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Journal of Helminthology (2015) 89, 317–325 q Cambridge University Press 2014

doi:10.1017/S0022149X14000108

Phylogenetics and systematics of Angiostrongylus lungworms and related taxa (Nematoda: Metastrongyloidea) inferred from the nuclear small subunit (SSU) ribosomal DNA sequences P. Eamsobhana1*, P.E. Lim2,3 and H.S. Yong2 1

Department of Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand: 2Institute of Biological Sciences, University of Malaya, 50603 Kuala Lumpur, Malaysia: 3 Institute of Ocean and Earth Sciences, University of Malaya, 50603 Kuala Lumpur, Malaysia (Received 17 September 2013; Accepted 4 February 2014; First Published Online 13 March 2014) Abstract The Angiostrongylus lungworms are of public health and veterinary concern in many countries. At the family level, the Angiostrongylus lungworms have been included in the family Angiostrongylidae or the family Metastrongylidae. The present study was undertaken to determine the usefulness and suitability of the nuclear 18S (small subunit, SSU) rDNA sequences for differentiating various taxa of the genus Angiostrongylus, as well as to determine the systematics and phylogenetic relationship of Angiostrongylus species and other metastrongyloid taxa. This study revealed six 18S (SSU) haplotypes in A. cantonensis, indicating considerable genetic diversity. The uncorrected pairwise ‘p’ distances among A. cantonensis ranged from 0 to 0.86%. The 18S (SSU) rDNA sequences unequivocally distinguished the five Angiostrongylus species, confirmed the close relationship of A. cantonensis and A. malaysiensis and that of A. costaricensis and A. dujardini, and were consistent with the family status of Angiostrongylidae and Metastrongylidae. In all cases, the congeneric metastrongyloid species clustered together. There was no supporting evidence to include the genus Skrjabingylus as a member of Metastrongylidae. The genera Aelurostrongylus and Didelphostrongylus were not recovered with Angiostrongylus, indicating polyphyly of the Angiostrongylidae. Of the currently recognized families of Metastrongyloidea, only Crenosomatidae appeared to be monophyletic. In view of the unsettled questions regarding the phylogenetic relationships of various taxa of the metastrongyloid worms, further analyses using more markers and more taxa are warranted.

Introduction Lungworms of the genus Angiostrongylus are bursate nematodes of the superfamily Metastrongyloidea. The Metastrongyloidea contains up to seven families,

*E-mail: [email protected]

depending on taxonomic authorities – Angiostrongylidae, Crenosomatidae, Filaroididae, Metastrongylidae, Protostrongylidae, Pseudaliidae and Skrjabingylidae – with over 180 species in 46 genera (Anderson, 2000). In the National Center for Biotechnology Information (NCBI) taxonomy, only six families are recognized, without Skrjabingylidae but with the genus Skrjabingylus treated as a member of the Metastrongylidae.

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Of the Angiostrongylus lungworms, the rat lungworm A. cantonensis (Chen, 1935) is a food-borne zoonotic parasite of public health importance in many countries of the tropics and subtropics (Eamsobhana & Tungtrongchitr, 2005; Eamsobhana, 2006). Its occurrence has now been reported in many countries worldwide (Eamsobhana, 2006; Cross & Chen, 2007; Foronda et al., 2010; Maldonado et al., 2012). Infection with this parasite causes eosinophilic meningitis or eosinophilic meningoencephalitis, with marked cerebrospinal fluid eosinophilia. Rats and snails have been implicated in the global dispersal of this zoonosis (Kliks & Palumbo, 1992). Based on the affected organ, three types of eosinophilic meningitis may be recognized, namely cerebrospinal angiostrongyliasis cantonensis, ocular angiostrongyliasis due to A. cantonensis and pulmonary angiostrongyliasis cantonensis. A related species, A. malaysiensis Bhaibulaya & Cross, 1971, in South-East Asia and Japan, has not been confirmed to infect humans but remains a possibility. It has been shown to produce neurological abnormality in infected rodent hosts (Cross, 1979). When first documented, it was referred to as A. cantonensis (Bhaibulaya & Cross, 1971). Another species in the Americas (from southern USA to northern Argentina in South America), A. costaricensis Morera and Ce´spedes, 1971, causes abdominal or intestinal angiostrongyliasis, which mimics appendicitis with eosinophilia (Graeff-Teixeira et al., 2009; Graeff-Teixeira, 2010). This species does not occur outside the Americas. In addition to the species of human public health importance, the heartworm A. vasorum (Baillet, 1866) is of veterinary concern. In contrast to A. cantonensis, A. costaricensis and A. malaysiensis, which infect rodents, the natural definitive hosts of A. vasorum are domestic dogs and various other carnivores. This parasite infects wild and domestic canids and causes canine angiostrongylosis (Morgan et al., 2005; Conboy, 2011). It is enzootic to Western Europe but has been found in many parts of the world. Infections in dogs tend to be chronic but can be fatal. Of the Angiostrongylus species, A. cantonensis has received great attention in laboratory and clinical studies (Eamsobhana, 2006; Cross & Chen, 2007; Graeff-Teixeira et al., 2009). In particular, immunological diagnosis of human angiostrongyliasis has been explored extensively (Eamsobhana & Yong, 2009). Polymerase chain reaction– restriction fragment length polymorphism (PCR-RFLP) has been employed to differentiate A. cantonensis, A. costaricensis and A. vasorum (Caldeira et al., 2003). The DNA sequences of a 66-kDa protein gene indicate a close relationship of A. cantonensis and A. malaysiensis compared to A. costaricensis (Eamsobhana et al., 2010a). Additionally, the DNA nucleotide sequences of internal transcribed spacer 2 (ITS-2) (Jefferies et al, 2009; Foronda et al., 2010), cytochrome c oxidase subunit I (COI) (Eamsobhana et al., 2010b; Tokiwa et al., 2012) and nuclear small subunit (18S) ribosomal RNA (Fontanilla & Wade, 2008; van Megan et al., 2009; Tokiwa et al., 2012) have been used to determine the relationship between various component taxa of Angiostrongylus. At the family level, the Angiostrongylus lungworms have been included in the family Angiostrongylidae (Anderson, 2000; NCBI Taxonomy) or the family Metastrongylidae (Bhaibulaya & Cross, 1971; Maldonado

et al., 2012; Myers et al., 2013). To date there appears to be no consensus on their family stautus. The present study was undertaken to determine the usefulness and suitability of the nuclear 18S (small subunit, SSU) rDNA sequences for differentiating various taxa of the genus Angiostrongylus, as well as to determine the systematics and phylogenetic relationship of Angiostrongylus species and other metastrongyloid taxa. The 18S rRNA gene is generally conserved. In a barcoding study of nematodes, it has been amplified most reliably from a range of taxa compared to the nuclear 28S rRNA as well as mitochondrial cytochrome c oxidase I (COI) and 16S rRNA genes (Bhadury et al., 2006). In the present study, with more extensive taxa sampling, the 18S (SSU) nucleotide sequences reveal a greater haplotype diversity in A. cantonensis than previously reported. The phylogenetic analyses also resolve the family status of Angiostrongylidae and Metastrongylidae, as well as the close relationship of A. cantonensis and A. malaysiensis and that of A. costaricensis and A. dujardini.

Materials and methods Parasite species and isolates The Angiostrongylus lungworms used in this study are summarized in Table 1. Adult male and female worms of A. cantonensis from Thailand and Hawaii were obtained from experimentally infected albino rats, as described by Eamsobhana et al. (2010a, b). Angiostrongylus cantonensis adult worms from Japan and China were obtained from wild-caught rodents. Angiostrongylus malaysiensis adult worms were obtained from the pulmonary arteries of wild-caught Rattus tiomanicus in Malaysia, and A. costaricensis adult specimens from Costa Rica were obtained from an experimentally infected cotton rat. DNA template preparation DNA extractions from individual adult female and male worms of A. cantonensis (Thailand, Hawaii, Japan and China isolates), A. costaricensis and A. malaysiensis, were prepared using the FTA (fast technology for analysis of nucleic acid) classic card method (Whatman BioScience, Newton Center, Massachusetts, USA) (Eamsobhana et al., 2010a, b). Captured nucleic acids on the FTA cards were purified following the FTA manufacturer’s instructions. Briefly, a 2.0 mm sample disc was removed from the dried spot of the individual worm sample on the FTA card using a Harris micro-punch. The punched disc was washed with FTA purification reagent (Whatman BioScience), rinsed with TE buffer (10 mM Tris– HCI, 0.1 mM EDTA, pH 8) and dried. Polymerase chain reaction (PCR) master mix was added directly to the DNA punch in a PCR tube, followed by amplification. PCR amplification and nucleotide sequencing The 18S RNA nuclear gene (SSU rRNA) was amplified using the primers SSU F07: 50 -AAAGATTAAGCCATGCATG-30 and SSU R09: 50 -AGCTGGAATTACCGCGGCTG-30 (Blaxter et al., 1998; Foronda et al., 2010). PCR amplification was carried out using a DNA thermal cycler (Perkin-Elmer

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Phylogenetics and systematics of Angiostrongylus lungworms Table 1. Nucleotide sequences of 18S rRNA gene for 42 taxa of Metastrongyloidea used in the present study. Dracunculus spp. of Dracunculoidea were used as outgroups. Specimens of A. cantonensis in the present study were obtained from experimentally infected rats in Thailand; the specimen of A. malaysiensis was recovered from a rat in Peninsular Malaysia; the specimen of A. costaricensis was from an experimentally infected cotton rat in Costa Rica. Family/Superfamily Samples from this study Angiostrongylidae

Samples from GenBank Angiostrongylidae

Crenosomatidae

Filaroididae

Metastrongylidae Protostrongylidae Pseudaliidae

Skrjabingylidae Dracunculoidea

Species

Code

Accession No.

Angiostrongylus cantonensis Angiostrongylus cantonensis Angiostrongylus cantonensis Angiostrongylus cantonensis Angiostrongylus cantonensis Angiostrongylus cantonensis Angiostrongylus costaricensis Angiostrongylus malaysiensis

AC-C-F1 AC-H-M1 AC-J-F1 AC-T-M1 AC-T-M2 AC-T-F1 ACOS-F1 AM-M2

JN663723 JN663725 JN663722 JN663728 JN663726 JN663724 JN663730 JN663729

Angiostrongylus cantonensis Angiostrongylus cantonensis Angiostrongylus cantonensis Angiostrongylus cantonensis Angiostrongylus costaricensis Angiostrongylus costaricensis Angiostrongylus dujardini Angiostrongylus dujardini Angiostrongylus malaysiensis Angiostrongylus vasorum Angiostrongylus vasorum Angiostrongylus vasorum Aelurostrongylus abstrusus Didelphostrongylus hayesi Crenosoma mephitidis Crenosoma striatum Crenosoma vulpis Otostrongylus circumlitus Troglostrongylus brevior Troglostrongylus wilsoni Filaroides martis Oslerus osleri Oslerus rostratus Parafilaroides decorus Metastrongylus elongatus Metastrongylus salmi Muellerius capillaris Parelaphostrongylus odocoilei Protostrongylus rufescens Halocercus invaginatus Pseudalius inflexus Stenurus minor Torynurus convolutus Skrjabingylus chitwoodorum Dracunculus insignis Dracunculus lutrae Dracunculus medinensis

Cetus, Waltham, Massachusetts, USA). Each 50 ml reaction mixture for PCR amplifications contained 5 ml of 10 £ PCR buffer (Tris–HCl, KCl, (NH4)2SO4, 15 mM MgCl2, pH 8.7) (QIAGEN, Hilden, Germany), 1 ml of each deoxynucleoside triphosphate (dNTP) mix (10 mM ), 1 ml of each primer (12.5 ng/ml), 0.2 ml of Taq DNA polymerase (5 U/ml), 42.8 ml of distilled water and a DNA sample disc (FTA card). Amplification conditions were: 948C for 2 min, followed by 38 cycles of 948C for 30 s, 458C for 30 s and 658C for 1 min. PCR products were analysed by 1.5% agarose gel electrophoresis stained by ethidium bromide. Amplification products were

AB683977 AB683978 AY295804 GQ181114 DQ116748 EF514913 AY542282 EF514915 EF514914 AJ920365 EF514916 EU915247 AJ920366 AY295806 AY295805 GU214747 AJ920367 AY295813 JX290562 AY295820 AY295807 AY295812 GU946678 AY295814 AJ920363 AY295809 AY295810 AY295815 AJ920364 AY295808 AY295816 AY295817 AY259818 AY295819 AY947719 JF934737 AY947720

purified using QIAquick PCR Purification Kit (QIAGEN). The purified PCR products were sent to a commercial company for sequencing. Samples were sequenced using an ABI PrismDyeTerminator Cycle Sequencing Core kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and analysed on an ABI PRISMH 377 Genetic Analyzer. SSU sequences from GenBank In order to elucidate the phylogenetic relationship among the different species of Angiostrongylus and related

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P. Eamsobhana et al. Table 2. Evolutionary divergence between sequences (number of base substitutions per site) of Angiostrongylus cantonensis based 18S rDNA nucleotide sequences. AB683978 (Japan: Miyagi; Japan: Aichi; China: Wenzhou); AB683977 (Japan: Tokyo, Kanagawa, Chiba, Ishikawa, Kagoshima, Okinawa; Taiwan: Taicung; China: Shenzhen, Lianjiang; Thailand: Bangkok); GQ181114 (Spain: Canary Islands); AY295804 (Japan: Akita University); JN663723 (China: Guangxi); JN663725 (Hawaii isolate); JN663722 (Japan: Okinawa); JN663724, JN663726, JN663728 (Thailand isolate). Accession No. 1. AB683978 2. AB683977 3. GQ181114 4. AY295804 5. JN663728 6. JN663726 7. JN663724 8. JN663722 9. JN663725 10. JN663723

1

2

3

4

5

6

7

8

9

– 0 0 0 0.0043 0.0022 0.0065 0.0022 0 0.0043

– 0 0 0.0043 0.0022 0.0065 0.0022 0 0.0043

– 0 0.0043 0.0022 0.0065 0.0022 0 0.0043

– 0.0043 0.0022 0.0065 0.0022 0 0.0043

– 0.0065 0.0065 0.0022 0.0043 0.0043

– 0.0086 0.0043 0.0022 0.0065

– 0.0043 0.0065 0.0022

– 0.0022 0.0022

– 0.0043

taxa, sequences generated from this study and some of the GenBank sequences (table 1) were used for construction of phylogenetic trees. Additonally, the nucleotide sequences of 12 protein-coding genes (cox1 – 3, nad1 – 6, nad4L, cytb and atp6) of the mitochondrial genome of A. cantonensis (NC_013065), A. costaricensis (NC_013067), A. vasorum (NC_018602), Aelurostrongylus abstrusus (NC_019571), Metastrongylus pudentotectus (NC_013813) and Metastrongylus salmi (NC_013815) were used for constructing the phylogenetic tree, with Dracunculus medinensis (NC_016069) and Syngamus trachea (NC_ 013821) as outgroups. The partial sequences (461 bp) of the nuclear 18S (SSU) rDNA were used to understand the phylogenetic relationship between species and genera, while the long sequences (1573 bp) were used to infer family-level relationships. Sequence alignment and data analysis The nucleotide sequences were initially aligned using BioEdit v. 7.0.9.0 (Hall, 1999) with the Clustal W program (Thompson et al., 1994) and then aligned manually. Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 5 (Tamura et al., 2011). Phylogenetic trees were generated by the maximum likelihood (ML) method based on the Tamura – Nei model (Tamura & Nei, 1993). Initial tree(s) for the heuristic search were obtained automatically by applying NeighborJoin and BioNJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. Analyses were conducted using the Kimura 2-parameter model (Kimura, 1980) to estimate the evolutionary divergence between sequences.

Results Genetic diversity of Angiostrongylus cantonensis The number of base substitutions per site between sequences are summarized in Table 2. The percentage of uncorrected ‘p’ distance ranged from 0 to 0.86%. Five of the 461 sites had different bases, namely sites 315, 324, 338, 443 and 445. This resulted in six haplotypes/genotypes

(table 3). Haplotype AC1 (site 443 with cytosine) was the most common, represented by isolates from China, Japan, Taiwan, Thailand and the Canary Islands. Haplotypes AC2 (site 315 with thymine), AC3 (site 338 with cytosine, site 445 with guanine) and AC4 (site 324 with guanine, site 443 with thymine and site 445 with guanine) were represented only by the Thailand isolate, while haplotype AC5 (site 445 with guanine) was represented by the Japan Okinawa isolate and haplotype AC6 (site 443 with thymine, site 445 with guanine) by the China Guangxi isolate.

Phylogeny of angiostrongylid and metastrongylid taxa Based on the partial nucleotide sequences (461 base pairs) of the 18S rRNA gene, the phylogenetic relationships of the metastrongyloid genera (Angiostrongylus, Aelurostrongylus, Didelphostrongylus, Metastrongylus and Skrjabingylus) separated the genera Angiostrongylus and Metastrongylus into distinct clades (fig. 1). The genera Aelurostrongylus, Didelphostrongylus and Skrjabingylus formed the basal group. The Angiostrongylus clade consisted of three groups: (1) A. cantonensis clustered with A. malaysiensis; (2) A. costaricensis with A. dujardini; and (3) A. vasorum. The [A. cantonensis–A. malaysiensis ] group

Table 3. Variable sites in 18S rDNA nucleotide sequences of Angiostrogylus cantonensis from various localities. Haplotype AC1: AB683978 (Japan), AB683977 (Japan), GQ181114 (Spain, Canary Islands), AY295804 (Japan), JN663725 (Hawaii); AC2: JN663726 (Thailand); AC3: JN663728 (Thailand); AC4: JN663724 (Thailand); AC5: JN663722 (Japan); AC6: JN663723 (China). Bold letters indicate unique bases. Haplotype AC1 AC2 AC3 AC4 AC5 AC6

Site 315

324

338

443

445

A T A A A A

A A A G A A

A A C A A A

C C C T C T

A A G G G G

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Angiostrongylus cantonensis AB683978 Angiostrongylus cantonensis AB683977 Angiostrongylus cantonensis GQ181114 59 Angiostrongylus cantonensis AY295804 Angiostrongylus cantonensis AC-H-M1_JN663725 Angiostrongylus cantonensis AC-T-M2_JN663726 Angiostrongylus cantonensis AC-T-M1_JN663728 93

Angiostrongylus cantonensis AC-J-F1_JN663722

59

Angiostrongylus cantonensis AC-T-F1_JN663724

69 Angiostrongylus cantonensis AC-C-F1_JN663723 Angiostrongylus malaysiensis AM-M2_JN663729 81 Angiostrongylus malaysiensis EF514914 Angiostrongylus costaricensis ACOS-F1_JN663730 59 Angiostrongylus costaricensis DQ116748 61 Angiostrongylus dujardini EF514915 96 Angiostrongylus dujardini AY542282 Angiostrongylus vasorum AJ920365 Angiostrongylus vasorum EU915247 93

Angiostrongylus vasorum EF514916

Metastrongylus elongates AJ920363 93 Metastrongylus salmi AY295809 Didelphostrongylus hayesi AY295806 Skrjabingylus chitwoodorum AY295819 Aelurostrongylus abstrusus AJ920366 Dracunculus medinensis AY947720 Dracunculus insignis AY947719 100 93 Dracunculus lutrae JF934737

0.05 Fig. 1. Phylogenetic tree of the metastrongyloid genera Angiostrongylus, Aelurostrongylus, Didelphostrongylus, Metastrongylus and Skrjabingylus (with Dracunculus spp. of Dracunculoidea as outgroups) generated by the maximum likelihood method based on partial 18S rDNA nucleotide sequences, conducted in MEGA5. The tree with the highest log likelihood (21427.5718) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 27 nucleotide sequences. Codon positions included were 1st þ 2nd þ 3rd. All positions containing gaps and missing data were eliminated. There was a total of 461 positions in the final dataset.

and the [A. costaricensis–A. dujardini ] group were, however, weakly supported. Likewise, the Angiostrongylus and Metastrongylus clades had weak support. Phylogeny of metastrongyloid families The phylogenetic relationships of the component families of the Metastrongyloidea inferred from long 18S rDNA nucleotide sequences (1573 bp) are depicted

in fig. 2. Only the family Crenosomatidae appeared to be monophyletic. The genera Angiostrongylus and Metastrongylus were distinctly separated, while the genera Aelurostrongylus, Didelphostrongylus and Skrjabingylus did not cluster with each other or with Angiostrongylus and Metastrongylus. Metastrongylus formed a cluster with the genus Oslerus of Filaroididae. The genus Skrjabingylus appeared most basal in the superfamily.

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Angiostrongylus constaricensis EF514913 65 53 Angiostrongylus constaricensis DQ116748 Angiostrongylus dujardini EF514915 86

Angiostrongylus vasorum AJ920365

ANG

99 Angiostrongylus vasorum EF514916 Angiostrongylus cantonensis AY295804 72

Angiostrongylus malaysiensis EF514914

Filaroides martis AY295807 91 Stenurus minor AY295817 Torynurus convolutus AY295818 Parafilaroides decorus AY295814 Halocercus invaginatus AY295808

FlL PSE FlL PSE

Pseudalius inflexus AY295816 99 Metastrongylus elongatus AJ920363 77

Metastrongylus salmi AY295809

MET

Oslerus osleri AY295812 FlL 99 Oslerus rostratus GU946678 77 Troglostrongylus brevior JX290562 Troglostrongylus wilsoni AY295820 Otostrongylus circumlitus AY295813 50

Crenosoma vulpis AJ920367

CRE

Crenosoma striatum GU214747 78 71 Crenosoma mephitidis AY295805 Parelaphostrongylus odocoilei AY295815 Aelurostrongylus abstrusus AJ920366 Muellerius capillaris AY295810 62

PRO ANG PRO

Protostrongylus rufescens AJ920364 Didelphostrongylus hayesi AY295806

Skrjabingylus chitwoodorum AY295819

ANG SKR

Dracunculus medinensis AY947720 99 Dracunculus insignis AY947719 79 Dracunculus lutrae JF934737

0.02 Fig. 2. Phylogenetic tree of the component families of Metastrongyloidea (with Dracunculus spp. of Dracunculoidea as outgroups) generated by the maximum likelihood method based on 18S rDNA nucleotide sequences, conducted in MEGA5. The tree with the highest log likelihood (25098.1772) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 32 nucleotide sequences. Codon positions included were 1st þ 2nd þ 3rd. All positions containing gaps and missing data were eliminated. There were a total of 1573 positions in the final dataset. ANG, Angiostrongylidae; CRE, Crenosomatidae; FIL, Filaroididae; MET, Metastrongylidae; PRO, Protostrongylidae; PSE, Pseudaliidae; SKR, Skrjabingylidae.

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Angiostrongylus costaricensis NC013067

70 100

Angiostrongylus vasorum NC018602

99

Angiostrongylus cantonensis NC013065

97

Aelurostrongylus abstrusus NC019571 Metastrongylus salmi NC013815 Metastrongylus pudendotectus NC013813

100

Syngamus trachea NC013821 Dracunculus medinensis NC016019

0.05 Fig. 3. Phylogenetic tree of six taxa of Metastrongyloidea (with Syngamus trachea and Dracunculus medinensis as outgroups) generated by the maximum likelihood method based on 12 protein-coding genes (cox1–3, nad1–6, nad4L, cytb and atp6) of the mitochondrial genome, conducted in MEGA5. The tree with the highest log likelihood ( –55, 523.3733) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved eight nucleotide sequences. Codon positions included were 1st þ2nd þ 3rd. All positions containing gaps and missing data were eliminated. There were a total of 10 ,121 positions in the final dataset.

Based on the 12 protein-coding genes of the mitochondrial genome, the genera Angiostrongylus and Aelurostrongylus were more closely related compared to the genus Metastrongylus (fig. 3).

Discussion The nuclear small subunit (SSU) ribosomal RNA (18S rRNA) gene has been widely used for phylogenetic studies of invertebrate organisms. It is the most conserved among the ribosomal encoding genes (Holterman et al., 2006). It was first used to infer the relationships of 53 species of nematodes (Blaxter et al., 1998). The partial sequences from the 18S (SSU) rRNA gene of A. cantonensis were among those used for phylogenetic analysis of the Metastrongyloidea (Carreno & Nadler, 2003). A phylo genetic tree of nematodes based on about 1200 full-length small subunit ribosomal DNA sequences indicated a considerable congruence with morphology at the family level and below (van Megan et al., 2009). The nematode 18S rDNA dataset has been proposed to be of greater use within major clades, where alignments are likely to be more robust (Smythe et al., 2006). The 18S (SSU) rDNA sequences have enabled the differentiation of closely related nematode species (Gasser & Newton, 2000). This gene has been used to identify the rat lungworm A. cantonensis in Tenerife, Canary Islands, Spain (Foronda et al., 2010), and as a genetic marker for identifying the infective third-stage A. cantonensis (Fontanilla & Wade, 2008). It has also been used to distinguish A. cantonensis from A. costaricensis, A. dujardini, A. malaysiensis and A. vasorum (Fontanilla & Wade, 2008; Foronda et al., 2010). The present study on 18S (SSU) rDNA sequences also unequivocally distinguishes the five Angiostrongylus species (fig. 1). Unlike the cytochrome c oxidase subunit I (COI) sequences which separated the isolates from Thailand, China and Hawaii (Eamsobhana et al., 2010b), the 18S sequences could not unequivocally separate the six geographical isolates (China, Hawaii, Japan, Spain – Canary Islands, Taiwan, Thailand) of

A. cantonensis. Nonetheless, in all cases the congeneric metastrongyloid species clustered together, indicating close genetic affinity (fig. 2). A recent study (Tokiwa et al., 2012) indicated extremely low intraspecific variation of the 18S (SSU) rRNA gene in A. cantonensis, with two genotypes in 13 localities in Japan and three localities in China; only a single specimen each from Taiwan and Thailand was included in that study. The present study uncovered four additional haplotypes (two from Thailand, one each from China and Japan), bringing the total to six (table 3). This is the result of a larger sample size. The uncorrected pairwise ‘p’ distances among A. cantonensis ranged from 0 to 0.86% (table 2). In the present study, based on a larger number of taxa and 18S (SSU) sequences of the genus Angiostrongylus, the phylogenetic analysis indicated a close relationship between A. cantonensis and A. malaysiensis (fig. 1). This concurs with the earlier findings based on 18S rDNA sequences (van Megan et al., 2009), 66-kDa protein gene (Eamsobhana et al., 2010a) and COI sequences (Eamsobhana et al., 2010b). It differs from the finding of A. malaysiensis being more closely related to A. costaricensis than to A. cantonensis (Fontanilla & Wade, 2008; Tokiwa et al., 2012), based on fewer sequences. The overall phylogenetic relationships of the five Angiostrongylus species in the present study are in agreement with the findings of van Megan et al. (2009). It is beyond reasonable doubt that A. costaricensis is more closely related to A. dujardini, compared to the findings of A. costricensis being more closely related to A. vasorum (Foronda et al., 2010) and A. dujardini being more closely related to A. vasorum (Fontanilla & Wade, 2008). Both the long and shorter 18S (SSU) nucleotide sequences in the present study unequivocally separated the genera Angiostrongylus and Metastrongylus (figs 1 and 2), rendering support to the placement of Angiostrongylus in Angiostrongylidae and Metastrongylus in Metastrongylidae. Indeed, the genus Metastrongylus appeared to be closer to the genus Oslerus of Filaroididae (fig. 2). The genera Aelurostrongylus and Didelphostrongylus, however,

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were not recovered with Angiostrongylus, indicating that the Angiostrongylidae, as presently constituted, may not be monophyletic. However, based on the 12 proteincoding genes of available mitochondrial genomes, Aelurostrongylus was closer to Angiostrongylus (fig. 3). Such non-concordance has also been reported for other organisms, for example in filarial worms (Eamsobhana et al., 2013). The need for employing multiple genes and extensive sampling of taxa is warranted to understand the phylogenetic relationships of the component taxa of the Metastrongyloidea. The higher taxonomic position of Skrjabingylus could not be resolved by the present study based on 18S (SSU) sequences. Until further evidence based on a larger number of congeneric taxa becomes available, it may be reasonable to treat it as belonging to Skrjabingylidae (Anderson, 2000; Carreno et al., 2005). At the family level, excepting Crenosomatidae, the other families appeared to be non-monophyletic (fig. 2). In summary, the present study revealed six 18S (SSU) haplotypes in A. cantonensis, indicating considerable intraspecific genetic diversity compared to earlier findings. The uncorrected pairwise ‘p’ distances among A. cantonensis ranged from 0 to 0.86%. This study confirmed the close relationship of A. cantonensis and A. malaysiensis and that of A. costaricensis and A. dujardini. The 18S (SSU) rDNA sequences support the family status of Angiostrongylidae and Metastrongylidae. There is no supporting evidence to include the genus Skrjabingylus as a member of Metastrongylidae. The genera Aelurostrongylus and Didelphostrongylus were not recovered with Angiostrongylus, indicating polyphyly of the Angiostrongylidae. Of the currently recognized families of Metastrongyloidea, only Crenosomatidae appeared to be monophyletic. In view of the unsettled questions regarding the phylogenetic relationships of various taxa of the metastrongyloid worms, further analyses using more markers and more taxa are warranted.

Acknowledgements The authors would like to thank Paibulaya Punthuprapasa and Adisak Yoolek for their excellent technical assistance. We also thank the Mahidol University and University of Malaya for support and facilities. We thank Dr Hongman Zhang (CDC, Guangxi), Dr Ichiro Miyagi and Dr T. Toma (University of the Ryukyus), Dr Akira Ishih (Hamamatsu University), Dr Elizabeth Abrahams and Dr Gabriela Solano (University of Costa Rica) for the gifts of Angiostrongylus materials. The constructive comments of the editor and two anonymous reviewers have helped to improve the manuscript.

Financial support This study was funded in part by the Department of Disease Control, Ministry of Public Health, Thailand, and the University of Malaya (YHS: grants H-50001-00A000025 and H-5620009).

Conflict of interest None.

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