New Genus of Blood Fluke (Digenea: Schistosomatoidea) from Malaysian Freshwater Turtles (Geoemydidae) and its Phylogenetic Position Within Schistosomatoidea | Journal of Parasitology
Sun, 11 Sep 2016 04:43
Jackson R. Roberts, Thomas R. Platt, Raphael Or(C)lis-Ribeiro, and Stephen A. Bullard (2016) New Genus of Blood Fluke (Digenea: Schistosomatoidea) from Malaysian Freshwater Turtles (Geoemydidae) and its Phylogenetic Position Within Schistosomatoidea. Journal of Parasitology: August 2016, Vol. 102, No. 4, pp. 451-462.SYSTEMATICS-PHYLOGENETICS
Jackson R. Roberts,Thomas R. Platt*,Raphael Or(C)lis-Ribeiro, andStephen A. BullardAquatic Parasitology Laboratory, School of Fisheries, Aquaculture, and Aquatic Sciences, College of Agriculture, Auburn University, 203 Swingle Hall, Auburn, Alabama 36849.
*Department of Biology, Saint Mary's College, Notre Dame, Indiana 46556.
Baracktrema obamai n. gen., n. sp. infects the lung of geoemydid turtles (black marsh turtle, Siebenrockiella crassicollis [type host] and southeast Asian box turtle, Cuora amboinensis) in the Malaysian states of Perak, Perlis, and Selangor. Baracktrema and UnicaecumStunkard, 1925 are the only accepted turtle blood fluke genera having the combination of a single cecum, single testis, oviducal seminal receptacle, and uterine pouch. Baracktrema differs from Unicaecum by having a thread-like body approximately 30''50 longer than wide and post-cecal terminal genitalia. Unicaecum has a body approximately 8''12 longer than wide and terminal genitalia that are anterior to the distal end of the cecum. The new genus further differs from all other accepted turtle blood fluke genera by having a cecum that is highly convoluted for its entire length, a spindle-shaped ovary between the cirrus sac and testis, a uterine pouch that loops around the primary vitelline collecting duct, a Laurer's canal, and a dorsal common genital pore. Phylogenetic analysis of the D1''D3 domains of the nuclear large subunit ribosomal DNA (28S) revealed, with high nodal support and as predicted by morphology, that Baracktrema and Unicaecum share a recent common ancestor and form a clade sister to the freshwater turtle blood flukes of Spirorchis, paraphyletic Spirhapalum, and Vasotrema and that, collectively, these flukes were sister to all other tetrapod blood flukes (Hapalorhynchus + Griphobilharzia plus the marine turtle blood flukes and schistosomes). Pending a forthcoming emended morphological diagnosis of the family, the clade including Spirorchis spp., paraphyletic Spirhapalum, Vasotrema, Baracktrema, and Unicaecum is a likely placeholder for ''Spirorchiidae Stunkard, 1921'' (type genus SpirorchisMacCallum, 1918; type species Spirorchis innominatusWard, 1921). The present study comprises the 17th blood fluke known to infect geoemydid turtles and the first proposal of a new genus of turtle blood fluke in 21 yr.
Received: September 17, 2015; Revised: March 4, 2016; Accepted: March 31, 2016;
Blood flukes (Schistosomatoidea; see Or(C)lis-Ribeiro et al., 2014) that mature in turtles (''turtle blood flukes,'' hereafter TBFs) have historically been included in paraphyletic ''Spirorchiidae'' (see Platt, 2002; Snyder, 2004; Or(C)lis-Ribeiro et al., 2014). They total 83 accepted species (53 freshwater; 30 marine) assigned to 19 genera and infect 40 turtle species (37 freshwater, 3 marine; Smith 1997a, 1997b; Platt, 1993, 2002; Tkach et al., 2009; Platt and Sharma, 2012). Nearly all (320 of 327; 98%) extant turtle species occur in freshwater, but a disproportionate number (36%) of TBFs have been described from only 3 of the 7 (43%) extant marine turtles: Caretta caretta (Linnaeus, 1758), Chelonia mydas (Linnaeus, 1758), and Eretmochelys imbricata (Linnaeus, 1766). Because (1) only 40 of 327 (12%) turtle species are documented as TBF hosts, (2) there are more than twice as many accepted TBF species (83) as documented turtle hosts, and (3) only 7 of 327 (2%) turtle species are marine (Smith, 1997a, 1997b; Platt, 2002; Snyder, 2004; van Dijk et al., 2014), we suspect that a large proportion of TBF biodiversity remains to be discovered, especially among freshwater turtles.
The iconic status and conservation rearing of marine turtles perhaps has hastened the study of their parasites and diseases (e.g., Witham, 1973; Glazebrook and Campbell, 1990a, 1990b), but freshwater turtles are less studied, despite a growing freshwater turtle aquaculture industry (Haitao et al., 2008). Like their marine counterparts, however, freshwater turtles are gaining protection and obtaining higher conservation status (IUCN, 2015) such that examination of these hosts for parasites may become increasingly prohibited (Platt and Sharma, 2012). As such, efforts toward TBF species discovery will likely focus on wide-ranging turtle species of low conservation concern or on aquacultured turtles. Yet, as with marine turtles, an understanding of host-parasite relationships, life cycles, immunology, and pathogenesis will become increasingly relevant due to the need for monitoring, management, and conservation of imperiled freshwater turtle populations (Benz and Collins, 1997). Specific details about how freshwater turtles are cultured are lacking (Haitao et al., 2008), but turtles confined in high densities and kept in extensive systems (e.g., earthen ponds) could develop intense TBF infections (e.g., Holliman and Fisher, 1968) in the presence of a susceptible snail host. This is analogous to sporadic aquaculture disease issues associated with fish blood flukes in cultured marine and freshwater fishes (Bullard and Overstreet, 2002, 2008). Moreover, in the face of scarcity of clean water and expansion of hydroelectric development, freshwater turtle habitats will be increasingly subjected to anthropogenic change globally, possibly leading to deleterious changes in the host-parasite relationship or local extinction of some blood flukes (depending on the biology of the intermediate host). Observations of marine TBF infections indicate that some TBFs can be pathogenic, but less is known about those aspects of the adult TBF-freshwater turtle relationship (Holliman and Fisher, 1968; Holliman et al., 1971), although previous work has focused on host cellular response to TBF eggs (Goodchild and Dennis, 1967).
Extant turtles are classified in the suborders Pleurodira (Chelidae + Pelomedusidae + Podocnemididae) and Cryptodira (11 families, including Geoemydidae) (van Dijk et al., 2014; Crawford et al., 2015). Eight of those 14 turtle families host TBFs: Geoemydidae (10 of 69; Table I), Chelidae (2 of 56 species infected), Cheloniidae (3 of 6), Chelydridae (1 of 4), Emydidae (10 of 53), Kinosternidae (3 of 26), Pelomedusidae (2 of 18), and Trionychidae (9 of 31). More than half of the species in each of these turtle families have not been examined for TBF infections. Regarding geoemydids, the host family of the present study, 10 of 69 (14%) species ranging from central Europe to southeast Asia host 16 TBFs assigned to 5 genera (Mehra, 1934, 1940; Sinha, 1934; Takeuti, 1942; Simha, 1958; Dwivedi, 1967; Rohde et al., 1968; Mehrotra, 1973a, 1973b; Tandon and Gupta, 1982, 1985; Tkach et al., 2009; Platt and Sharma, 2012; van Dijk et al., 2014). That 10 geoemydids are infected by species assigned to 5 TBF genera indicates a proportionally high level of generic, and perhaps species-level, TBF diversity among geoemydids.
Herein we propose a new TBF genus based on specimens collected from 2 previously reported geoemydid hosts'--the black marsh turtle, Siebenrockiella crassicollis (Gray, 1830), and the southeast Asian box turtle, Cuora amboinensis (Riche in Daudin, 1801)'--as well as conduct a phylogenetic analysis including the new genus and based on sequence data for the partial D1''D3 domains of 28S rDNA. This is the first proposal of a new TBF genus in 21 yr (Platt and Pichelin, 1994), i.e., since UterotremaPlatt and Pichelin, 1994.
Turtles were purchased from commercial turtle trappers during January and February 2008. Turtles were injected with a lethal dose of sodium pentabarbitol and necropsied by TRP using standard procedures (Platt, 1988, 1998) after the animal failed to respond with an eye blink when touched with a camel-hair paint brush. TBFs were killed by placing them in a small vial with enough saline to prevent desiccation, followed by the addition of steaming 5% neutral buffered formalin before the vial was capped and gently shaken. TBFs were stored in formalin, rinsed in water to remove formalin before staining, stained with Ehrlich's hematoxylin, cleared in methyl salicylate, prepared as whole mounts in Canada balsam, and studied using a compound microscope with differential interference contrast (DIC) optics. Illustrations of stained, whole-mounted specimens were made with the aid of a Leica DM-2500 microscope (Leica Microsystems, Wetzlar, Hesse, Germany) equipped with DIC and a drawing tube. Measurements are herein reported in micrometers (Î¼m), followed by the number measured in parentheses. Turtle scientific names and taxonomic authorities follow van Dijk et al. (2014). Higher-level turtle classification and nomenclature follows Guillon et al. (2012) and Crawford et al. (2015), and anatomical terms for turtles follow Ashley (1974). Classification and anatomical terms for TBFs follow Byrd (1939: uterine pouch), Luhman (1935: genital sucker), Platt (1998, 2002: genital spines, plicate organ, and most other terms; 1993: median esophageal pouch), and Yamaguti (1971: Manter's organ).
Specimens for molecular analyses were preserved in 95% EtOH and processed for sequencing the D1''D3 domains of the nuclear large subunit ribosomal DNA (28S). Total genomic DNA was extracted using DNeasyTM Blood and Tissue Kit (Qiagen, Valencia, California). Polymerase chain reaction (PCR) was carried out using the forward primer ''U178'' (5'²-GCA CCC GCT GAA YTT AAG) (where Y is C or T) and the reverse primer ''L1642'' (5'²-CCA GCG CCATCC ATT TTC A) (Lockyer et al., 2003). PCR amplifications were performed in a total volume of 25 Î¼l, consisting of approximately 25''40 ng of template of gDNA, 0.2 Î¼M of each primer along with 2.5 Î¼l of 10 buffer with MgCl2, 2 Î¼l of dNTP mixture and 0.6 units of Taq polymerase as provided in the Takara Ex Taq kit (Takara Biomedicals, Otsu, Japan). The cycling profile consisted of an initial 4 min at 94 C for denaturation, followed by 40 repeating cycles of 94 C for 30 sec for denaturation, 50''56 C for 30 sec for annealing, and 72 C for 2 min for extension, followed by a final 5 min at 72 C for extension. All PCR reactions were carried out in a Perkin Elmer GeneAmp 2400 thermocycler (Perkin Elmer, Waltham, Massachusetts). PCR products were purified using the PCR Product Pre-Sequencing Kit (USB Corporation, Cleveland, Ohio) following the manufacturer's protocols. Sequences were determined directly from PCR templates using a Beckman/Coulter CEQ2000XL DNA sequencer and dye-terminator chemistry. Primers used in sequencing reactions included a forward primer on the 5'² end of the fragment, Dig12 (5'²-AAG CAT ATC ACT AAG CGG), and a reverse primer on the 3'² end of the fragment, LSU1500R (5'²-GCT ATC CTG AGG GAA ACT TCG) (Tkach et al., 1999, 2000). Internal primers included the forward primers 300F (5'²-CAA GTA CCG TGA GGG AAA GTT G) and 900F (5'²-CCG TCT TGA AAC ACG GAC CAA G) and the reverse primers 300R (5'²-CAA CTT TCC CTC ACG GTACTT G) (Lockyer et al., 2003) and ECD2 (5'²-CTT GGT CCGTGT TTC AAG ACG GG) (Olson et al., 2003).
Sequences obtained were aligned with those for blood flukes available in GenBank. Homologous sequences from representatives of Diplostomoidea were used as outgroups (Or(C)lis-Ribeiro et al., 2014). The ingroup consisted of the sequence data from the new species (GenBank No. KX061500) plus 73 taxa from Or(C)lis-Ribeiro et al. (2014) (GenBank accession numbers reported therein), excluding short sequences from Aporocotyle spinosicalis (AF167094) and Cardicola coeptus (JF803976) and blood fluke cercariae. Sequences were aligned using MAFFT (Katoh and Toh, 2010) with default settings implemented in the CIPRES Science Gateway ver. 3.3 (Miller et al., 2010). The alignment was refined by eye using MEGA version 5.2.2 (Tamura et al., 2011), and ends of each fragment were trimmed to match the shortest sequence. Ambiguous positions were identified and removed using the Gblocks server (Castresana, 2000) with settings for a less stringent selection and the following parameters: minimum number of sequences for a conserved position = 40; minimum number of sequences for a flanking position = 40; maximum number of contiguous non-conserved positions = 8; minimum length of a block = 5; allowed gap positions = with half. Bayesian inference (BI) was performed using the Metropolis-coupled Markov chain Monte Carlo method (MC3) in MrBayes version 3.2.6 (Huelsenbeck et al., 2001; Ronquist and Huelsenbeck, 2003; Huelsenbeck and Ronquist, 2005) and run on CIPRES (Miller et al., 2010). Model of evolution was selected based on the Akaike Information Criterion (Posada and Buckley, 2004) as implemented in the jModelTest version 2.1.4 (Darriba et al., 2012; Guindon and Gascuel, 2003). The GTR + I + G (proportion of invariable sites = 0.288 and gamma distribution = 1.352) model was inferred as the best estimator; therefore, BI used the following parameters: nst = 6, rates = invgamma, ngammacat = 4, and default priors. Analyses were run in duplicate each containing 4 independent chains (3 heated and 1 cold chain) (nchains = 4) for 1.0 107generations (ngen = 10,000,000) sampled at intervals of 1,000 generations (samplefreq = 1,000). Results of the first 2,500 sampled trees were discarded as ''burn-in'' based on the stationarity of the likelihood values, assessed by plotting the log-likelihood values of the sample points against generation time using Tracer version 1.5 (Rambaut and Drummond, 2009). All retained trees were used to estimate posterior probability of each node. A majority rule consensus tree with average branch lengths was constructed for the remaining trees using ''summarize the trees'' (sumt) in MrBayes. Resulting phylogenetic trees were visualized using FigTree v1.3.1 (Rambaut, 2009). Branch supports were considered as significant when posterior probabilities were >0.95.
Baracktreman. gen. Roberts, Platt, and Bullard
View larger version (13K)Figure 1.Body of holotype (USNM Coll. No. 1254763) of Baracktrema obamai n. gen., n. sp. from black marsh turtle, Siebenrockiella crassicollis (Gray, 1831), (Testudines: Geoemydidae) from Perak, Perlis, and Selangor states of Malaysia. Scale value aside bar. Anterior sucker (as), esophagus (es), esophageal-cecal junction (ecj), anterior extent of testis (at), posterior extent of testis (pt), ovary (ov), cecal termination '(ct), and common genital pore (cgp). Roman numerals and dashed lines indicate body segments that are illustrated at higher magnification (see Figs. 2''7). Principally ventral view; anterior portion is ventrolateral view.
View larger version (59K)Figures 2''7.Holotype (USNM Coll. No. 1254763) of Baracktrema obamai n. gen., n. sp. from black marsh turtle, Siebenrockiella crassicollis (Gray, 1831), (Testudines: Geoemydidae) from Perak, Perlis, and Selangor states of Malaysia. Scale value aside bar, all figures are the same scale. (2) Anterior body segment (II) showing anterior sucker (as), pharynx (ph), nerve commissure (nc), esophagus (es), esophageal gland (eg), esophageal swelling (sw), cecum (ce), and vitellarium (v). Ventrolateral view. (3) Body segment (III). Ventral view. (4) Body segment (IV). Ventral view. (5) Body segment (V). Ventral view. (6) Body segment (VI). Ventral view. (7) Body segment (VII) showing ovary (ov), junction of vitellarium and primary vitelline collecting duct (jvc), vas deferens (vd), junction of ovary and oviduct (joo), vitelline duct (vt), cecal termination (ct), o¶type (oo), oviducal seminal receptacle (osr), ascending portion of uterine pouch (aup), junction of uterine pouch with uterus (jup), descending portion of uterine pouch (dup), seminal vesicle (sv), cirrus (ci), distal portion of uterus (diu), common genital pore (cgp), and excretory pore (ep). Ventral view.
View larger version (56K)Figures 8''9.Baracktrema obamai n. gen., n. sp. (8) Genitalia of holotype (USNM Coll. No. 1254763) from lung of black marsh turtle, Siebenrockiella crassicollis (Gray, 1831), (Testudines: Geoemydidae) from Perak, Perlis, and Selangor states of Malaysia. Junction of vitelline duct with oviduct (jvo), oviduct (od), Laurer's canal (Lc), Laurer's canal pore (Lcp), vitelline duct (vt), junction of uterine pouch with uterus (jup), descending portion of uterine pouch (dup), distal portion of uterus (diu), vas deferens (vd), o¶type (oo), uterus (u), oviducal seminal receptacle (osr), ascending portion of uterine pouch (aup), seminal vesicle (sv), cirrus (ci), common genital pore (cgp), and excretory pore (ep). Ventral view. (9) Genitalia of paratype (USNM Coll. No. 1254764) from lung of Southeast Asian box turtle, Cuora amboinensis (Daudin, 1801), Geoemydidae, from the Selangor state of Malaysia, showing comparable features as in Figure 8. Dorsal view.
View larger version (30K)Figure 10.Phylogenetic relationships of blood flukes reconstructed by Bayesian inference and based on partial D1''D3 domains of 28S from 79 taxa (majority rules consensus tree). Numbers aside tree nodes indicate posterior probability. Definitive hosts are indicated by icons aside tree nodes.
Diagnosis.Body cylindrical (not flattened or ventrally concave), thread-like, extremely elongate (approximately 30''50 longer than wide), with posterior end tapering more sharply than anterior end, lacking tegumental spines, body protuberances, and ventral sucker. Anterior sucker terminal, comprising an apparently protrusive and eversible hemisphere directing anteriad. Pharynx present, enveloping anterior extremity of esophagus, centered on mouth, aspinous. Esophagus short (approximately 5% of body length), ventral to anterior nerve commissure, lacking diverticula, straight anteriorly and slightly sinuous posteriorly, expanding laterally to form esophageal swelling immediately anterior to esophageal-intestinal junction; esophageal gland surrounding esophagus from level of nerve commissure to esophageal-intestinal junction, strongly basophilic anterior to esophageal-intestinal junction. Plicate organ and median esophageal pouch absent. Intestine comprising a single cecum, smooth (lacking diverticula or secondary rami), extremely elongate (up to 90% of body length), terminating posterior to gonads, extensively convoluted along entire length. Testis dorsal or lateral to cecum, occupying middle to posterior portion of body, exceeding 1/4''1/3 of body length. Cirrus sac present. Genital sucker and genital spines absent. Ovary single, longer than wide, spindle-shaped, lacking lobes, dextral, dorsal to cecum, anterior to distal tip of cecum, post-testicular. Oviduct extending posteriad from ovary, medial; middle portion of oviduct functioning as oviducal seminal receptacle. Laurer's canal post-cecal, post-gonadal, opening dorsally near level of o¶type. Vitellarium follicular, filling space surrounding cecum between esophageal-intestinal junction and ovary. O¶type a strongly glandular and thick-walled spheroid chamber, post-cecal, post-gonadal, anterior to common genital pore. Uterus post-gonadal, post-cecal, extensively convoluted, with associated uterine pouch; uterine pouch ventral to uterus and all other genitalia, post-cecal, post-gonadal, inverse U-shaped, comprising distinctive ascending (proximal) and descending (distal) portions; ascending portion extending dorsoanteriad from uterus, ventral to vas deferens, curving mediad before connecting with descending portion of uterine pouch; descending portion of uterine pouch laterally expanded, massive, extensively convoluted, extending posteriad before ending blindly. Common genital pore dorsal, sinistral. Excretory vesicle y-shaped; excretory pore terminal. Manter's organ absent.
Differential diagnosis.Body cylindrical, extremely elongate (approximately 30''50 longer than wide), lacking spines and ventral sucker. Anterior sucker a protrusive and eversible hemisphere directing anteriad. Esophagus short (approximately 5% of body length). Intestine comprising a single cecum, extremely elongate, terminating posterior to gonads, extensively convoluted. Testis exceeding 1/4''1/3 of body length. Ovary spindle-shaped, anterior to distal tip of cecum, post-testicular, occupying posterior 1/5 of body. Laterally expanded medial portion of oviduct an oviducal seminal receptacle. Laurer's canal post-cecal, post-gonadal, opening dorsally near o¶type. O¶type a glandular and thick-walled spheroid chamber, post-cecal, post-gonadal, anterior to common genital pore. Uterus post-gonadal, post-cecal, convoluted along entire length; uterine pouch post-cecal, post-gonadal, inverse U-shaped, with ascending and descending portions. Common genital pore dorsal, sinistral. Manter's organ absent.
Type species.Baracktrema obamai n. sp.
Baracktrema obamain. sp. Roberts, Platt, and Bullard
Description of adult (based on 17 individuals comprising 5 and 12 intact and partial specimens, respectively; number in parentheses indicates number of specimens measured).Body 3,550''6,530 (5) long, 58''130 (11) wide or 1''2% (4) body length at level of esophageal-intestinal junction, 68''158 (13) wide or 2''3% (4) body length at level of testis (typically maximum width of specimen), 43''75 (11) wide or 1''2% (5) body length at level of genital pore (Figs. 1''7). Anterior sucker 10''23 (10) long or 0.95. With high nodal support and as predicted by morphology, Baracktrema and Unicaecum share a recent common ancestor and form a clade sister to the freshwater turtle blood flukes of Spirorchis, paraphyletic Spirhapalum, and Vasotrema and that, collectively, these flukes were sister to all other tetrapod blood flukes (Hapalorhynchus + Griphobilharzia plus the marine turtle blood flukes and schistosomes). The sequences for Baracktrema and Unicaecum produced relatively long branch lengths (comparable to those of the freshwater TBF Hapalorhynchus and of the crocodilian blood fluke Griphobilharzia), perhaps reflective of the marked morphological differences between these taxa; further justifying their assignment to distinct genera. The present phylogenetic analysis also reiterated support for monophyly of Schistosomatidae and paraphyly of TBFs (as well as Spirhapalum), with the blood flukes of freshwater turtles sister to all other tetrapod blood flukes (TBFs of marine turtles and schistosomes). We noted that some schistosome interrelationships differed from those previously reported (Or(C)lis-Ribeiro et al., 2014), e.g., Bivitellobilharzia spp. were sister to the clade comprising all non-Schistosoma schistosomatids. Interrelationships of the fish blood flukes (Aporocotylidae Odhner, 1912) echoed that of Or(C)lis-Ribeiro et al. (2014) wherein Aporocotylidae was monophyletic and sister to all tetrapod blood flukes (TBFs + schistosomes), and the euteleostean aporocotylids were monophyletic and sister to the chondrichthyan blood fluke Chimaerohemecus trondheimensis van der Land, 1967.
The life cycle of B. obamai should resemble that of other TBFs and schistosomes, requiring a snail first intermediate host and having a furcocercous cercariae that directly penetrates the vertebrate definitive host. Adults of B. obamai infect the lung arterioles, where clusters of tens to hundreds of eggs infected lung alveoli of specimens of both turtle host species. The mechanism involved for the transit of the eggs from the circulatory system to the alveoli could rely on the immune system of the host, as demonstrated for some schistosomes (Damian, 1987; Pearce, 2005). Alternatively, or in addition, eggs of B. obamai may be deposited directly into the luminal space of the alveoli by the fluke. How eggs travel from the lung to the external environment is not known to us; however, we speculate that some combination of active expulsion (coughing) and the bronchial escalator are involved.
The life cycles of TBFs and schistosomes differ regarding egg development: schistosome eggs are fully developed before they are released from the definitive host and hatch shortly after contact with water (Tanabe, 1923; Stunkard and Hinchliffe, 1952; Wu, 1953; Najim, 1956; Sinha and Srivastava, 1960; Greene, 1962; Lee and Wykoff, 1966; Yamaguti, 1975); however, TBF eggs apparently require a period of development in the external environment before hatching (Goodchild and Kirk, 1960; Holliman and Fisher, 1968; Wall, 1941a, 1941b, 1951). Examination of the eggs of B. obamai in saline at the time of necropsy by TRP revealed larvated eggs each having a fully developed, ciliated miracidium occupying 1/3''1/2 of the egg lumen. Miracidia swam actively inside the egg, immediately emerged upon the addition of freshwater and swam in the observation dish. Although the function of the uterine pouch in the new species is unknown, it is possible that the transit (or placement) of the eggs from the circulatory system into the lung occurs too quickly to allow for completion of larval development. Fertilized eggs could be held in the uterine pouch until development is complete such that eggs would be capable of hatching upon contact with environmental water. This scenario is highly speculative, and we did not observe a mass of eggs in the uterine pouch of any live specimen; however, the notion is intuitive based upon available facts regarding the life cycle of this and related species.
The morphological and molecular phylogenetic affinities between Baracktrema (1 species infecting Geoemydidae in the Indomalayan region only) and Unicaecum (2 species infecting Emydidae in North America only) (see Remarks) are intriguing regarding co-diversification of blood flukes and their turtle hosts. Baracktrema obamai infects C. amboinensis and S. crassicollis; U. ruszkowskii infects G. pseudogeographica and T. scripta; and U. dissimilis infects Graptemys geographica (Le Sueur, 1817) and T. scripta. These turtle families as well as Testudinidae plus monotypic Platysternon (Gray, 1831) comprise superfamily Testudinoidea, which has an extensive fossil record in Asia, where most extant species range (Crawford et al., 2015). Crawford et al. (2015) hypothesized that the Emysternia (Emydidae + Platysternon) entered the nearctic via high-latitude dispersal routes during the Paleogene (65''1.5 mya; a warming period) (see also Estes and Hutchison, 1980; Holroyd et al., 2001; Zachos et al., 2001; Eberle et al., 2010; Hutchison, 2013). In light of hypothesized Paleogene dispersal, the close morphological and molecular similarities among Baracktrema and Unicaecum are noteworthy. This also potentially applies to other TBF genera of the Indo-Malayan, Palearctic, and Nearctic regions (Or(C)lis-Ribeiro et al., 2014; Crawford et al., 2015). Platysternon megacephalum (Gray, 1831), the monotypic extant member of Emysternia (Crawford et al., 2015), and other unsampled geoemydids are critical hosts to examine for testing such biogeographic hypotheses.
Molecular phylogenetics of turtle and fish blood flukes remains, unsurprisingly, in a state of flux. Tree topologies differ, sometimes markedly, among the various laboratories studying them; perhaps related to outgroup choice, taxon sampling, fragment length, analysis method, alignment parameters, treatment of gaps, and model of evolution used. Pending a revised morphological diagnosis, we considered only existing 28S sequence data (see Or(C)lis-Ribeiro et al.  for a complete list) from adult specimens with confirmed identities (i.e., excluding sequences derived from cercariae, which we think are not defensibly morphologically identifiable, as of yet, to the level of species, genus, or blood fluke family). As a result, we view the clade including Spirorchis spp., paraphyletic Spirhapalum, Vasotrema, Baracktrema, and Unicaecum as a likely placeholder for ''Spirorchiidae Stunkard, 1921'' (type genus SpirorchisMacCallum, 1918; type species Spirorchis innominatusWard, 1921; see MacCallum, 1918; Stunkard, 1921; Ward, 1921; Platt, 2002) (Fig. 10). Accepting the current definition of monophyletic Schistosomatidae (crown group within Schistosomatoidea [see Or(C)lis-Ribeiro et al., 2014]), this system would leave 2 TBF clades (i.e., the Hapalorhynchus and Griphobilharzia clade plus the marine TBF clade comprising Carettacola, Learedius, and Hapalotrema) in ''systematic limbo,'' without a family assignment. Along with generating additional sequence data from other TBF species and genera, morphological diagnoses of these lineages/clades are in progress, which will help alleviate the nomenclatural ambiguity resulting from Snyder's (2004) indication of TBF paraphyly. Alternatively, perhaps additional TBF sequence data from 28S and other loci plus discovery of TBF species representing novel genera may demonstrate TBF monophyly.
The ''Spirorchis clade'' (Fig. 10), excluding H. gracilis, comprises all TBFs that infect freshwater cryptodirans (sensu Bickham and Carr ; Guillon et al. ; for sub-order Cryptodira, i.e., the ''hidden neck turtles,'' freshwater turtles that retract their neck vertically [Bonin et al., 2006]) and for which 28S sequences are publically available. Although many more TBFs (49 species) infect cryptodirans, molecular data for those species currently is lacking. All extant marine turtles are assigned to Cryptodira, but their blood flukes comprise the sister lineage to schistosomatids not those of freshwater cryptodirans (Fig. 10). Further in regard to the lack of basic knowledge of TBF biodiversity and biogeography, the present phylogenetic analysis further highlights the need for increased molecular taxonomic coverage among freshwater TBFs in general: only 9 of 57 (14%) freshwater TBF species and 6 of 10 (60%) freshwater TBF genera are associated with published 28S sequence data (Fig. 10). This predicament is even worse among the fish blood flukes, which are almost exclusively represented by marine taxa (Or(C)lis-Ribeiro et al., 2014). The present phylogenetic analysis clustered as sister taxa 2 genera that occur on opposite sides of the planet: Unicaecum spp. reportedly range in North America only (Stunkard, 1925, 1927; Byrd, 1939; Brooks, 1979), and B. obamai reportedly ranges in the Indomalayan region only (present study); further suggestive of the fact that unnamed TBF lineages remain to be studied, especially among freshwater turtles outside of North America.
We thank Dr. Reuben S.K. Sharma (Faculty of Veterinary Medicine, Universiti Putra Malaysia [UPM]) for hosting T.R.P.'s sabbatical stay and supporting collections in Malaysia; UPM for providing sabbatical funds to T.R.P.; and Scott Snyder (University of Nebraska Omaha, Omaha, Nebraska) and Sara Brant (Museum of Southwestern Biology, University of New Mexico, Albuquerque, New Mexico) for furnishing molecular sequence data originally derived from one of T.R.P.'s specimens of B. obamai. This is a contribution of the Southeastern Cooperative Fish Parasite and Disease Project (AU-SFAAS) and was supported in part by National Science Foundation Division of Environmental Biology grant nos. 1112729, 1051106, and 1048523 awarded to S.A.B.
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