephala) and explored its phylogenetic relationship within Syndermata (Acanthocephala and Rotifera) and among major metazoan groups. The complete mtDNA genome contains 36 genes including 12 protein coding genes (lacking atp8), 22 transfer RNA (tRNA) genes and 2 ribosomal RNA genes (rrnL and rrnS). Results from phylogenetic analyses of amino acid sequences for 10 protein-coding genes from 41 representatives of major metazoan groups (including O. luehei) supported monophyly of Acanthocephala, Syndermata, and paraphyly of Eurotatoria (Bdelloidea + Monogononta from Rotifera). Considering that Acanthocephala is composed of obligatory endoparasitic species, the relationship of the monophyletic acanthocephalans within the Syndermatan clade suggests that obligate parasitism within this group evolved in the common ancestor of the acanthocephalans after this lineage diverged from its common ancestor with the Bdelloidea.

Additional comparison of complete mtDNA genomes from unstudied acanthocephalan lineages, such as the classes Polyacanthocephala and Eoacanthocephala is required to fully assess the phylogenetic relationships and evolutionary patterns of different life histories represented in the syndermatan groups. Supplementary materials related to this article can be found online at doi:10.1016/j.parint.2011.12.001. Acknowledgment This work was supported by the Ministry of Education, Science and Technology (2009-0073882) and a grant from Marine Biotechnology Program funded by Ministry of Land, Transport and Maritime Affairs (MMRBK; Marine Mollusk Resource Bank of Korea) of Korean Government to J.-K. Park. References [1] Bush AO, Fernandez JC, Esch GW, Seed JR. Parasitism: diversity and ecology of animal parasites. Cambridge: Cambridge University Press; 2001. p. 566. [2] Kennedy CR. Ecology of the Acanthocephala. Cambridge: Cambridge University Press; 2006. p. 249. [3] Amin OM. Key to the families and subfamilies of Acanthocephala with the erection of a new class (Polyacanthocephala) and a new order (Polyacanthorhynchida). Parasitol 1987;73:1216–9. [4] Monks S. Phylogeny of the Acanthocephala based on morphological characters. Syst Parasitol 2001;48:81–116. [5] García-Varela M, Cummings MP, Pérez-Ponce de León G, Gardner SL, Laclette JP. Phylogenetic analysis based on 18S ribosomal RNA gene sequences supports the existence of class Polyacanthocephala (Acanthocephala). Mol Phylogenet Evol 2002;23:288–92. [6] Garcia-Varela M, Nadler SA. Phylogenetic relationships among Syndermata inferred from nuclear and mitochondrial gene sequences. Mol Phylogenet Evol 2006;40:61–72. [7] Bullock WL. Morphological features as tools and pitfalls in acanthocephalan systematics. In: Schmidt GD, editor. Problems in systematics of parasites. Maryland, Baltimore: University Park Press; 1969. p. 9–43. [8] Nickol BB, Crompton DWT. Biology of the Acanthocephala. Cambridge: Cambridge University Press; 1985. p. 307. [9] Amin OM. Classification. In: Crompton DWT, Nickol BB, editors. Biology of the Acanthocephala. Cambridge: Cambridge University Press; 1985. p. 22–71. [10] Near TJ, Garey JR, Nadler SA. Phylogenetic relationships of the Acanthocephala inferred from 18S ribosomal DNA sequences. Mol Phylogenet Evol 1998;10:287–98. [11] Near TJ. Acanthocephalan phylogeny and the evolution of parasitism. Integr Comp Biol 2002;42:668–77. [12] Garcia-Varela M, Nadler SA. Phylogenetic relationships of Palaeacanthocephala (Acanthocephala) inferred from SSU and LSU rDNA gene sequences. Parasitol 2005;91:1401–9.

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