Phylogeny and paraphyly among tetrapod blood flukes (Digenea: Schistosomatidae and Spirorchiidae)

International Journal for Parasitology 34 (2004) 1385–1392 www.parasitology-online.com

Phylogeny and paraphyly among tetrapod blood flukes (Digenea: Schistosomatidae and Spirorchiidae)* Scott D. Snyder* University of Nebraska at Omaha, Omaha, NE 68182, USA Received 12 May 2004; received in revised form 13 August 2004; accepted 17 August 2004

Abstract The blood flukes of turtles (Digenea: Spirorchiidae) and the blood flukes of crocodilians, birds and mammals (Digenea: Schistosomatidae) have long been considered as closely related, but distinct evolutionary lineages. Recent morphological and molecular studies have considered these families as sister taxa within the Schistosomatoidea. Representatives of both families have similar furcocercous cercariae and similar two-host life cycles, but have different definitive hosts, distinct reproductive patterns and different morphologies. Sequences including approximately 1800 bases of the small subunit ribosomal DNA and 1200 bases of the large subunit ribosomal DNA were generated from representatives of eight spirorchiid genera. These sequences were aligned with pre-existing sequences of Schistosomatidae and other representatives of the Diplostomida and analysed for phylogenetic signal using maximum parsimony and Bayesian inference. These analyses revealed that the Spirorchiidae is paraphyletic and that the turtle blood flukes are basal to the highly derived schistosomatids. Three genera of spirorchiids from marine turtles form a sister group to the Schistosomatidae and five genera of spirorchiids from freshwater turtles occupy basal positions in the phylogeny of tetrapod blood flukes. Marine turtles are considered to be derived from freshwater turtles and the results of the current study indicate that the spirorchiid parasites of marine turtles are similarly derived from a freshwater ancestor. The close relationship of the marine spirorchiids to schistosomatids and the basal position of the marine transmitted Austrobilharzia and Ornithobilharzia in the schistosomatid clade suggests that schistosomatids arose after a marine turtle blood fluke ancestor successfully colonised birds. q 2004 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Digenea; Spirorchiidae; Schistosomatidae; Blood fluke; Turtles; Tetrapods

1. Introduction Members of Spirorchiidae (Platyhelminthes: Digenea) live in the vascular, lymphatic and nervous systems of turtles worldwide. Nearly 100 species (Smith, 1997) are currently circumscribed within 19 genera (Platt, 2002). However, less than 15% of turtle species have been examined for spirorchiids (Smith, 1997), indicating that the majority of spirorchiid species have yet to be discovered. Research on spirorchiids has been plagued by poorly described species and disputes over nomenclature *

Nucleotide sequence data reported in this paper are available in the GenBanke and EMBL databases under the accession numbers AY604704– AY604711. * Tel.: C1 402 554 2469; fax: C1 402 554 3532. E-mail address: [email protected]

and synonymy that have hindered the identification of species and their placement among genera (Platt, 1992, 1993). Systematic confusion within the family is such that the use of subfamilies has been determined to be ‘untenable’ (Platt, 2002). Such confusion is largely the result of divergent morphology and an inadequate specimen base. Morphological characters are difficult to evaluate for systematic relevance and many specimens are simply unavailable for examination (Platt, 2002) or, when available, are in such poor condition that they are of little value for systematic study. Morphological (Brooks et al., 1985) and molecular (Olson et al., 2003) data have placed spirorchiids as the sister taxon to the Schistosomatidae, blood flukes of crocodilians, birds and mammals. Another morphological analysis (Cribb et al., 2001) placed the Sanguinicolidae, blood flukes of fish, as the sister taxon to the Schistosomatidae with the spirorchiids

0020-7519/$30.00 q 2004 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2004.08.006

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immediately basal. Although occupying similar habitats within their respective hosts and possessing similar brevifurcate cercariae and two-host (gastropod–vertebrate) life cycles, the monoecious spirorchiids are quite morphologically distinct from the dioecious and sexually dimorphic schistosomatids (Khalil, 2002; Platt, 2002). Relationships within the Schistosomatidae, and especially within the medically important Schistosoma, have been the subject of numerous recent molecular analyses (Snyder and Loker, 2000; Agatsuma et al., 2001, 2002; Lockyer et al., 2003). However, the relationships among spirorchiids and schistosomes have received scant attention, with a single spirorchiid species used as an outgroup (Snyder and Loker, 2000) or as the lone representative of the family in a broader phylogeny (Olson et al., 2003). Similarly, the relationships among spirorchiid genera largely have been overlooked. Mehra (1934) and Byrd (1939) presented evolutionary trees of purported spirorchiid relationships, but provided few details as to how those trees were derived. In the only generic-level study to use methods of phylogenetic systematics, Platt (1992) analysed a matrix of morphological characters of Spirorchiinae with the resultant synonymy of seven genera into three. Inadequate phylogenetic examination not only limits understanding of the turtle blood flukes, but also hinders understanding of the evolution of the medically important Schistosomatidae. Platt and Brooks (1997) present several competing hypotheses for the evolution of dioecious schistosomes from a monoecious, spirorchiid-like ancestor, but the hypotheses cannot be tested for lack of a robust phylogeny including multiple species of both spirorchiids and schistosomes. The present study generated DNA sequence data from the nuclear large subunit rDNA (LSU) and the nuclear small subunit rDNA (SSU) from representatives of eight genera of spirorchiids from freshwater and marine turtles. The data were incorporated into a phylogenetic analysis along with pre-existing data from 10 genera of schistosomatids and other selected diplostomidan digeneans (sensu Olson et al., 2003).

2. Materials and methods 2.1. Specimen collection Adult spirorchiids were collected from European and North American freshwater turtles and marine turtles between 2001 and 2003. Specimens were either provided to the author or collected by the author according to Snyder and Clopton (2005). Voucher specimens, as available, are preserved in the collection of the Harold W. Manter Laboratory (HWML) of the University of Nebraska, Lincoln, USA. Taxon names, definitive hosts, collection localities, HWML accession numbers and GenBank accession numbers of specimens are provided in Table 1.

2.2. DNA extraction, amplification and sequencing Genomic DNA was extracted from single adult worms or from worm fragments using DNeasy tissue kits (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions or according to the techniques of Tkach and Pawlowski (1999). Polymerase chain reaction (PCR) was used to amplify LSU rDNA using a forward primer, U178 (5 0 -GCA CCC GCT GAAYTT AAG) (where Y is C or T) and a reverse primer, L1642 (5 0 -CCA GCG CCA TCC ATT TTC A) (Lockyer et al., 2003). Amplification of SSU rDNA used a forward primer, 18SE (5 0 -CCG AAT TCG TCG ACA ACC TGG TTG ATC CTG CCA GT) and a reverse primer, WORMB (5 0 -CTT GTT ACG ACT TTT ACT TCC) (Littlewood and Olson, 2001). Reactions were performed in a total volume of 25 ml and consisted of 25–40 ng of gDNA, 0.2 mM of each primer along with 2.5 ml of 10! buffer with MgCl2, 2 ml of dNTP mixture and 0.6 units of Taq polymerase as provided in the Takara Ex Taq kit (Takara Biomedicals, Otsu, Japan). Reaction volume was brought to 25 ml with sterile deionised water. Amplification was performed on a Perkin Elmer GeneAmp 2400 thermocycler under the following conditions: 94 8C for 4 min, followed by 40 cycles of 94 8C for 30 s, 50–56 8C for 30 s, and 72 8C for 2 min, followed by one cycle of 72 8C for 5 min. Unincorporated PCR primers and nucleotides were removed from PCR products using exonuclease I and shrimp alkaline phosphatase from a PCR Product Pre-Sequencing Kit (USB Corporation, Cleveland, OH, USA). Sequences were determined directly from PCR templates using a Beckman/ Coulter CEQ2000XL DNA sequencer and dye-terminator chemistry. Primers used in sequencing reactions for LSU rDNA included a forward primer on the 5 0 end of the fragment, Dig12 (5 0 -AAG CAT ATC ACT AAG CGG) and a reverse primer on the 3 0 end of the fragment, LSU1500R (5 0 -GCT ATC CTG AGG GAA ACT TCG) (Tkach et al., 1999, 2000). Internal primers included the forward primers 300F (5 0 -CAA GTA CCG TGA GGG AAA GTT G) and 900F (5 0 -CCG TCT TGA AAC ACG GAC CAA G) and the reverse primers 300R (5 0 -CAA CTT TCC CTC ACG GTA CTT G) (Lockyer et al., 2003) and ECD2 (5 0 -CTT GGT CCG TGT TTC AAG ACG GG) (Olson et al., 2003). Primers used in sequencing SSU rDNA included the PCR primers and the internal forward primers 388F (5 0 -AGG GTT CGA TTC CGG AG) and 1100F (5 0 -CAG AGT TTC GAA GAC GAT C) and the reverse primers CEST1R (5 0 -TTT TTC GTC ACT ACC TCC CC) and 1270R (5 0 -CCG TCA ATT CCT TTA AGT) (Littlewood and Olson, 2001). 2.3. Sequence analysis Sequence data from taxa collected for the current study (Table 1) were aligned with sequences taken from GenBank. The ingroup included representatives of the Spirorchiidae, Schistosomatidae, Sanguinicolidae and Clinostomidae. Outgroups were selected from other

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Table 1 Parasite taxa, hosts and geographical origin, along with GenBank and museum accession numbers Digenean taxa

Host species

Geographical origin

Large subunit GenBank no.

Small subunit GenBank no.

Harold W. Manter Laboratory no.

Spirorchiidae Carettacola hawaiiensisa Hapalorhynchus gracilisa

Chelonia mydas Chelydra serpentina

AY604717 AY604718

AY604709 AY604710

45789 45790

Hapalotrema mehraia Learedius learedia Spirhapalum polesianuma Spirorchis artericolaa

Chelonia mydas Chelonia mydas Emys orbicularis Chrysemys picta

AY604716 AY604715 AY604713 AY604712

AY604708 AY604707 AY604705 AY604704

45791 45792 – 45793

Unicaecum sp.a

Trachemys scripta

AY604719

AY604711

45794

Vasotrema robustuma

Apalone spinifera

Pacific Ocean, HI, USA Reelfoot Lake, Lake County, TN, USA Pacific Ocean, HI, USA Pacific Ocean, HI, USA Lesniki, Kyiv Region, Ukraine Reelfoot Lake, Lake County, TN, USA Reelfoot Lake, Lake County, TN, USA Nishnabotna River, Floyd County, IA, USA

AY604714

AY604706

45795

Schistosomatidae Austrobilharzia terrigalensis

Batillaria australis

Bilharziella polonica Dendritobilharzia pulverulenta Gigantobilharzia huronensis Heterobilharzia americana Ornithobilharzia canaliculata Orientobilharzia turkestanicum Schistosoma mansoni Schistosomatium douthitti Trichobilharzia ocellata Brachylaimidae Brachylaima thompsoni Clinostomidae Clinostomum sp. Diplostomatidae Alaria alata Leucochloridiidae Urogonimus macrostomus Leuchocloridium perturbatum Sanguinicolidae Aporocotyle spinosicanalis Plethorchis acanthus Sanguinicola inermis Strigeidae Cardiocephaloides longicollis a

AY157249

AY157223

–

Anas platyrhynchos Gallus gallus domesticus Agelaius phoeniceus Mesocricetus auratus Larus delewarensis Ovis sp. Mus musculus Mesocricetus auratus Lymanea stagnalis

Sydney Harbour, NSW, Australia Kherson Region, Ukraine Bernallio County, NM, USA Winnebago County, WI, USA Laboratory infection, UK Donley County, Texas, USA Iran Laboratory infection, UK USA Germany

AY157240 AY157241 AY157242 AY157246 AY157248 AY157254 AY157173 AY157247 AY157243

AY157214 AY157215 AY157216 AY157220 AY157222 AF442499 M62652 AY157221 AY157217

– – – – – – – – –

Blarina brevicaudata

Wisconsin, USA

AF184262

AY222085

–

Rana catesbeiana

Reelfoot Lake, Lake County, TN, USA

AY222176

AY222095

–

Nyctereutes procyonoides

Kherson Region, Ukraine

AF184263

AY222091

–

Anas platyrhynchus Turdus merula

Kherson Region, Ukraine Za´hlinice, Czech Republic

AY222168 AY222169

AY222086 AY222087

– –

Merluccius merluccius Mugil cephalus Lymnaea stagnalis

North Atlantic Ocean Brisbane River, Qld, Australia Warminia-Mazury Region, Poland

AY222177 AY222178 AY222180

AJ287477 AY222096 AY222098

– – –

Larus ridibundus

Kherson Region, Ukraine

AY222171

AY222089

–

Sequences generated as part of the present study.

representatives of the Diplostomida of Olson et al. (2003) and included Brachylaimidae, Leucochloridiidae, Diplostomidae, and Strigeidae (Table 1). Sequences were assembled using Contig Express (v. 8.0, InforMax) and provisionally aligned using Clustal W (Thompson et al., 1994), followed by alignment by eye in MacClade v. 4.06 (Maddison, D.R., Maddison, W.P., 2003. MacClade 4: analysis of phylogeny and character evolution. Sinauer, Sunderland, MA). Alignment files are available by anonymous FTP from ftp.ebi.ac. uk in directory/pub/databases/embl/align or via the EMBLALIGN database via SRS at http://www3.ebi.ac.uk/ Services/webin/help/webin-align/align_SRS_help.html; under accessions ALIGN_000693 and ALIGN_000694.

Positions for which alignment was ambiguous were removed before analysis. Maximum parsimony analysis of these data was performed using the heuristic search (1000 replicates), random sequence addition, and TBR branch-swapping options of PAUP* (v. 4.0b10) (Swofford, D.L., 2001. PAUP*: Phylogenetic Analysis Using Parsimony (and other methods), Version 4.0b10. Sinauer, Sunderland, MA). Gaps were treated as missing data and characters were unordered with equal weight. Nodal support was assessed using bootstrap resampling (Felsenstein, 1985) (1000 bootstrap replicates, 100 heuristic searches/replicate). Phylogenetic analysis of sequence data using Bayesian inference was completed with MrBayes (v. 3.0b4)

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(Huelsenbeck and Rongquist, 2001) after evaluation of models of nucleotide substitution using Modeltest (v. 3.06) (Posada and Crandall, 1998). The combined data set and the partitioned SSU and LSU data were best fit by a generaltime-reversible model with gamma distributed among-site rate variation and estimates of invariant sites (nst, 6; rates, invgamma; ncat, 4; shape, estimate; inferrates, yes; basefreqs, empirical). Posterior probabilities were generated over 1,000,000 generations using the parameters of Olson et al. (2003). Constraint analyses were used to test for differences among schistosomatid phylogenies from the current and previous studies. Fig. 1 of Snyder and Loker (2000) and fig. 3 of Lockyer et al. (2003) were drawn as constraint trees using MacClade and imported into PAUP*. Trees were tested against results from the current analysis using parsimony criteria and the Kishino–Hasegawa test (Kishino and Hasegawa, 1989) in PAUP*.

3. Results Bayesian analysis of the SSU data (1806 characters) produced a tree with the topology shown in Fig. 1A.

Parsimony analysis of the SSU data (1806 characters, 346 parsimony informative) produced five equally parsimonious trees (tree lengthZ1311) that exhibited considerable differences in the topology of the tetrapod blood flukes. A strict consensus of all five trees produced a poorly resolved topology, although within this topology the schistosomatids and spirorchiids remain a monophyletic group with the schistosomatids as the most derived lineage. Nodal support for the relationships among genera within the Schistosomatidae generally was strong (Fig. 1A), however, support for relationships among the spirorchiid genera was weak. Examination of the LSU data (1236 characters) using Bayesian inference produced a tree (Fig. 1B) with considerably better nodal support than the tree produced by analysis of SSU data (Fig. 1A). Two equally parsimonious trees were produced upon analysis of the LSU data with maximum parsimony. Both trees had a length of 2207 based on 551 parsimony informative characters. These tree differ form one another only in the placement of Carettacola. In one topology the relationship of Carettacola to Learedius and Hapalotrema is identical to that seen in Fig. 1B. In the second equally parsimonious tree Carettacola is removed from the Learedius/Hapalotrema clade and placed as the single sister taxon to the Schistosomatidae,

Fig. 1. Phylograms based on Bayesian inference of ribosomal DNA data. (A) Results of analysis of small subunit rDNA data. Nodal support above the line or left of the ‘/’ is based on posterior probabilities (Bayesian inference) and below the line or right of the ‘/’ is based on bootstrapping (maximum parsimony) (C, !50%). Branch lengths were calculated during Bayesian analysis and summarised using the ‘sumt’ command of MrBayes. (B) Results of analysis of large subunit rDNA data.

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a result consistent with Bayesian analysis of the SSU data (Fig. 1A). Parsimony analysis of the LSU data retains the monophyly of the sanguinicolids in the analysis by placing Aporocotyle, Plethorchis and Sanguinicola as members of a single clade, a result that is again consistent with Bayesian analysis of the SSU data (Fig. 1A). Sanguinicolid monophyly is at odds with Bayesian analysis of the LSU data in which Sanguinicola is the weakly supported sister taxon of Clinostomum (Fig. 1B). Maximum parsimony and Bayesian inference analyses of the combined SSU and LSU data produced single trees of identical topology (Fig. 2). These analyses included 3042 characters, 1841 of which were invariant, and 857 of which were parsimony informative. Parsimony analysis produced a single most parsimonious tree with a tree length of 3523, a consistency index of 0.513 and a retention index of 0.598. Nodal support as inferred from both posterior probabilities and bootstrapping is high throughout the tree (Fig. 2).

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Relationships among tetrapod blood flukes inferred by analysis of the combined data set (Fig. 2) are identical to those revealed by Bayesian inference analysis of LSU data (Fig. 1B). The only difference between the topologies presented in Figs. 1B and 2 is that sanguinicolids are monophyletic in the combined analysis and not in the LSU analysis. One of two equally parsimonious trees produced by analysis of the LSU data is identical to the topology produced by both parsimony and Bayesian analyses of the combined data set (Fig. 2). Differences are substantial among the topologies produced by Bayesian analysis of SSU data (Fig. 1A) and analyses of the combined SSU and LSU data (Fig. 2), however, as in the analyses of the LSU (Fig. 1B) and combined data sets (Fig. 2) the spirorchiids are basal to and form a monophyletic group with the Schistosomatidae. Constraint analyses demonstrated no significant differences in schistosomatid topology between the results of the current study (Fig. 2) and fig. 1 of Snyder and Loker (2000) (tZ1.414, PZ0.157). Similarly, the differences between Fig. 2 of the current study and fig. 3 of Lockyer et al. (2003) were not significant (tZ1.947, PZ0.052).

4. Discussion

Fig. 2. Phylogram based on Bayesian inference of the combined small subunit ribosomal DNA and large subunit ribosomal DNA data. Tree topology is identical to that produced from a maximum parsimony analysis of the combined data. Nodal support above the line or left of the ‘/’ is based on posterior probabilities (Bayesian inference) and below the line or right of the ‘/’ is based on bootstrapping (maximum parsimony). Branch lengths were calculated during Bayesian analysis and summarised using the ‘sumt’ command of MrBayes. The families to which the genera of fish and tetrapod blood flukes belong are indicated to the right of the figure; Clinostomum belongs to Clinostomidae. Definitive hosts are indicated by icons to the right of the generic names.

The Spirorchiidae and Schistosomatidae have long been recognised as distinct, but closely related families (Stunkard, 1921; Byrd, 1939; Brooks et al., 1985; Olson et al., 2003) easily differentiated by discrete reproductive patterns (monoecious vs. dioecious), different morphologies (monomorphic vs. dimorphic), and different definitive hosts (turtles vs. crocodilians, birds and mammals). Examination of molecular data from representatives of these groups calls into question the distinct nature of these families. Although estimates of phylogeny differ upon analysis of SSU (Fig. 1A), LSU (Fig. 1B) and the combined data set (Fig. 2), all three data sets place spirorchiids as the basal members of a clade in which schistosomatids are the most derived members. The Spirorchiidae is therefore paraphyletic and is of suspect taxonomic validity, although the Schistosomatidae remains as a monophyletic group within the tetrapod blood flukes. The spirorchiids previously were considered to be sister to the schistosomatids based on a morphological analysis that accepted a priori the validity of both families (Brooks et al., 1985). The only previous molecular examination of relationships between these two families (Olson et al., 2003) used only a single spirorchiid species, a sample size too small to differentiate between spirorchiids as sister or basal to the schistosomatids. The relationships among schistosomatids and spirorchiids is perhaps best examined in the context of previous studies of schistosomatid phylogeny. The topology of the Schistosomatidae produced by the combined data set in the current study (Fig. 2) is different from that produced by two of the most recent examinations of relationships within this

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family (Snyder and Loker, 2000; Lockyer et al., 2003), although constraint analysis does not find these differences to be statistically significant. All three studies report the existence of four major clades of schistosomatids (Bilharziella, Trichobilharzia, Dendritobilharzia, and Gigantobilharzia [BTDG]; Heterobilharzia and Schistosomatium; Austrobilharzia and Ornithobilharzia [AO]; Schistosoma and Orientobilharzia [SO]). However, Lockyer et al. (2003) found the BTDG clade to be basal, whereas Snyder and Loker (2000) found the SO clade basal. The current study finds strong support from both bootstrapping (maximum parsimony) and posterior probabilities (Bayesian inference) for the basal position of the AO clade (Fig. 2), a topology that matches that of Olson et al. (2003). Difference among schistosomatid topologies may reflect differences in the numbers of outgroups used to root the schistosomatid trees. Lockyer et al. (2003) used a single sanguinicolid species to root the Schistosomatidae, whereas Snyder and Loker (2000) used one spirorchiid and one sanguinicolid. The current study uses five outgroup taxa and also include three sanguinicolids and one clinostome as basal members of the ingroup. The addition of taxa to the outgroup has been found to improve the rooting of the ingroup (Graham et al., 2002). In addition, Snyder and Loker (2000) used LSU data to produce their topology, but when only LSU data are analysed in the current study the AO clade remains basal (Fig. 1B). Similarly, Lockyer et al. (2003) used SSU, LSU and cytochrome oxidase data, but their analysis of only LSU data retains the basal position of the BDGT clade. Spirorchiids of the genera Carettacola, Hapalotrema and Learedius are parasites of marine turtles that form a well supported clade (Fig. 2) sister to the schistosomatids. The sister taxon status of the marine turtle spirorchiids provides additional perspective on the basal position of the AO clade. Cercariae of Austrobilharzia and Ornithobilharzia species possess eyespots, as do all spirorchiids, and have been reported to develop in marine coenogastropod intermediate hosts (Lockyer et al., 2003). Although intermediate hosts of marine turtle spirorchiids are unknown, the life cycles of seven species representing three freshwater spirorchiid genera used in the current study (Hapalorhynchus, Spirorchis, Vasotrema) have been determined (Smith, 1997; Snyder, unpublished observation). These species parasitise freshwater pulmonate snails, suggesting that the more derived marine spirorchiids are parasites of marine gastropods. The use of a marine gastropod host by both derived marine-transmitted spirorchiids and basal marinetransmitted schistosomatids is indicative of the capture of avian hosts by an ancestral spirorchiid. The notion of such a host capture is surprising given that marine spirorchiids are monoecious and that both Austrobilharzia and Ornithobilharzia are dioecious with pronounced sexual dimorphism. The only indications of sexual dimorphism in the Spirorchiidae have been reported in two putative species of Uterotrema (Platt and

Blair, 1996). Uterotrema burnsi appears to be a fully functional hermaphrodite whereas the more robust Uterotrema kreffti has greatly reduced female reproductive anatomy, but apparently possesses a fully functional testis. Platt and Blair (1996) suggest that these putative species may, in fact, represent a single androdioecious species and Platt and Brooks (1997) view this phenomenon as an indication that the evolutionary foundations of dioecy and sexual dimorphism were laid in the Spirorchiidae. No Uterotrema spp. were available for the current study and their evolutionary affinities remain unknown. However, as parasites of Australian freshwater turtles, they are not expected to be closely related to the cosmopolitan marine turtle parasites Carettacola, Hapalotrema and Learedius that form the sister taxon to the Schistosomatidae. This gap in our knowledge emphasises the need for the incorporation of additional spirorchiid taxa into examinations of schistosomatid evolution. Hapalorhynchus, Unicaecum, Spirorchis, Spirhapalum and Vasotrema occupy positions basal to the marine spirorchiids and schistosomatids (Fig. 2). Representatives of all five genera are exclusively parasites of freshwater turtles, suggesting that marine spirorchiids arose from a freshwater ancestor. Marine turtles are among the most derived of all turtles and are thought to have themselves arisen from a freshwater ancestor in a major radiation of cryptodiran turtles some 90–120 million years ago (Shaffer et al., 1997). The limited number of spirorchiid taxa available for the current study makes it impossible to determine if marine spirorchiids coevolved with their hosts as these turtles diverged from extant freshwater lineages or if the parasitism of marine turtles by spirorchiids arose as a result of host capture. Similarly, limited spirorchiid taxon sampling prevents anything more than speculation about the origin and divergence of freshwater spirorchiids. About 20 species of Hapalorhynchus have been reported from pelomedusid, batagurid, trionychid, chelydrid and kinosternid turtles in Africa, Asia and North America (Byrd, 1939; Brooks and Sullivan, 1981; Bourgat and Kulo, 1987; Bourgat, 1990; Platt, 1988, 2002). These hosts represent both side-neck turtles (Pleurodira) and hidden-neck turtles (Cryptodira), two lineages encompassing all extant turtles that are thought to have arisen on the supercontinent Pangea during the late Triassic (Gaffney and Kitching, 1994; Rougier et al., 1995). The diverse hosts and widespread geographical distributions of Hapalorhynchus suggests that this genus may also have been Pangean in origin and that it diversified along with its hosts. Unicaecum is basal to Hapalorhynchus yet the two species circumscribed within Unicaecum are found only in North American emydids (Platt, 2002), one of the most highly derived lineages of cryptodiran turtles (Shaffer et al., 1997). If Hapalorhynchus did originate in Pangea the lineage represented by Unicaecum surely did as well and the lineage should be expected to have a much wider

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geographic distribution than is currently described. However, as only some 15% of the world’s turtle species reportedly have been examined for spirorchiids (Smith, 1997) and only eight of the 19 recognised spirorchiid genera are represented in the current study a large diversity of blood flukes representing the Unicaecum lineage may remain undiscovered or unanalysed. The basal spirorchiids in the current analysis are also restricted in both host and geographical distribution when compared to Hapalorhynchus. Spirorchis is represented by eight to 10 known species (Platt, 1993) that are largely restricted to emydid turtles, although two species have also been reported from the chelydrid, Chelydra serpentina. Sister to Spirorchis is Spirhapalum, a Eurasian genus circumscribing two species that parasitise the derived emydids and batagurids (Shaffer et al., 1997; Platt, 2002). In Platt’s (1992) morphological analysis of relationships within the Spirorchiinae Plasmiorchis, parasites of Indian batagurids, were found to be sister to Spirorchis with Spirhapalum basal to these two genera. Plasmiorchis was not available for the current study. Vasotrema, the genus basal to Spirorchis and Spirhapalum (Fig. 2), is comprised of five species that parasitise North American trionychids. Trionychids are of Laurasian distribution and arose before the emydids and batagurids (Shaffer et al., 1997), turtle families that are parasitised by Spirorchis and Spirhapalum. The sister status of Spirorchis and Spirhapalum and their respective distributions in North America and Eurasia indicate that these lineages, along with Vasotrema, arose prior to the breakup of Laurasia some 100 million years ago. A Laurasian origin of this basal lineage implies that the derived Hapalorhynchus colonised African turtles after the breakup of Pangea. This alternative to the hypothesis that Hapalorhynchus arose on Pangea as, by extension, did all of the more basal spirorchiids cannot be tested given our limited understanding of spirorchiid distribution and diversity. Like Unicaecum, Vasotrema is only represented in North America although its basal position in a Laurasian clade indicates that it might be expected in Eurasian trionychids. As mentioned previously relationships within the Schistosomatidae differ among the current study (Fig. 2) and previous examinations (Snyder and Loker, 2000; Lockyer et al., 2003). Within the current study, nodal support for the basal position of the marine transmitted avian parasites Austrobilharzia and Ornithobilharzia is strong (Figs. 1B and 2), although analysis of the SSU data alone does not support this position (Fig. 1A). Previous work in the Platyhelminthes has indicated that the use of SSU data alone (Fig. 1A) produces less reliable estimates of phylogeny than the combination of SSU and LSU data (Olson et al., 2003). The basal position of Austrobilharzia and Ornithobilharzia indicates that an ancestral marine-transmitted bird

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parasite was successful in colonising both freshwater snails and mammals (hosts to more derived Orientobilharzia, Schistosoma, Heterobilharzia and Schistosomatium). Cercariae of one of these lineages (Orientobilharzia and Schistosoma) lost their eyespots whereas descendents in the other lineage (Bilharziella, Trichobilharzia, Dendritobilharzia, Gigantobilharzia) maintained freshwater snail hosts, but reacquired birds as definitive hosts. One of the most recent attempts to arrange spirorchiid genera in higher taxonomic groups reported nine subfamilies, six of which were monotypic (Yamaguti, 1971). Of taxa in the current study Carettacola (Carettacolinae), Hapalorhynchus (Hapalorhynchinae), Hapalotrema (Hapalotrematinae), Unicaecum (Unicaecuminae) and Vasotrema (Vasotrematinae) all belong to subfamilies circumscribing members of a single genus. The remaining three genera belong to the Spirorchiinae. Of these the freshwater Spirorchis and Spirhapalum form a monophyletic group at the base of the tetrapod blood flukes (Fig. 2), but the marine Learedius is highly derived and most closely related to other marine spirorchiids. Smith (1972) presented an alternative systematic that is no better supported by the current work than the scheme of Yamaguti (1971). The current study demonstrates that the Spirorchiidae is paraphyletic and therefore taxonomically invalid. If taxonomy is to reflect evolutionary history the blood flukes of tetrapods (SpirorchiidaeCSchistosomatidae) should either be classified as a single family or the Spirorchiidae broken apart into numerous smaller families. Given the major morphological and life history features that unite the Schistosomatidae the latter alternative seems the most likely course. Morphological dissimilarity has precluded a comprehensive systematic within the Spirorchiidae and the reciprocal illumination provided by both molecules and morphology may be the only way to make sense of this taxonomic tangle. Unfortunately, such decisions must await the availability of fresh specimens of these fascinating worms.

Acknowledgements I wish to thank Dr Thierry Work of the United States Geological Service, Honolulu, HI and Dr Vasyl Tkach of the University of North Dakota for specimens collected in support of this work. I also gratefully acknowledge use of the Reelfoot Lake Research and Teaching Center and advice on Bayesian analysis from Dr Peter Olson of the Natural History Museum (London). This work was funded by the University Committee on Research of the University of Nebraska at Omaha, the National Research Council (USA) and NIH Grant 1 P20 RR16469 [BRIN].

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