DOI: 10.2478/s11686-008-0029-4 © 2008 W. Stefañski Institute of Parasitology, PAS Acta Parasitologica, 2008, 53(2), 133–144; ISSN 1230-2821 In search of mitochondrial markers for resolving the phylogeny of cyclophyllidean tapeworms (Platyhelminthes, ) – a test study with

D. Timothy J. Littlewood1*, Andrea Waeschenbach1 and Pavel N. Nikolov2 1Department of Zoology, Natural History Museum, Cromwell Road, London SW7 5BD, UK; 2Central Laboratory of General Ecology, Bulgarian Academy of Sciences, 2 Gagarin Street, 1113 Sofia, Bulgaria

Abstract The most species rich order of tapeworms is the and prior to wide-scale sampling of these worms for phylo- genetics, we wished to develop reliable PCR primers that would capture fragments of mitochondrial (mt) DNA with phyloge- netic utility across the order. Nuclear ribosomal RNA gene sequences are well-established and valuable markers for resolving interrelationships spanning a wide range of taxonomic divergences, but fail to provide resolution amongst recently diverged lineages. Entire mt genomes of selected cyclophyllidean tapeworms are available on GenBank, and we used these to design PCR primers to amplify mtDNA from cox1, rrnL and nad1 for a range of cyclophyllideans (7 davaineids, 1 hymenole- pidid and 1 dilepidid) and selected outgroups (Tetrabothrius sp. and sp.). A combined nuclear and mt gene data set was used to estimate a reference phylogeny and the performance of the individual genes was compared to this. Although nuclear and mt genes each contributed to the structure and stability of the phylogenetic estimate, strongest nodal support was provided by nuclear data amongst the basal lineages and by mt data amongst the most recently diverged lineages. The appar- ent complementarity afforded by combining nuclear and mt data was compromised by these data partitions providing conflicting signal at poorly supported nodes. Nevertheless, we argue for a combined evidence approach. PCR primers that amplify rrnL were designed and tested successfully against a diversity of cyclophyllideans; rrnL and nad1 appeared to be more informative than the fragment of cox1. The was not supported by molecular evidence. The new primers will likely pro- vide considerable resolution to estimates of cyclophyllidean interrelationships in future studies.

Keywords mtDNA, phylogeny, Cyclophyllidea, Davaineidae

Introduction The family Davaineidae Braun, 1900 is one of the most species-rich groups of cyclophyllideans. According to our es- Of the 17 recognised orders of tapeworm, the most species- timate, it includes about 450 species parasitising mainly rich, by far, is the Cyclophyllidea. Some 3,100 described (about 425 species, 127 of which are reported from gallina- species (Georgiev 2003) make this order as diverse in terms of ceous birds) as well as (including 6 species report- species number as the remaining cestode orders combined (in ed from humans). total, ~5,200 cestode species are known). Nevertheless, the Historically, the group was regarded as a family (Fuhr- estimate is likely to represent only a small proportion of the mann 1932, Wardle and McLeod 1952, Yamaguti 1959, true diversity of these tapeworms that infect predominantly Schmidt 1986, Jones and Bray 1994) or a suborder (Artyukh tetrapods (mostly birds and mammals but also amphibians and 1966) including two (Artyukh 1966, Jones and Bray 1994) or reptiles), as many potential hosts and biogeographic provinces three (Fuhrmann 1932; Wardle and McLeod 1952; Yamaguti have remained unsampled. Cyclophyllideans include well- 1959; Schmidt 1986; Movsesyan 2003a, b) subfamilies or known parasites of humans and livestock (e.g. species of families, respectively. and Echinococcus), but their abundance and wide distribution Among the davaineid genera, the most species-rich is Rail- in the wild suggests they play important roles in all terrestri- lietina Fuhrmann, 1920 with about 190 species reported from al, most limnetic and many coastal ecosystems. birds and mammals (including humans), (Movsesyan 2003a).

*Corresponding author: [email protected] 134 D. Timothy J. Littlewood et al.

Œl¹ski

Fuhrmann (1920) subdivided the genus into four subgenera scored and analysed cladistically, remains the best treatment but it was later subjected to various changes concerning both of the relationships between the families of Cyclophyllidea to the composition and rank of the subordinated taxa (Fuhrmann date. However, with the propensity for many of these features 1924, Stiles and Orleman, cited after Artyukh 1966, López- to evolve convergently, independent molecular phylogenies Neyra 1931, Fuhrmann 1932, Movsesyan 1966). The gener- are much needed in order to bring stability and order to the ally accepted system was that proposed by Fuhrmann (1932) group. Moreover, additional characters are needed to add great- (Wardle and McLeod 1952, Yamaguti 1959, Artyukh 1966, er resolution to the estimates of inter- and intra-familial rela- Schmidt 1986). According to it, the genus Raillietina includ- tionships. ed the subgenera Raillietina Fuhrmann, 1920, Paroniella Fuhr- Building on our previous studies on the molecular sys- mann, 1920, Fuhrmannetta Stiles et Orleman, 1926 and Skrja- tematics of cestodes, we investigated the phylogenetic utility binia Fuhrmann, 1920. The last comprehensive taxonomic of potential gene markers in estimating the interrelationships works on davaineids (Jones and Bray 1994, Movsesyan 2003a) of selected members of a cyclophyllidean family, as a precur- elevated the subgenera of Raillietina to the generic level; this sor to broad-scale sampling and molecular phylogenetic analy- concept is followed in the present study. ses at higher taxonomic levels. To date, commonly used mo- The characters used for distinguishing the taxa at the fam- lecular markers for the Cestoda include complete small sub- ily level were the structure of the uterus and (or) presence of unit and complete and partial large subunit ribosomal RNA paruterine organ, and at the generic level these were the scolex genes, i.e. ssrDNA and lsrDNA, respectively. These genes armament, the position and number of the genital pores per have been used extensively to estimate ordinal level relation- proglottis, the position of the genital organs and, if paruterine ships (Kodedová et al. 2000, Olson et al. 2001, Waeschenbach organ is lacking, the number of the eggs within an egg capsule et al. 2007) and relationships within orders (e.g. Brabec et al. (Wardle and McLeod 1952; Yamaguti 1959; Artyukh 1966; 2006) but hardly amongst cestode families; one order that has Schmidt 1986; Jones and Bray 1994; Movsesyan 2003 a, b). been investigated more than most with these genes is the As a result, the available of the Davaineidae is arti- Proteocephalidea (e.g. Hypsa et al. 2005). Additional popular ficial rather than representing the phylogenetic relationships markers include mitochondrial genes such as partial cyto- within the group. The davaineids have never been subjected to chrome oxidase subunit I (cox1; e.g. Hu et al. 2005, Wickström extensive phylogenetic studies on the basis of molecular data. et al. 2005), partial small subunit ribosomal RNA gene rrnS Representatives of two davaineid genera (Raillietina and O- (12S; von Nickisch-Rosenegk et al. 1999) and partial large phryocotyle) were included in analysis on the basis of molec- subunit ribosomal RNA gene rrnL (16S; Zehnder and Ma- ular data to study the interordinal phylogenetic relationships riaux 1999), which have been used with varying success for of the cestodes (Mariaux 1998). The few proposed hypotheses within order/family phylogenetic resolution and the recogni- within the order Cyclophyllidea did not include davaineids tion of cryptic taxa. Preliminary analyses with cyclophylli- (von Nickisch-Rosenegk et al. 1999) or gave controversial re- deans (Tkach and Littlewood, unpublished) suggest that genes sults for their relationships with other cyclophyllidean groups evolving faster than the nuclear ribosomal genes used so far (Foronda et al. 2004). are required to differentiate more recent divergence events. In summary, the family Davaineidae was chosen as a mod- However, rather than choosing from the limited number of el group for several reasons. First, it is a speciose family with available PCR primer combinations, of which few appear to controversial classification. The genus Raillietina and related hold much promise (Olson and Tkach 2005), we have decided genera form a compact and morphologically homogeneous to evaluate a rich resource of orthologous genes and gene group (e.g., the genera Raillietina and Fuhrmannetta could be regions, from available completed mitochondrial (mt) ge- distinguished by the position of genital pores: unilateral in the nomes. former and irregularly alternating in the latter), thus repre- Interest in comparative mitogenomics and mt genomes, as senting a suitable model taxon for testing the reliability of a source of molecular markers in of biomedical and morphological criteria by molecular phylogenetic evidence. economic importance (e.g. see Le et al. 2002, Johnston 2006), In particular, the Raillietina spp. from provide has resulted in a number of completed mt genomes being fully a good opportunity to test the phylogenetic relatedness of par- characterized for key cyclophyllidean taxa. Currently on Gen- asites from resident hosts in Europe and North America. Sec- Bank there are full sequences for members of two families ond, developing molecular phylogenetic markers for the da- only, one hymenolepidid and nine taeniids: Hymenolepis di- vaineids contributes to a wider molecular methodology aimed minuta (NC_002767), Taenia crassiceps (NC_002547), T. so- at understanding the phylogenetic relationships within the lium (NC_004022), T. asiatica (NC_004826), T. saginata order Cyclophyllidea as a whole; this has the further potential (NC_009938), Echinococcus granulosus (NC_008075), of enhancing tools for biomedical and veterinary studies on E. multilocularis (NC_000928), E. oligarthrus (NC_009461), cyclophyllideans. Also importantly, representatives of the E. shiquicus (NC_009460) and E. vogeli (NC_009462). We Davaineidae allow independent estimates of phylogeny to be have taken these mt genomes as a starting point in devising evaluated in the light of morphology and host ranges. novel molecular phylogenetic markers with potential for re- The seminal publication of Hoberg et al. (1999) in which solving interrelationships across the wider Cyclophyllidea. In adult morphological and life cycle features were compiled, order to assess the phylogenetic utility of various mt gene re- Molecular systematic markers for Cyclophyllidea 135

Stanis³a * * gions for within family studies, we have chosen species of the * * Davaineidae using individual gene and combined (concat- enated) gene analysis of mt and nuclear ribosomal genes for EU665484 EU665480 EU665481 EU665483 comparison. Mitochondrial genes were assessed in terms of resolving phylogenies congruent with combined evidence so- * * * * lutions, nodal support, proportion of alignable variable sites Mitochondrial and hence likely utility for expanding molecular assessments EU665469 EU665475*EU665476* EU665486* EU665487* EU665479* EU665490* EU665477* EU665488* EU665478* EU665489*

of cyclophyllidean interrelationships. Finally, we test PCR cox1 _ trnT rrnL nad1_trnN primers spanning selected gene regions across a diversity of cyclophyllidean species and make recommendations for future gene and taxon sampling. ssrDNA F286981 EU665471* EU665482* EF095248 EU665463* EU665474* EU665485* AF286980 EU665473 AF286982 EU665472 AJ287582 EU665470 AF286983 AF314223 AF314223

Materials and methods Nuclear

Primer design from mt genomes lsrDNA EF095263 EU665458*EU665459* EU665464* EU665465* EU665457 AF286914 EU665462* EU665468* AF286916 AF286952 EU665460*EU665461* EU665466* EU665467* AF286917 Entire mt genomes of the following species were aligned: Hy- AF286915 menolepis diminuta (NC_002767), Taenia crassiceps (NC_ 002547), T. solium (NC_004022), T. asiatica (NC_004826), Echinococcus granulosus (NC_008075), and E. multilocu- laris (NC_000928); it was not deemed necessary to include all the available sequences from Taenia or Echinococcus as con- servation of alignable positions between genera was more Winnebago important for primer design. Alignments were achieved ini- tially using ClustalX (Thompson et al. 1997) with default set- tings, followed by refinement by eye using MacClade (Mad- Guanacaste, Costa Rica Guanacaste, Costa Rica Khaskovo Region, Bulgaria Laboratory strain Parasitological Institute, King Boris’ Garden, Sofia, Bulgaria Werribee Park, Victoria, Australia Victoria, Park, Werribee St. Kilda, Victoria, Australia Victoria, Kilda, St. Wimbourne, Dorset, UK Wimbourne, Slovak Academy of Sciences Slovak County, Wisconsin, USA Wisconsin, County,

dison and Maddison 2005). Nucleotides for protein coding Location genes were aligned with reference to translation frame and all other genes were aligned to minimize indels. As the purpose of this alignment was to identify conserved regions for PCR primer design, little attention was paid to refining highly vari- able regions. Using MacClade, a strict consensus sequence

was generated in order to determine regions of 100% se- sequence generated in this study *

quence identity. Lengths of consensus sequence were consid- (Australian emu);

ered further for primer design if they were 17bp length with (Syrian );

$ (Short-tailed shearwater) (Mallard duck); Anseriformes(Mallard duck); Lake Butte des Morts, (Black-striped field mouse); Nova Cherna, Silistra, Bulgaria (Downy woodpecker); Cedar Point, Nebraska, USA (White-tipped dove); (lab. rat); Rodentia (Common quail); Galliformes #3 degenerate positions (i.e. multistate nucleotides occupying (Hairy woodpecker); Piciformes Cedar Point, Nebraska, USA (Blackbird); Passeriformes

the same position in the alignment), and > 30% GC. Two re- (Great curassow); Galliformes gions were selected: cox1+trnT+rrnL and nad1+trnN. Prim- ers were edited using PrimerExpress v. 1.0 (ABI) or MacVector Dendrocopos syriacus Dendrocopos Crax rubra laboratory mouse culture Apodemus agrarius Piciformes pubescens Columbiformes Picoides villosus Struthioniformes Puffinus tenuirostris Turdus merula Turdus Anas platyrhynchos v. 9.5.2 (MacVector, Inc.) until hairpins and primer dimers Host were minimized or eliminated, and had suitable melting tem- peratures (#55°C) for routine PCR amplification. Primer sp. pairs were chosen for testing such that normal (not long) PCR sp. sp. sp. nov. 1 sp. nov. sp. nov.3 could be employed, amplifying fragments #2000 bp. In turn, 2 sp. nov. amplicons would need to be sequenced with the same 5’ and Species Raillietina Mesocestoides Raillietina Raillietina Raillietina tunetensis Leptotila verreauxi Fuhrmannetta malakartis Coturnix coturnix Raillietina sonini Hymenolepis diminuta Rattus norvegicus Fimbriaria Raillietina australis novaehollandiae Dromaius Tetrabothrius 3’primers, and with as few internal primers as possible for full Dilepis undula double-stranded coverage.

Taxon and outgroup selection for phylogeny Seven species belonging to two davaineid genera (6 Raillie- Hymenolepididae Davaineidae tina spp. and Fuhrmannetta malakartis) from galliform (2 Family species), struthioniform (1 species), columbiform (1 species) and piciform (3 species) birds were selected along with Di-

lepis undula (Dilepididae) and Fimbriaria sp. (Hymenolepi- including parasite hosts and location; List of taxa and gene sequences used in this study, didae) in accordance with the phylogenetic hypothesis about Mesocestoidata Mesocestoididae Tetrabothriidea Tetrabothriidae Cyclophyllidea Dilepididae Phylogeny Outgroups Table I. Table the relationships among dilepidids, hymenolepidids and da- Ingroup 136 D. Timothy J. Littlewood et al.

Roborzyñski rosbœŸæv fjad kadsææ¿æ vaineids (Hoberg et al. 1999); see Table I. The taxonomy of Sequence alignment and phylogenetic analyses the avian hosts follows Peterson (2002). Based on recent mo- Alignments for phylogenetic analysis were performed using lecular phylogenetic analyses of the Cestoda (Waeschenbach ClustalX (Thompson et al. 1997) with default settings, fol- et al. 2007), two species, Mesocestoides sp. and Tetrabothrius lowed by refinement by eye using MacClade (Maddison and sp. were chosen as outgroups to root the phylogenies. Tetra- Maddison 2005). Nucleotides for mt protein coding genes bothrius is the type genus from the order Tetrabothriidea. Al- were aligned, in frame, with respect to amino acid transla- though the family Mesocestoididae is recognised as a member tions, and tRNAs were aligned with respect to putative sec- of the Cyclophyllidea in morphologically based classification ondary structure models established with tRNA-scan-SE 1.21 schemes, it is known to represent a distinct but closely relat- (www.genetics.wustl.edu/eddy/tRNAscan-SE/; Lowe and Eddy ed lineage, the Mesocestoidata; see Hoberg et al. (2001) for 1997). Regions that could not be unambiguously aligned were additional information. excluded from the analysis. A single data matrix of concat- enated genes was assembled with MacClade. Only unam- DNA amplification and sequencing biguously alignable positions were included in phylogenetic Total genomic DNA (gDNA) was extracted from ethanol pre- analyses. served specimens using DNeasy tissue kit (QIAGEN) follow- Nuclear ribosomal (ssrDNA and lsrDNA) and mt (cox1 ing the manufacturer’s instructions. PCR and sequencing prim- 890–932 bp, 3’ end; rrnL 917–954 bp, 5’ end; nad1 626–801 ers are listed in Table II. ZX-1 was designed by van der bp, 3’ end) genes were each analysed separately and in combi- Auwera et al. (1994); all other primers have originated from nation (total nuclear ribosomal, total mitochondrial including this research group. For each reaction, 2 µl gDNA was used as tRNAs, and total molecular data), using Bayesian inference a template in 25 µl reactions using Ready-To-GoTM PCR (BI) with MrBayes, version 3.1.2 (Huelsenbeck and Ronquist beads (Amersham Pharmacia Biotech). Cycling conditions 2001), maximum likelihood (ML) and maximum parsimony for partial (D1-D3) lsrDNA (~1662 bp) were as follows: de- (MP) criterion with PAUP* version 4.0b10 (Swofford 2002). naturation for 5 min at 95°C, followed by 40 cycles of 30 s at Partition homogeneity tests as implemented in PAUP* (Incon- 95°C, 30 s at 55°C, 2 min at 72°C; and 7 min extension at gruence Length Difference [ILD] test of Farris et al. 1995), 72°C. Cycling conditions for complete ssrDNA (~2185 bp) were conducted to determine whether individual data parti- were as follows: denaturation for 2 min at 94°C, followed by tions were not significantly heterogeneous from one another. 40 cycles of 30 s at 94°C, 30 s at 54°C, 2 min at 72°C and Modeltest version 3.7macX (Posada and Crandall 1998) completed by 7 min at 72°C. Cycling conditions for cox1+ was used to select a model of evolution using the Akaike In- trnT+rrnL rDNA (1889–1943 bp) were as follows: denatura- formation Criterion. For the majority of data sets Modeltest tion for 3 min at 94°C, followed by 40 cycles of 30 s at 94°C, selected the GTR+I+G model, and this was chosen for all 30 s at 52°C, 3 min at 72°C; and 7 min extension at 72°C. ML and BI analyses; for cox1 and lsrDNA Modeltest chose Cycling conditions for nad1+trnN (~850 bp) were as follows: TVM+I+G and GTR+G models respectively, each with denaturation for 3 min at 94°C, followed by 40 cycles of 30 s GTR+I+G as second best models. As such, for BI, likelihood at 94°C, 30 s at 55°C, 1.5 min at 72°C and completed by 7 min settings were set to nst = 6, rates = invgamma, ngammacat = 4 at 72°C. Cycling conditions for rrnL only (~930 bp) were as (equivalent to the GTR+I+G). For combined gene analyses, for cox1+trnT+rrnL, except the extension step was reduced parameters were estimated separately for each gene. Four to 2 min. All mt fragments represent partial gene fragments chains (temp = 0.2) were run for 5,000,000 generations and at both the 5’ and 3’ ends. PCR amplicons were either gel-ex- sampled every 1000 generations. 500,000 generations were cised using QIAquickTM Gel Extraction Kit (QIAGEN) or pu- discarded as ‘burnin’. rified directly using QIAquickTM PCR Purification Kit (QIA- Maximum likelihood analyses were performed using suc- GEN) following the standard manufacturer-recommended cessive approximation: model parameters were estimated based protocol. Cycle-sequencing from both strands was carried out on a starting tree determined by neighbor-joining (NJ). A heu- on an ABI 3730 DNA Analyser, Big Dye version 1.1. using ristic search was performed implementing the estimated model ABI BigDyeTM chemistry using manufacturer’s instructions. parameters using nearest-neighbor-interchange (NNI) branch Products for the mt fragment cox1-rrnL rDNA were cloned swapping. Model parameters were estimated on the best tree using TOPO TA Cloning® Kit with pCR®2.1-TOPO® vector and a heuristic search performed using subtree-pruning-reg- (Invitrogen), following manufacturer’s instructions. Positive rafting (SPR) branch swapping. After estimating model pa- clones were grown for 15 h in 3 ml volumes of LB at 37°C at rameters, heuristic searches using tree-bisection-reconnection 200 rpm in a shaking incubator. Plasmid DNA was purified (TBR) branch swapping were performed until the topology using QIAprep Spin Miniprep Kit (QIAGEN) following the remained unchanged. standard manufacturer-recommended protocol, and cycle- MP analyses were performed using the heuristic search sequenced from both strands using M13 primers and internal method (100 search replicates) and TBR branch-swapping primers (see Table II). Contiguous sequences were assembled algorithm. All characters were unordered and were weighted and edited using SequencherTM (GeneCodes Corp., Ver. 4.6) equally. Gaps were treated as missing data. A starting tree was and sequence identity checked using the Basic Local Align- obtained via stepwise addition, and addition of sequences was ment Search Tool (BLAST) (www.ncbi.nih.gov/BLAST/). random. One tree was held at each step during stepwise addi- Molecular systematic markers for Cyclophyllidea 137

Table II. Primers used for amplification and sequencing of (a) partial lsrDNA, (b) ssrDNA, (c) partial cox1+trnT+partial 16S rDNA, (d) partial nad1+trnN. F = Forward, R = Reverse, all primers are 5’ to 3’

(a) lsrDNA PCR and sequencing primers ZX-11 F ACCCGCTGAATTTAAGCATAT 1500R R GCTATCCTGAGGGAAACTTCG Additional sequencing primers 300F F CAAGTACCGTGAGGGAAAGTTG 400R R GCAGCTTGACTACACCCG ECD2 R CTTGGTCCGTGTTTCAAGACGGG 1090F F TGAAACACGGACCAAGG (b) ssrDNA PCR and sequencing primers WormA F GCGAATGGCTCATTAAATCAG WormB (21mer) R CTTGTTACGACTTTTACTTCC 18S-8 (nested PCR primer) F GCAGCCGCGGTAATTCCAGC A27’ (nested PCR primer) R CCATACAAACGTCCCCGCCTG Additional sequencing primers 300F F AGGGTTCGATTCCGGAG 600R R ACCGCGGCKGCTGGCACC 930F F GCATGGAATAATGGAATAGG 1200F F CAGGTCTGTGATGCCC 1270F F ACTTAAAGGAATTGACGG 1270R R CCGTCAATTCCTTTAAGT (c) cox1+trnT+rrnL rDNA PCR and sequencing primers Cyclo_cox1Fa F CARCATATGTTTTGRTTTTTTGG Cyclo_16SRc R AATAGATAAGAACCGACCTGGC Cyclo_16SF (16S only) F TGCCTTTTGCATCATGCT Cyclo_16SR (16S only) R AATAGATAAGAACCGACCTGG Additional sequencing primers Cyclo_cox1Rb R CCTAAYGACATAACATAATGRAAATG Cyclo_16SRa R RATGCAAAAGGCAAATAAACC Cyclo_16SFa F ATTTGCCTTTTGCATYATGCT Cyclo_16SRb R TTATGCTACCTTYGCACAGTC Cyclo_16SFb F TTGACTGTGCRAAGGTAGC (d) nad1+trnN mtDNA PCR and sequencing primers Cyclo_nad1F F GGNTATTSTCARTNTCGTAAGGG Cyclo_trnNR R TTCYTGAAGTTAACAGCATCA Additional sequencing primers Cyclo_nad1Fb F AGGTTTGARGCKTGTTTTATG

1Original ZX-1 (ACCCGCTGAAYTTAAGCATAT) (van der Auwera et al. 1994); Y was replaced with T. tion. Nodal support was estimated by bootstrapping (1000 with indels, often present in rDNA) prevents unambiguous replicates) using MP. Maximum likelihood bootstrap values alignment. In order to visualise the amount of variability with- for 100 replicates were obtained using Genetic Algorithm for in and between the various genes and gene regions sampled, Rapid Likelihood Inference (GARLI) Version 0.942 (Zwickl we exported the final alignment from MacClade back to 2006) using default settings, except setting ‘Genthreshforto- ClustalX in order to retrieve alignment scores for each align- poterm’ to 10,000 generations. ment position. ClustalX score parameters were held as fol- lows: scoreplot = 5, residue exception cutoff = 1, DNA weight matrix = IUB. Score values were subsequently exported to a Comparing sequence variability and information content spreadsheet and plotted as a function of alignment position, within and between genes indicating gene boundaries and regions that could not be Sequence variability brings with it the opportunity of detect- unambiguously aligned (and therefore useless for phyloge- ing phylogenetic signal, but too much variability (especially netic estimation). 138 D. Timothy J. Littlewood et al.

Testing PCR primers across a diversity of Cyclophyllidea allow primer design. As a result of sequence conservation var- From the resulting alignments of cox1+trnT+rrnL data set, ious tRNA genes were also highlighted initially as possible PCR primers were designed to amplify a fragment of rrnL. primer sites, including trnN, trnI, trnK, trnW, trnL2 and trnR, These were tested on gDNA from a variety of cyclophyllidean but unsurprisingly these regions were found to coincide with tissue samples, including Eurycestus avoceti (Dilepididae), stem-loop regions, which in turn would likely form stable hair- Anomotaenia microphallos (Dilepididae), an unidentified gy- pins within the primer sequences. We used MacVector v. 9.5.2 rocoeliid, an unidentified species of Biuterina (Paruterinidae) (MacVector Inc.) to find suitable PCR primers in regions of and Pseudocatenotaenia matovi (Catenotaeniidae). high sequence conservation across the reference alignment, and tested candidate primers against individual reference se- quences. Primers forming hairpin loops, with low GC content Results and the propensity to form primer dimers were excluded using the primer settings within the program. Suitable primers were Suitable PCR primers were successfully designed from pub- designed from nad1, trnN, cox1 and rrnL regions, and were lished mt genome sequences, and utilized to amplify contigu- tested successfully for the selected davaineids and outgroup ous fragments of mtDNA from a diversity of davaineid ces- taxa (see Table II). For each taxon, two mt fragments were todes and outgroups. Individual gene fragments were tested readily amplified for the taxa under study; one fragment in- for their ability to provide structure and strong nodal support cluded partial cox1, trnT and partial rrnL (cox1+trnT+rrnL), to inferred phylogenies that were in turn compared against and the other included partial nad1 and trnN (nad1+trnN). phylogenetic estimates from established nuclear ribosomal Internal sequencing primers were designed for each fragment; gene markers. Two new phylogenetic markers were developed see Table II. Primers worked well for all templates. from the preliminary data. Phylogenetic analyses Mitochondrial primer design from mt genomes Ten cox1+trnT+rrnL, ten nad1+trnN, 6 partial lsrDNA and 6 Across the alignment of mt genomes, few regions were suit- complete ssrDNA sequences were generated from this study. ably conserved to allow primer design. Although at the amino A full list of taxa and GenBank sequences used in the study is acid level many of the protein coding genes appear to be con- shown in Table I. Figure 1 shows a representation of the align- served, only nad1 and cox1 provided useful stretches of con- ments for each gene fragment for all taxa used (including out- served sequence with the necessary features (see below) to groups), taken from the final ClustalX output (after refine-

Fig. 1. ClustalX alignment scores for nuclear (ssrDNA, partial lsrDNA) and mitochondrial (cox1_trnT_rrnL and nad1_trnN) genes for select- ed cestodes (see Table I), indicating regions that could not be unambiguously aligned (black boxes) and regions of high and low variation; sequence conservation is indicated by a high alignment score. ClustalX score settings: scoreplot = 5, residue exception cutoff = 1, DNA weight matrix = IUB Molecular systematic markers for Cyclophyllidea 139

ment in MacClade), indicating regions of variability for each tial cox1 (33.6%), in contrast to lsrDNA (23.7%) and ssrDNA position in the alignment. Regions that could not be aligned (15.0%). unambiguously are also indicated, and these mostly represent For each individual data partition there was strong agree- regions of high variability (loops in the secondary structures) ment in final tree topologies between different methods of of RNA and tRNA genes and regions with high proportions of analysis, although MP tended to yield multiple parsimonious indels. The greatest proportion of unalignable sites came from solutions reflecting poor or low nodal support in many of the ribosomal sequences, but in particular those from the V4 and individual mt gene analyses. Figure 2 shows the BI trees with V7 variable regions of ssrDNA, various regions of the D2 var- nodal support from posterior probabilities and bootstrap ML iable region of lsrDNA and the mt tRNA genes. Each of these and MP analyses. Nodes were considered to be of strong sup- regions is characterized by extensive loop structures. Very few port if BI posterior probabilities were $0.95, ML bootstrap regions of the mt protein coding genes were not alignable as >90% and MP bootstrap >90%. Nuclear ribosomal genes tend- there were few indels of inferred amino acids. Alignments of all ed to provide greater nodal support at deeper divergent nodes data sets used as the basis for phylogenetic analyses have been and mt genes provided greater support amongst more recent deposited with EBI under accession numbers: ALIGN_001247 divergences. Combined data sets provided increasingly great- (ssrDNA), ALIGN_001248 (lsrDNA), ALIGN_001249 (mt er nodal support throughout the phylogeny, with the fully con- cox1_trnT_rrnL), ALIGN_001250 (mt nad1_trnN). catenated data set offering a resolved phylogeny, congruent All permutations of partition homogeneity tests indicated amongst different methods of analysis and with high nodal insignificant heterogeneity between data partitions; all indi- support throughout. The fact that nodal support at the deeper vidual genes against one another (P = 0.08), individual mt nodes is greater in the nuclear ribosomal genes trees and genes against one another (P = 0.39), ssrDNA vs. lsrDNA greater for the more recent divergences in the mt genes trees, (P = 0.68), mitochondrial vs. nuclear (P = 0.96). Phylogenetic is reflected in the topology of the ‘all data’ tree, where the analyses were performed for each of the following individual basal and recent divergences are identical (or almost identical) and concatenated partitions: complete ssrDNA, partial lsrDNA, to their corresponding topologies in the ssrDNA and lsrDNA cox1, rrnL and nad1, combined nuclear (ssrDNA+lsrDNA), and ‘mt only’trees respectively. Differences in tree topologies combined mt data (mt only) and combined nuclear+mt data amongst different data sets were mostly around poorly sup- (all data). Results summarising the phylogenetic analyses are ported nodes. presented in Figure 1 and Table III (alignment details and tree Table IV provides results of tests comparing individual data statistics). A total number of 5649 unambiguously alignable sets against different tree topologies: Shimodaira-Hasegawa nucleotide positions were available in the final alignment; (S-H) test results, with probabilities and log likelihood scores. 1515 positions were excluded. Of the included positions, 2084 S-H tests were run under a RELL distribution with 1000 boot- were variable and 1384 were phylogenetically informative strap replicates, using likelihood parameters taken from the under the principles of parsimony. Although nuclear ribo- ML analysis for each individual test data set; e.g. for nad1 data somal data contributed more positions to the alignment, mt set, likelihood scores for trees were estimated using the model genes provided almost twice as many variable and twice as determined for the nad1 ML analysis. A number of tree topol- many parsimony informative sites. As a proportion of unit ogies show significant differences in phylogenetic signal be- length of alignable sequence, genes with the most informative tween data sets, but the poor nodal support for many nodes (see characters were partial rrnL (38.6%) > nad1 (37.0%) > par- Fig. 2) suggests that many of the topologies were not actual-

Table III. Summary of statistics from alignments and phylogenetic analyses

Alignment MP ML BI (branch and bound) (succ. approx.) no. of no. of no.of unambiguous variable phylogenetically positions positions informative no. of -ln -ln positions (MP) length CI RI trees likelihood likelihood partial rrnL 886 474 342 1210 0.609 0.49 1 5825.55 5829.23 partial cox1 879 401 296 1167 0.567 0.362 2 5571.18 5576.65 partial nad1 756 403 280 1073 0.56 0.395 1 5042.75 5047.61 mt only 2631* 1325* 950* 3566 0.583 0.414 1 17144.10 17067.36 partial lsrDNA 1166 481 277 804 0.766 0.689 5 5514.26 5157.01 ssrDNA 1852 278 157 449 0.804 0.734 7 4887.25 4891.56 nuclear only 3018 759 434 1254 0.779 0.704 4 10224.05 10228.18 all data 5649 2084 1384 4821 0.634 0.492 2 28345.65 27614.01

*Includes positions for trnT and trnN. Abbreviations: MP – maximum parsimony; CI – consistency index; RI – retention index; ML – maxi- mum likelihood; BI – Bayesian inference. 140 D. Timothy J. Littlewood et al.

Fig. 2. Summary of phylogenetic analyses for individual and combined gene partitions. Partitions were: individual mt genes (cox1, nad1, rrnL), individual ribosomal RNA genes (lsrDNA, ssrDNA), combined mt (cox1_trnT_rrnL and nad1_trnN), combined nuclear (lsrDNA+ssrDNA) and combined mt and nuclear (all) data. Regions homologous to previously published mt primers discussed in the text are marked as follows: 16S-5’ and 16S-3’ (rrnL; Zehnder and Mariaux 1999) ly robust to begin with. In other words, differences between (all data) tree, although individual gene partitions contributed poorly supported tree topologies are not considered important to the resolution of different regions of the tree. To reiterate, (in spite of their statistical significance) unless there is accom- the S-H test does not account for nodal support. Thus, topolo- panying high nodal support amongst the different trees under gies with nodes of low support were considered compatible consideration. Nuclear ribosomal and mitochondrial data sets with alternative trees if nodes with high support (as indicated conflicted most with each other suggesting each locus was in Fig. 2) were not compromised. With individual nuclear arguing for significantly different phylogenetic solutions, al- ribosomal genes, most signal was provided for deeper branch- though the distribution of well-supported nodes differed be- ing nodes, although lsrDNA and lsrDNA+ssrDNA provided tween these data partitions. Individual mitochondrial genes additional support for the more derived nodes amongst more and the combined mt data were not in conflict with the com- closely related taxa. A comparison of total nuclear (lsrDNA bined evidence solution (all data). When considering nodal +ssrDNA) and total mt gene phylogenies indicates a few topo- support and the robustness of the tree topologies, only with all logical differences. The sister taxon to the Fimbriaria and data combined were nodes well-supported throughout the phy- Hymenolepis clade is Dilepis with nuclear data, and unre- logeny. All trees were compatible with the combined evidence solved with mt data. Relationships within the Raillietina clade Molecular systematic markers for Cyclophyllidea 141

Table IV. Results of Shimodaira-Hasegawa tests, giving probability/-ln likelihood respectively for each data set and tree topology compari- son. Data sets were considered to argue for significantly different tree topologies when P < 0.05*, < 0.01**, or < 0.001***. However, the test provides indications of differences between tree topologies only, and not the robustness of the topologies (indicated by nodal support; see Fig. 2). Nuclear versus mitochondrial results are boxed; nuclear only is lsrDNA+ssrDNA

Tree topologies rrnL cox1 nad1 mt only lsrDNA ssrDNA nuclear all data Data sets rrnL -5825.551 -5833.367 -5830.443 -5825.964 -5853.33 -5852.627 -5852.627 -5830.684 1 0.319 0.481 0.450 ** ** ** 0.501 cox1 -5576.298 -5571.177 -5575.385 -5572.521 -5577.968 -5579.251 -5579.061 -5573.743 0.381 1 0.453 0.772 0.264 0.206 0.223 0.698 nad1 -5049.362 -5051.239 -5042.749 -5050.569 -5075.813 -5080.042 -5080.04 -5046.226 0.362 0.276 1 0.050 *** *** *** 0.591 mt only -17145.001 -17146.333 -17148.433 -17142.478 17203.393 -17206.180 -17206.180 -17144.103 0.739 0.685 0.577 1 *** *** *** 0.830 lsrDNA -5196.127 -5171.021 -5190.587 -5168.753 -5154.262 -5158.263 -5157.564 -5159.776 ** 0.132 ** 0.064 1 0.66 0.686 0.626 ssrDNA -4904.886 -4896.456 -4911.329 -4895.851 -4894.48 -4887.253 -4889.706 -4892.747 0.067 0.294 * 0.140 0.387 1 0.164 0.472 nuclear only -10284.374 -10244.151 -10284.374 -10242.253 -10224.833 -10224.239 -10224.051 -10231.244 *** 0.146 *** * 0.834 0.868 1 0.593 all data -28398.914 -28363.946 -28389.145 -28357.64 -28409.146 -28415.397 -28415.391 -28345.647 ** 0.341 * 0.108 *** *** *** 1 are unresolved with nuclear data but far better resolved with being largely identical to the combined mt data tree (except of mt data. In summary, mt genes tended to provide robust sup- the position of Tetrabothrius sp. and D. undula). Hence, this port for nodes subtending more recently diverged lineages short region of ~930 bp seems to encapsulate the phylogenetic with nad1 and rrnL providing consistently more support signal present in the overall mt data of ~2700 bp. In order to test across the phylogenies than cox1 alone. this pair of primers for their applicability in other cyclophyl- lidean families, PCRs were successfully performed on DNA New mitochondrial primers and their performance amongst samples from 4 cyclophyllidean families (Dilepididae, Gyro- other cyclophyllideans coeliidae, Paruterinidae and Catenotaeniidae) (Fig. 3). Similar- In an attempt to design mt markers that can be sequenced with- ly, but showing slightly less overall nodal support than rrnL out cloning and with only few, if any, internal primers that and the combined mt data, nad1 (amplified with primers Cy- amplify fragments with a large proportion of phylogenetic sig- clo_nad1F+Cyclo_trnNR; partial trnR makes up only the last nal, we designed a primer pair (Cyclo_16SF+Cyclo_16SR; 60 bp at the 3’ end of the ~850 bp fragment) seems to be a fur- Table II) that amplifies ~930 bp of rrnL. The choice for this ther promising candidate in assessing recent cyclophyllidean region was based on the fact that overall nodal support was divergences, although its amplification and sequencing was equal to the combined mt data topology, with the topology somewhat hampered by the degenerate nature of the primers.

Fig. 3. Agarose gel indicating successful PCR amplification of rrnL from 4 cyclophyllidean families; lane 0 – Hyperladder I marker (Bioline); lane 1 – Eurycestus avoceti (Dilepididae); lane 2 – Anomotaenia microphallos (Dilepididae); lane 3 – Gyrocoeliidae; lane 4 – Biuterina sp. (Paruterinidae); lane 5 – Pseudocatenotaenia matovi (Catenotaeniidae) 142 D. Timothy J. Littlewood et al.

Discussion ed the well-supported nodes from each of the data sets. No sin- gle gene provided the same level of resolution as the com- It is clear from the phylogenetic analyses conducted with com- bined (all data) estimate. plete ssrDNA and partial lsrDNA that whilst these nuclear Regarding the phylogenetic relationships of the davaine- genes have contributed significantly to our understanding of ids, our results do not support the erection of Fuhrmannetta as deeper divergences amongst Cestoda (e.g. Olson et al. 2001, a distinct genus, which is in contrast with the current taxon- Waeschenbach et al. 2007), they fail to provide much resolu- omic arrangement of Raillietina and related genera (Jones and tion amongst closely related taxa (see Fig. 2). In contrast, and Bray 1994, Movsesyan 2003a) and questions the reliability of as expected from a diversity of similar studies on metazoan the position of genital pores (unilateral or alternating) as a interrelationships, the present study shows that mitochondri- character for distinguishing genera. The davaineids from al genes provided a greater number of variable positions and woodpeckers resident for Europe and North America formed more phylogenetic signal amongst closely related taxa, yield- a well-supported clade, and on this basis we consider that the ing consistently higher nodal support for individual and com- present results generally corroborate the “Fuhrmann’s Rule” bined data partitions. (defined by Ass 1938, cited after Dogel’ 1962), which states An alignment of published sequences of complete mt “...each avian order has its specific cestode fauna” (Fuhrmann genomes of cyclophyllidean tapeworms provided sufficient 1932). The “rule” could not be applied unambiguously to regions of sequence conservation to develop new PCR prim- host-parasite associations since it does not take into consider- ers and new molecular markers for cyclophyllidean phylo- ation other factors related to the ecology of the hosts and the genetics. Using selection criteria that would encapsulate large dynamics of the host-parasite assemblages. However, it could fragments of homologous sequences for all cyclophyllideans, be refined and interpreted in the light of the contemporary the program MacVector provided a rapid means of designing cladistic tools combined with the molecular methods used. and evaluating potential PCR primers in silico. However, only Therefore, we consider the present results support the gener- two regions of mtDNA were identified as likely candidates al approach to testing this rule in the future; additional parasite providing suitable amplicons for evaluation. Other gene re- sampling to compare phylogenetic reconstructions and host/ gions were far too variable. Although this does not discount geographic distributions are required. the likely phylogenetic utility of other genes, it suggests that Zehnder and Mariaux (1999) working with partial lsrDNA specific (rather than ubiquitous) primers would need to be and a smaller fragment of rrnL (see Fig. 1 for region of rrnL developed for smaller sub-groups of cyclophyllideans if dif- used) also found that these genes yielded significantly differ- ferent mitochondrial genes were to be targeted. This study fo- ent phylogenetic hypotheses for estimates of proteocephali- cussed on an evaluation of closely related species within the dean relationships. However, just as we have found in the Davaineidae, with other cyclophyllidean and non-cyclophyl- present study, the genes provide high nodal support at differ- lidean taxa providing the reference for evaluating the potential ent, albeit complementary regions of the inferred trees and for new markers across the order. what appeared to be topological conflict (as indicated by a The two regions of mtDNA identified and subsequently failed incongruence length difference test) may simply reflect amplified were a fragment including partial cox1 to partial differences in resolving power at different taxonomic levels in rrnL (including trnT), and a fragment including partial nad1 the trees. Zehnder and Mariaux (1999) chose not to combine and trnN. After sequencing these regions, individual genes their data sets as they failed an ILD test, and they restricted (excluding tRNA genes) were evaluated for their phylogenet- analyses to MP only. Such an approach may have been over ic utility with a view to providing markers for routine sequenc- cautious and their data seem worthy of reanalysis, particular- ing and resolving interrelationships within cyclophyllidean ly with a model-based method. Yoder et al. (2001) noted that families in light of available nuclear ribosomal markers al- a failed ILD test was not a good measure of data combinabil- ready widely available. Amongst the davaineids selected, dif- ity, and the present study demonstrates that nuclear and mito- ferent genes gave different phylogenetic estimates with vary- chondrial gene partitions are contributing complementary sig- ing degrees of nodal support. Tree topologies were congruent nal in different parts of the tree. amongst nuclear or mitochondrial gene estimates, but gener- It seems clear that a mixture of nuclear and mitochondri- ally nuclear phylogenies were significantly different from al gene markers are required to provide robust phylogenetic mitochondrial estimates in terms of topology. However, dif- resolution within the Cyclophyllidea, and perhaps also more ferences in topology between nuclear and mitochondrial esti- generally for other cestode orders. Ideally, all fragments ex- mates were not accompanied by strongly supported nodes; in amined in the present analysis would be characterized for other words strongly supported nodes in one data set were wider and denser taxonomic sampling of cyclophyllidean ces- likely in regions that were poorly supported in the other data todes. We recognise that denser taxon sampling might require set. Consequently, a combined nuclear+mitochondrial (all the addition of still more markers as the proportion of phylo- data) analysis yielded a topology similar to the nuclear esti- genetically informative positions becomes limiting. Never- mates in terms of deeper divergence patterns, and similar to theless, based on individual gene performance, phylogenetic the mitochondrial estimates amongst more divergent taxa; resolution and nodal support, we would suggest that the par- thus providing a consensus phylogenetic estimate that reflect- tial rrnL gene characterized herein, and possibly the protein Molecular systematic markers for Cyclophyllidea 143

coding gene nad1, might prove most useful initially, as the to use more informative genes. We suggest that separate analy- best mt genes to add to one of the nuclear markers. Given the ses of independent data sets should be assessed in the light of higher number of variable positions per unit length, we sug- maximizing complementary signal at different taxonomic lev- gest that lsrDNA should be preferred over ssrDNA if only one els of inferred phylogenies, and that combined evidence were to be chosen. Our sampling purposely included very solutions are likely to provide best estimates of phylogeny. closely related taxa and we expect that a broad sampling of Clearly, there are limitations in combined evidence phyloge- cyclophyllideans can yield a reasonably resolved phylogeny netics (e.g. in phylogenomics; Rokas and Carroll 2005, Jef- with these new mitochondrial markers in combination with froy et al. 2006), but in cestode systematics we are a long way one or more of the nuclear ribosomal genes. short of saturating phylogenies with molecular data or taxa. Until additional nuclear markers can be developed for resolving inter- and intra-familial relationships of cestodes, it Acknowledgments. We thank Dr F. Agustin Jimenez-Ruiz, Terry may be worth pursuing these same mitochondrial genes in Havercost (Manter Laboratory of Parasitology, University of Ne- resolving the interrelationships of other cestode orders. To this braska, Lincoln, USA), Georgi Maslarov, Ivan Ivanov, Nikolay Tenev (Regional Department of Forests, Kardzhali, Bulgaria) and Ivan Yan- end, additional lengths of mitochondrial genes and genomes chev (Central Laboratory of General Ecology) for their assistance in need to be characterized so that we might apply a similar strat- the collection of the cestodes from birds. We thank also Dr Ian Bev- egy in developing and testing ubiquitous markers. Additional- eridge (Veterinary Clinical Centre, University of Melbourne, Aus- ly, it may be worth testing the primers designed herein across tralia), Dr Jean Mariaux (Natural History Museum, Geneva, Switzer- a diversity of cestode orders, although we suspect they would land), Dr Boyko Georgiev (Central Laboratory of General Ecology, Sofia, Bulgaria) and Gabriela Hrckova (Parasitological Institute, begin to fail when sampling earlier divergent lineages of ces- Slovak Academy of Sciences) for providing cestode specimens. todes. It is noteworthy that the rrnL PCR primers of Zehnder Thanks to two anonymous reviewers for their helpful comments. AW and Mariaux (1999), designed for Proteocephalidea, would and DTJL were funded through a NERC award (NER/A/S/2003/ not work well for Cyclophyllidea, due to numerous base 00313). PN was funded by an EU SYNTHESYS grant (GB-TAF- changes in the priming regions. 600). The fragment of cox1 used in the present study was not the same region popularly chosen for ‘barcoding’ in other meta- zoan taxa (a fragment of approx. 650 bp towards the 5’ end of References the gene; Hajibabaei et al. 2007), but is instead the latter half of the gene toward the 3’end. Indeed, finding conserved prim- Artyukh E.S. 1966. Foundations of cestodology. Vol. VI. Davaineata – helminths of wild and domestic . Nauka, Moscow, ers in the ‘barcoding’ region of flatworm mt cox1 seems to 511 pp. (In Russian). be particularly difficult (Littlewood, unpublished; Zarowiecki Brabec J., Kuchta R., Scholz T. 2006. Paraphyly of the Pseudophyl- et al. 2007). Instead, in order to amplify the fragment in a lidea (Platyhelminthes: Cestoda): circumscription of mono- short product it seems that PCR primers with a significant phyletic clades based on phylogenetic analysis of ribosomal number of degenerate bases are required, and in turn this in- RNA. International Journal for Parasitology, 36, 1535–1541. DOI: 10.1016/j.ijpara.2006.08.003. troduces the need for cloning, nested PCR amplifications or Cunningham C.W. 1997. Can three incongruence tests predict when more complicated sequencing protocols. Additional sequences data should be combined? Molecular Biology and Evolution, of mt genomes and flanking regions of the cox1 barcoding 14, 733–740. region may provide means of designing more suitable PCR Dogel’ V.A. 1962. General Parasitology. Izdatel’stvo Leningradsko- primers that complement the growing data for this region, but go Universiteta, 464 pp. (In Russian). Farris J.S., Källersjö M., Kluge A.G., Bult C. 1995. Testing signifi- most likely such primers will be restricted in the taxonomic cance of incongruence. Cladistics, 10, 315–319. range they can be successfully applied. Foronda P., Casanova J.C., Valladares B., Martinez E., Feliu C. 2004. This study highlights the potential problems in choosing Molecular systematics of several cyclophyllid families (Ces- additional genes for phylogeny reconstruction. If we were to toda) based on the analysis of 18S ribosomal DNA gene choose a gene that was not statistically different from the sequences. Parasitology Research, 93, 279–282. DOI:10.1007/ s00436-004-1130-8. nuclear genes when constrained against their inferred topolo- Fuhrmann O. 1920. Considérations générales sur les Davainea. gies (i.e. using non-parametric tests such as the Shimodaira- Festschrift für Zschokke, Basel, 27, 19 pp. Hasegawa test), then from this study we would elect the frag- Fuhrmann O. 1924. Questions de nomenclature concernant le genre ment of cox1 and reject rrnL or nad1. However, the S-H test Raillietina Fuhrmann (syn.: Davainea Bl.). Annales de Para- was passed because the cox1 solution provided little nodal sitologie Humaine et Comparée, 2, 312–313. Fuhrmann O. 1932. Les ténias des oiseaux. Mémories de l’Université support throughout the tree, and where it existed (at the base de Neucha$ tel, 381 pp. of the tree) was not in topological conflict with the nuclear Georgiev B.B. 2003. Cestoda (Tapeworms). In: (Eds. D.A. Thoney gene topologies. Instead, the greater added phylogenetic sig- and N. Schlager) Lower Metazoans and Lesser Deuterosto- nal came from mitochondrial genes that appeared to be in con- mes. Grzimek’s Life Encyclopedia. Second Edition. flict with the nuclear topologies. Failure of conditional com- Volume 1. Gale, Detroit, 225–243. Hajibabaei M., Singer G.A.C., Hebert P.D.N., Hickey D.A. 2007. bination tests of independent data sets (e.g. see Cunningham DNA barcoding: how it complements taxonomy, molecular 1997) and significant differences in topology testing (Yoder et phylogenetics and population genetics. Trends in Genetics, al. 2001) may therefore each bias a researcher from choosing 23, 167–172. DOI: 10.1016/j.tig.2007.02.001. 144 D. Timothy J. Littlewood et al.

Hoberg E.P., Jones A., Bray R.A. 1999. Phylogenetic analysis among Olson P.D., Littlewood D.T.J., Bray R.A., Mariaux J. 2001. Interre- the families of the Cyclophyllidea () based on com- lationships and evolution of the tapeworms (Platyhelminthes: parative morphology, with new hypotheses for co-evolution Cestoda). Molecular Phylogenetics and Evolution, 19, 443– in . Systematic Parasitology, 42, 51–73. 467. DOI: 10.1006/mpev.2001.0930. Hoberg E.P., Mariaux J., Brooks D.R. 2001. Phylogeny among or- Olson P.D., Tkach V.V. 2005. Advances and trends in the molecular ders of the Eucestoda (Cercomeromorphae): Integrating mor- systematics of the parasitic Platyhelminthes. Advances in phology, molecules and total evidence. In: (Eds. D.T.J. Lit- Parasitology, 60, 167–246. DOI: 10.1016/S0065-308X(05) tlewood and R.A. Bray) Interrelationships of the Platyhel- 60003-6. minthes. Taylor & Francis, London and New York, 112–126. Peterson A.P. 2002. Zoonomen Nomenclatural data. http://www. Hu M., Gasser R.B., Chilton N.B., Beveridge I. 2005. Genetic vari- zoonomen.net. ation in the mitochondrial cytochrome c oxidase subunit 1 Posada D., Crandall K.A. 1998. Modeltest: testing the model of DNA within three species of Progamotaenia (Cestoda: Anoploce- substitution. Bioinformatics, 14, 817–818. phalidae) from macropodid marsupials. Parasitology, 130, Rokas A., Carroll S.B. 2005. More genes or more taxa? The relative 117–129. DOI: 10.1017/S0031182007002752. contribution of gene number and taxon number to phyloge- Huelsenbeck J.P., Ronquist F. 2001. MRBAYES: Bayesian inference netic accuracy. Molecular Biology and Evolution, 22, 1337– of phylogenetic trees. Bioinformatics, 17, 754–755. 1344. DOI: 10.1093/molbev/msi121. Hypsa V., Skerikova A., Scholz T. 2005. Phylogeny, evolution and Schmidt G.D. 1986. CRC handbook of tapeworm identification. host–parasite relationships of the order Proteocephalidea (Eu- CRC Press Inc., Boca Raton, Florida, 675 pp. cestoda) as revealed by combined analysis and secondary Swofford D.L. 2002. PAUP*. Phylogenetic analysis using parsimony structure characters. Parasitology, 130, 359–371. DOI: 10. (*and other methods). Version 4. Sinauer Associates, Sunder- 1017/S0031182004006638. land, Massachusetts. Jeffroy O., Brinkmann H., Delsuc F., Philippe H. 2006. Phylogeno- Thompson J.D., Gibson T.J., Plewniak F., Jeanmougin F., Higgins mics: the beginning of incongruence? Trends in Genetics, 22, D.G. 1997. The ClustalX windows interface: flexible strate- 225–231. DOI: 10.1016/j.tig.2006.02.003. gies for multiple sequence alignment aided by quality analy- Johnston D.A. 2006. Genomes and genomics of parasitic flatworms. sis tools. Nucleic Acids Research, 25, 4876–4882. In: (Eds. A.G. Maule and N.J. Marks) Parasitic flatworms: van der Auwera G., Chapelle S., de Wachter R. 1994. Structure of the molecular biology, biochemistry, immunology and physiolo- large ribosomal subunit RNA of Phytophthora megasperma, gy. CAB International, Wallingford, 37–80. and phylogeny of the oomycetes. FEBS Letters, 338, 133–136. Jones A., Bray R.A. 1994. Family Davaineidae Braun, 1900. In: von Nickisch-Rosenegk M., Lucius R., Loos-Frank B. 1999. Contri- (Eds. L.F. Khalil, A. Jones and R.A. Bray) Keys to the cestode butions to the phylogeny of the Cyclophyllidea (Cestoda) parasites of vertebrates. CAB International, Wallingford, inferred from mitochondrial 12S rDNA. Journal of Molecular 407–441. Evolution, 48, 586–596. Kodedová I., Dolezel D., Broucková M., Jirku M., Hypsa V., Lukes Waeschenbach A., Webster B.L., Bray R.A., Littlewood D.T.J. 2007. J., Scholz T. 2000. On the phylogenetic positions of the Added resolution among ordinal level relationships of tape- Caryophyllidea, Pseudophyllidea and Proteocephalidea (Eu- worms (Platyhelminthes: Cestoda) with complete small and cestoda) inferred from 18S rRNA. International Journal for large subunit nuclear ribosomal RNA genes. Molecular Phy- Parasitology, 30, 1109–1113. DOI: 10.1016/S0020-7519(00) logenetics and Evolution, 45, 311–325. DOI: 10.1016/j.ympev. 00090-4. 2007.03.019. Le T.H., Blair D., McManus D.P. 2002. Mitochondrial genomes of Wardle R.A., McLeod J.A. 1952. The zoology of the tapeworms. parasitic flatworms. Trends in Parasitology, 18, 206–213. University of Minnesota Press, Minneapolis, 780 pp. DOI: 10.1016/S1471-4922(02)02252-3. Wickström L.M., Haukisalmi V., Varis S., Hantula J., Henttonen H. López-Neyra C.R. 1931. Revisión del género Davainea. Obra Pre- 2005. Molecular phylogeny and systematics of anoplocepha- miada por la Academia de Ciencias Exactas, Físicas y Natu- line cestodes in rodents and lagomorphs. Systematic Para- rales, de Madrid, 177 pp. sitology, 62, 83–99. DOI: 10.1007/s11230-005-5488-5. Lowe T.M., Eddy S.R. 1997. tRNAscan-SE: a program for improved Yamaguti S. 1959. Systema helminthum. Vol. II. The cestodes of ver- detection of transfer RNA genes in genomic sequences. Nu- tebrates. Interscience Publishers Inc., New York & London, cleic Acids Research, 25, 955–964. 860 pp. Maddison W.P., Maddison D.R. 2005. MacClade. Version 4.07. Si- Yoder A.D., Irwin J.A., Payseur B.A. 2001. Failure of the ILD to nauer Associates, Sunderland, Massachusetts. determine data combinability for slow loris phylogeny. Sys- Mariaux J. 1998. A molecular phylogeny of the Eucestoda. Journal tematic Biology, 50, 408–424. of Parasitology, 84, 114–124. DOI: 10.2307/3284540. Zarowiecki M.Z., Huyse T., Littlewood D.T.J. 2007. Making the Movsesyan S.O. 1966. On the reorganisation of the cestodes of the most of mitochondrial genomes – markers for phylogeny, genus Raillietina Fuhrmann, 1920 (Cestoda: Davaineidae). molecular ecology and barcodes in Schistosoma (Platyhel- Tematicheskiy Sbornik Rabot po Gel’mintologii Sel’skokho- minthes: Digenea). International Journal for Parasitology, zyaystvennykh Zhivotnykh. Kolos, Moscow, 12, 5–10 (In Rus- 37, 1401–1418. DOI: 10.1016/j.ijpara.2007.04.014. sian). Zehnder M.P., Mariaux J. 1999. Molecular systematic analysis of the Movsesyan S.O. 2003a. Foundations of cestodology. Vol. XIII, Pt. I. order Proteocephalidea (Eucestoda) based on mitochondrial Davaineata – helminths of animals and human. Nauka, Mos- and nuclear rDNA sequences. International Journal for cow, 395 pp. (In Russian). Parasitology, 29, 1841–1852. DOI: 10.1016/S0020-7519(99) Movsesyan S.O. 2003b. Foundations of cestodology. Vol. XIII, Pt. II. 00122-8. Davaineata – helminths of animals and human. Nauka, Mos- Zwickl D.J. 2006. Genetic algorithm approaches for the phylogenet- cow, 262 pp. (In Russian). ic analysis of large biological sequence datasets under the maximum likelihood criterion. PhD Thesis, The University of Texas at Austin, USA.

(Accepted April 21, 2008)