Syst Parasitol (2015) 91:231–239 DOI 10.1007/s11230-015-9576-x
A species pair of Bivesicula Yamaguti, 1934 (Trematoda: Bivesiculidae) in unrelated Great Barrier Reef fishes: implications for the basis of speciation in coral reef fish trematodes Nancy Trieu . Scott C. Cutmore . Terrence L. Miller . Thomas H. Cribb
Received: 7 March 2015 / Accepted: 16 April 2015 Ó Springer Science+Business Media Dordrecht 2015
Abstract Combined morphological and molecular analysis shows that a species of Bivesicula Yamaguti, 1934 from four species of Apogonidae Gu¨nther [Nectamia fusca (Quoy & Gaimard), Ostorhinchus angustatus (Smith & Radcliffe), O. cookii (Macleay) and Taeniamia fucata (Cantor)] on the Great Barrier Reef is morphologically similar to, but clearly distinct from B. unexpecta Cribb, Bray & Barker, 1994 which infects a sympatric pomacentrid, Acanthochromis polyacanthus (Bleeker). Bivesicula neglecta n. sp. is proposed for the form from apogonids. Novel ITS2 rDNA sequences generated for the two species differ at just one consistent base position, implying that the two species are closely related. The combination of their close relationship, high but distinct specificity and co-occurrence suggests that speciation was driven by a recent host switching event enabled by similar dietary ecomorphology. Introduction The Bivesiculidae Yamaguti, 1934 is a family of trematodes which predominantly infect the intestines
N. Trieu S. C. Cutmore (&) T. H. Cribb School of Biological Sciences, The University of Queensland, St Lucia, QLD 4072, Australia e-mail: [email protected]
T. L. Miller School of Marine and Tropical Biology, James Cook University, PO Box 6811, Cairns, QLD 4870, Australia
of marine fishes (Cribb, 2002). The five recognised genera containing 26 species have been reported mainly in the Tropical Indo-west Pacific region but are also known from the Caribbean and off east coast of the United States. Six bivesiculid species are known in Australian waters, four of which are reported from the Great Barrier Reef (GBR): Bivesicula claviformis Yamaguti, 1934, B. unexpecta Cribb, Bray & Barker, 1994, Bivesiculoides fusiformis Cribb, Bray & Barker, 1994 and Paucivitellosus fragilis Coil, Reid & Kuntz, 1965 (see Pearson, 1968; Cribb et al., 1994, 1998). Here we report on two evidently closely related species of Bivesicula Yamaguti, 1934 from the GBR. Bivesicula unexpecta is the only bivesiculid known as a sexual adult from pomacentrids, having been reported from just one species, Acanthochromis polyacanthus (Bleeker), from both the southern and northern GBR (Cribb et al., 1994, 1998). Here we analyse an expanded data set of pomacentrids examined for bivesiculids. We also report a new species of Bivesicula from four species of Apogonidae Gu¨nther, the first known from that family, from the GBR using a combined morphological and molecular approach. The nature of the genetic similarity and host-specificity of the two species allows us to speculate about the nature of speciation in this group.
Materials and methods Apogonid and pomacentrid fishes were collected at four Australian sites: Heron Island, southern GBR
(23°270 S, 151°550 E), Lizard Island, northern GBR (14°400 S, 145°270 E), Moreton Bay, off south-east Queensland (27°230 S, 153°260 E) and Ningaloo Reef off Point Cloates, Western Australia (22°400 S, 113°410 E). Fish were collected by line, spear fishing and by clove oil as an anaesthetic and were kept in aerated saltwater tanks until required for dissection. Fish were killed by cranial pithing and the gastrointestinal tract was examined for parasites using the gutwash approach as described by Cribb & Bray (2010). Specimens were fixed by pipetting them into near boiling saline solution followed by immediate preservation in 70% ethanol to allow either morphological or molecular analysis. Worms for morphological analysis were stained using Mayer’s haematoxylin, destained in 1% hydrochloric acid, neutralised with 0.5% ammonia solution, dehydrated through a graded ethanol series, cleared in methyl salicylate and mounted on slides in Canada balsam. Measurements were made using a SPOT InsightTM digital camera (Diagnostic Instruments, Inc.) mounted on an Olympus BH-2 compound microscope using SPOTTM imaging software. Measurements are in micrometres (lm) and are presented as a range, followed by the mean in parentheses. Typespecimens are lodged in the Queensland Museum (QM), Brisbane, Australia. Drawings were made with the aid of a camera lucida and then digitised using Adobe Illustrator CS4. Specimens for molecular analysis were processed according to the protocols used by Cribb et al. (2014a). Cycle sequencing was carried out for the ITS2 rDNA region using the primers 3S (Bowles et al., 1993) and ITS2.2 (Cribb et al., 1998). The start and end of the ITS2 rDNA region were determined by annotation through the ITS2 Database (Koetschan et al., 2012) using the ‘Metazoa’ model. Muscle implemented in MEGA version 5.2 (Tamura et al., 2011) was used to align the sequences. The numbers of base pair differences between sequences was calculated within MEGA 5.2. The Unconditional Exact Test implemented in Quantitative Parasitology version 3.0 (Rozsa et al., 2000) was used to compare the prevalence of bivesiculid parasites in various combinations of fishes.
Results For the Pomacentridae, 48 species from 14 genera were examined for bivesiculids (Table 1).
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Acanthochromis polyacanthus from Heron and Lizard Islands were heavily infected with specimens consistent with B. unexpecta (prevalence 57 and 64% respectively); this pomacentrid species was not observed at the two other sites. No other pomacentrid was infected with adult bivesiculids. The only other bivesiculid infections were sporadic appearances of immature worms in Abudefduf whitleyi Allen & Robertson, Pomacentrus amboinensis Bleeker and P. coelestis Jordan & Starks; these were interpreted as juveniles of B. claviformis which matures in serranid fishes (see Cribb et al., 1998). For the Apogonidae, 580 individuals representing 11 genera and 30 species were examined (Table 2). Bivesiculid infections were found in four species belonging to three genera [Nectamia fusca (Quoy & Gaimard), Ostorhinchus angustatus (Smith & Radcliffe), O. cookii (Macleay) and Taeniamia fucata (Cantor)] at Heron Island only. Of the four infected species, T. fucata was the only one examined in substantial numbers at another location (with substantial examinations at both Heron and Lizard Islands). Prevalence of infection of T. fucata with bivesiculids at Heron Island (7/19) differed significantly from that at Lizard Island (0/18) (p = 0.003). The specimens from apogonids differ morphologically from those infecting pomacentrids (B. unexpecta), with specimens from apogonids possessing vitelline follicles that extend from the level of the eyespots to midway between the testis and the posterior end of body. Bivesicula unexpecta possesses vitelline follicles that are distributed only in the anterior half of the body. In addition, the new specimens from apogonids are widest close to the middle of the body, whereas B. unexpecta is widest distinctly posterior to the middle of the body. In light of these data (and comparison with other species discussed below), a new species is proposed for the new specimens from GBR apogonids. Family Bivesiculidae Yamaguti, 1934 Genus Bivesicula Yamaguti, 1934
Bivesicula neglecta n. sp. Type-host: Nectamia fusca (Quoy & Gaimard) (Perciformes: Apogonidae). Other hosts: Ostorhinchus angustatus (Smith & Radcliffe), O. cookii (Macleay) and Taeniamia fucata (Cantor) (Perciformes: Apogonidae).
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Table 1 Prevalence of Bivesicula unexpecta in Australian Pomacentridae. Abbreviations: HI, Heron Island, Great Barrier Reef (GBR); LI, Lizard Island, GBR; MB, Moreton Bay, south-east Queensland; Ning., Ningaloo Reef, Western Australia. Numbers for infected fish are shown in bold Genus Abudefduf
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Table 1 continued Genus
Description (Fig. 1) [Measurements based on 14 specimens.] Body elliptical, usually longer than wide, 437–675 9 288–439 (542 9 350). Length/width ratio 1.44–1.78:1 (1.55:1). Tegument covered with fine spines throughout. Eyespots persistent or dispersed laterally on each side of oesophagus. Prepharynx short. Pharynx 25–47 9 38–52 (33 9 45). Oesophagus sinuous, 45–78 (69) long. Intestinal bifurcation midway between eye-spots and cirrus-sac. Caeca blind, both arms of equal length, reaching well posterior to posterior margin of testis to 68–144 (96) from posterior extremity of body, 245–352 (272) long. Testis single, entire, post-equatorial, 73–115 9 67–107 (103 9 89). Cirrus-sac median, orientated antero-posteriorly, filled with prostatic cells, 81–123 9 65–87 (101 9 77). Internal seminal vesicle entire, 30–50 (40) in diameter. External seminal vesicle rounded, inconspicuous. Pars prostatica complex and poorly defined. Genital pore small, adjacent to posterior margin of cirrus-sac. Ovary subspherical, distinctly dextral, dorso-dextral to cirrus-sac, 55–96 9 51–75
Type-locality: Heron Island, southern Great Barrier Reef (23°270 S, 151°550 E). Site in host: Intestine. Prevalence: N. fusca: 36%; O. angustatus: 43%; O. cookie: 31%; T. fucata: 37% (see Table 2 for details). Material examined: 14 specimens ex N. fusca, 12 specimens ex O. cookii, three specimens ex O. angustatus (all immature) and three specimens ex T. fucata. Type-material: Holotype (QM G 234720) and 13 paratypes (QM G 234721–33) lodged in the QM. Deposition of molecular data: Six identical ITS2 rDNA sequences generated, three submitted to GenBank (see Table 3). Etymology: The name neglecta is proposed in recognition of the fact that we first detected this species in collections made over 20 years ago.
(70 9 82). Seminal receptacle not detected. Vitelline follicles large, densely distributed from level of eyespots to close to posterior end of body. Uterine path not determined definitively, restricted to posterior to cirrussac, appears to circle testis. Eggs 69–83 9 38–57 (76 9 45). Excretory vesicle V-shaped; arms pass ventrally to testis 344–546 (445) long, terminating at level of intestinal bifurcation, 70–126 (86) from anterior extremity of body. Excretory pore single, prominent, at posterior extremity. Molecular results ITS2 rDNA sequences were generated for specimens of B. unexpecta infecting A. polyacanthus from Heron Island (n = 3) and for B. neglecta n. sp. from apogonids [N. fusca (n = 2), O. cookii (n = 1) and T. fucata (n = 3)]. No samples from O. angustatus were available for molecular characterisation. The sequences were 430 bp long, comprising 123 bp of 5.8S, 258 bp of ITS2 and 49 bp of flanking 28S. All three sequences of B. unexpecta were identical as were all six sequences of B. neglecta n. sp. Sequences of B. unexpecta differed from those of B. neglecta n. sp. at a single consistent base. GenBank and QM accession numbers for bivesiculid specimens sequenced during this study are listed in Table 3.
Discussion Taxonomy Based on morphological and molecular comparisons, the bivesiculid from apogonids reported here clearly represents a new species of Bivesicula. Prior to the proposal of B. neglecta n. sp., Bivesicula had 16 accepted species (Cribb, 2015). Bivesicula neglecta n. sp. is easily distinguished from B. australis Crowcroft,
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Table 2 Prevalence of Bivesicula neglecta n. sp. in Australian Apogonidae. Abbreviations: HI, Heron Island, Great Barrier Reef (GBR); LI, Lizard Island, GBR; MB, Moreton Bay, SE Queensland; Ning., Ningaloo Reef, Western Australia. Numbers for infected fish are shown in bold Genus Apogon
rueppellii taeniophorus Pristiapogon
kallopterus Rhabdamia Siphamia
0/2 0/3 0/25 7/19
Table 3 GenBank and Queensland Museum (QM) accession numbers for Bivesicula spp. sequenced during this study. All specimens were collected from off Heron Island, southern Great Barrier Reef (23°270 S, 151°550 E) Species
Number of replicates
GenBank accession number
QM accession number
Bivesicula neglecta n. sp.
QM G 234720–7
QM G 234728–32
QM G 234733
1947, B. auxisae Gu & Shen, 1983, B. claviformis, B. congeri Yamaguti, 1970, B. fistulariae Shen, 1982, B. lutiani Shen, 1983, B. ostichthydis Shen, 1982,
B. palauensis Shimazu & Machida, 1995 and B. tarponis Sogandares-Bernal & Hutton, 1959 on the basis that the latter have distinctly elongate bodies (more
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Fig. 1 Bivesicula neglecta n. sp. from intestine of Nectamia fusca. A, Ventral view of holotype; B, Same with vitelline follicles and eggs removed. Scale-bars: A, B, 100 lm
than twice long as wide) in contrast to the relatively squat body outline of B. neglecta n. sp. Relative to species with comparably rounded bodies, B. neglecta n. sp. can be distinguished from B. caribbensis Cable & Nahhas, 1962, B. gymnothoracis Shimazu & Machida, 1995, B. hepsetiae Manter, 1947, B. megalopis Shen, 1982, B. obovata Shimazu & Machida 1995, B. synodi Yamaguti, 1938 and B. unexpecta in having vitelline follicles distributed well posterior to the testis in contrast to not exceeding the posterior margin of the testis in all the other species. The distribution of the vitelline follicles, which range from the level of the eye-spots to the bifurcation of the excretory vesicle, is the greatest for any species of Bivesicula. Geographical distribution Whereas B. unexpecta and B. claviformis have been found commonly at both Heron and Lizard Islands on
the GBR, our data suggest that B. neglecta n. sp. is restricted to the southern GBR. Of the hosts demonstrated to be susceptible, 41 of 128 examined were infected at Heron Island, differing significantly from the 0 of 28 infected at Lizard Island (p = 0.0001). This finding adds to a growing body of records demonstrating distinctions in the trematode fauna of the southern and northern GBR in the same fish species (Cribb et al., 2014b). Species level host-specificity The systematic sampling reported here allows us to be confident that B. unexpecta is oioxenous (infecting a single host species) and that B. neglecta n. sp. is stenoxenous (infecting a narrow range of phylogenetically related hosts). This is consistent with the findings of the review by Miller et al. (2011) which showed that such host-specificity was characteristic of the great majority of trematodes of GBR fishes
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whereas euryxenicity (infection of hosts linked only by ecology or physiology) was exceptional. It is of interest that another GBR bivesiculid, Paucivitellosus fragilis, is one of the rare GBR species for which hosts from multiple fish orders have been reported (Pearson, 1968; Cribb et al., 1994). Notably, however, the specificity of that species has not yet been tested by molecular approaches. Perusal of the literature suggests that oioxenicity or stenoxenicity is the rule for species of Bivesicula. All but three of the 17 species have each only been reported from just a single family of fishes. The three exceptions in which multiple host families have been reported are not especially convincing. Bivesicula australis was reported initially from a scorpaenid from Tasmania (Crowcroft, 1947) and subsequently from congrid, gempylid, lophiid, pentacerotid and pleuronectid fishes by Koryakovtseva (1984) and an unknown species by Hafeezullah & Dutta (1980). In the case of the work of Koryakovtseva (1984), there are no figures of the species provided, thus critical assessment of the records is difficult. Bivesicula ostichthydis was originally recorded from a holocentrid (Shen, 1982), and subsequently recorded from an ophidiid by Shimazu & Machida (1995) who drew attention to several differences that might easily relate to species level distinctions. Bivesicula claviformis, the type-species of the genus, is the most problematic. Although the species was described from a carangid (type-host) and a lutjanid by Yamaguti (1934), the overwhelming majority of records are from serranids (Yamaguti, 1938, 1939; Nagaty, 1948; Manter, 1961; Fischthal & Kuntz, 1965; Machida et al., 1970; Gu & Shen, 1983; Shen, 1985; Lester & Sewell, 1990; Cribb et al., 1994; Shimazu & Machida, 1995; Rigby et al., 1997; Cribb et al., 1998; Nahhas et al., 2004) but there are also reports from a holocentrid (Koryakovtseva, 1984) and a possible scombrid, although there is uncertainty over the identification of the latter host which might have been a serranid (Shimazu & Machida, 1995). Thus, the records for this species are at best enigmatic and it is possible that multiple species are involved. Overall, although the host-specificity of these three species of Bivesicula certainly need further consideration, especially with the use of comparative molecular data, we do not see compelling evidence that any species of Bivesicula is shared by multiple fish families.
Genus level host-specificity With the addition of the records for B. neglecta n. sp., the 17 recognised species of Bivesicula have now been reported from 24 families and 13 orders of fishes. This diversity of hosts is made more remarkable by the fact that only five families have been reported with two (Congridae, Megalopidae, Scombridae and Serranidae) or three (Holocentridae) species of Bivesicula. Subject to the reporting of more species, this distribution suggests that the overall pattern of evolution of the genus has been driven either by radiation followed by widespread extinction or by recent hostswitching. Examination of B. neglecta n. sp. and B. unexpecta offers an opportunity to resolve the issue. Although B. unexpecta and B. neglecta n. sp. are readily distinguished morphologically, they differ by only a single base across the ITS2 rDNA region. This marker typically differs by far more base pairs in most combinations of congeners of trematodes of coral reef fishes (e.g. Chambers & Cribb, 2006; Miller & Cribb, 2007a, b; Downie et al., 2011) although differences as low as a single base have been reported for some combinations of aporocotylids (Nolan & Cribb, 2006). The close relationship between these two species, despite their infection of unrelated fishes, suggests that their speciation was driven by a recent host-switching rather than extinction. We suspect that this paradigm may be applicable for many combinations of species of Bivesicula. In this context, it is of interest that hostspecificity apparently becomes fixed rapidly after the host-switching events as demonstrated by the mutual exclusivity of B. neglecta n. sp. and B. unexpecta in large numbers of co-occurring apogonids and pomacentrids. It seems likely that the host-switching event inferred above was facilitated by the similarity of the dietary ecomorphology of the two host groups. Of the 11 genera of apogonid fishes sampled for bivesiculids, only species of Nectamia Jordan, Ostorhinchus Lace´pe`de and Taeniamia Waite were infected. The ecomorphology of the species of these three genera is similar and contrasts significantly with that of species of the other apogonid genera sampled. Barnett et al. (2006) found that species of Nectamia, Ostorhinchus and Taeniamia all have large-gaped mouths, and that their lower jaws have great flexibility and protrude. The morphological similarities correlate with the feeding ecology of these species which are nocturnal,
midwater foragers, consuming primarily planktonic and free-swimming prey (Marnane & Bellwood, 2002; Barnett et al., 2006). In contrast, species of Cheilodipterus Lace´pe`de have longer jaws covered in large teeth and the upper jaw protrudes. These characteristics are strongly correlated with a piscivorous diet. Species of Apogon Lace´pe`de and Pristiapogon Klunzinger are nocturnal and have a wider mouth with an increased jaw opening lever ratio, suggesting that they forage on the benthos for crustaceans (Barnett et al., 2006). Segregation of feeding microhabitat and prey items in apogonids is likely to be a significant factor in determining which apogonid species have the potential to encounter B. neglecta n. sp. Acanthochromis polyacanthus is phylogenetically distant from species of Nectamia, Ostorhinchus and Taeniamia, but it is similar in its ecomorphology. It forages in the water column for planktonic prey and its distribution around coral reefs is remarkably similar to other diurnal and nocturnal planktivorous fish species (Thresher, 1983). We argue that similar feeding ecology and cooccurrence of apogonids and A. polyacanthus enabled host-switching and cladogenesis in the common ancestor of B. unexpecta and B. neglecta n. sp. Several other studies have shown putatively sister species of trematodes to be associated with distinct patterns of host-specificity (e.g. Jousson et al., 2000; Hunter et al., 2010; Calhoun et al., 2013; Curran et al., 2013). Especially where the species occur in sympatry, we suspect that circumstances comparable to those discussed above have underpinned trematode speciation. In the case of two combinations of Mediterranean sparid trematodes, Jousson et al. (2000) were able to infer that, as here, the host distribution was not the result of co-evolutionary interactions but rather was driven by host diet (and presumably host-switching). In the case of the complex of transversotrematids characterised by Hunter et al. (2010), infection was independent of diet and could be inferred to be at least partially the result of host-switching. Although some trematode taxa have clearly radiated in association with groups of hosts, host-switching of the kind evident in this study appears to be an important component of the evolution of this host-parasite system. Acknowledgements We thank the staff of the Heron and Lizard Island Research stations for long-term support of our work.
Syst Parasitol (2015) 91:231–239 Funding We thank the Australian Research Council and the Australian Biological Resources Study for ongoing support to T. H. Cribb. Collection was funded in part by the Australian node of the CReefs global research initiative, a partnership between the Australian Biological Resource Study (ABRS), BHP Billiton, the Great Barrier Reef Foundation, the Census of Marine Life, the Australian Institute of Marine Science (AIMS) and the Alfred P. Sloan Foundation. T. L. Miller is supported by a Queensland Government Smart Futures Fellowship. Conflict of interest The authors declare that they have no conflict of interest. Compliance with ethical standards All applicable institutional, national and international guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.
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