Parasitology International 63 (2014) 285–294
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Biogeography of tropical Indo-West Pacific parasites: A cryptic species of Transversotrema and evidence for rarity of Transversotrematidae (Trematoda) in French Polynesia Thomas H. Cribb a,⁎, Robert D. Adlard b, Rodney A. Bray c, Pierre Sasal d,e, Scott C. Cutmore a a
School of Biological Sciences, The University of Queensland, Brisbane, Queensland 4072, Australia Biodiversity Program, Queensland Museum, P.O. Box 3300, South Brisbane, Queensland 4101, Australia c Department of Life Sciences, Natural History Museum, Cromwell Road, London SW7 5BD, UK d USR 3278 CNRS EPHE, Centre de Recherches Insulaires et Observatoire de l'Environnement (CRIOBE), BP 1013, Papetoai, Moorea, French Polynesia e Laboratoire d'Excellence CORAIL, French Polynesia b
a r t i c l e
i n f o
Article history: Received 5 September 2013 Received in revised form 13 November 2013 Accepted 20 November 2013 Available online 28 November 2013 Keywords: Trematoda Transversotrematidae Biogeography French Polynesia Dispersal Richness
a b s t r a c t We sought transversotrematid trematodes from French Polynesian fishes by examining 304 individual scaled fishes of 53 species from seven families known to harbour the family elsewhere. A single species was found at two locations in the Tuamotus Archipelago on two species of Chaetodontidae (Chaetodon auriga and Chaetodon ephippium) and one species of Lutjanidae (Lutjanus gibbus). The species closely resembles Transversotrema borboleta Hunter & Cribb, 2012 from chaetodontids and lutjanids of the northern Great Barrier Reef (GBR) but differs from it consistently in 8 base positions of ITS2 rDNA. This level of variation exceeds that between some clearly morphologically distinct pairs of species of Transversotrema and the form from French Polynesia is thus interpreted as a distinct, though cryptic, species and named Transversotrema polynesiae n. sp. The new species forms part of a complex of species, here characterised as the T. borboleta complex, associated with chaetodontids and lutjanids in the tropical Indo-West Pacific. Most of the putative species within this complex are yet to be described. Comparison of identical numbers of matched samples of fishes from French Polynesia, Heron Island (southern GBR) and Lizard Island (northern GBR) revealed 1, 4 and 10 species of Transversotrema respectively suggesting that the French Polynesian fauna is depauperate for this family. In addition to those species apparently missing from suitable hosts in French Polynesia, several species from further west infect fishes (especially Nemipteridae) that are themselves absent from French Polynesia. This dramatic east–west decline in richness contrasts strongly with what is known for monogeneans, which appear to maintain their richness over the same scale, and is more precipitate than is known for other groups of trematodes. The decline might be explained in part by the absence of the as yet unknown first intermediate hosts in French Polynesia. However, we predict that it is explained by other life cycle traits. We hypothesise that the characters of large short-lived cercariae, short-lived miracidia, the absence in the life-cycle of second intermediate hosts that are capable of transporting the species, and definitive and first intermediate hosts that have limited vagility combine to give marine Transversotrematidae limited dispersal capacity and a propensity for localised speciation. © 2013 Published by Elsevier Ireland Ltd.
1. Introduction It is well understood that marine biological richness in the tropical Indo-West Pacific (TIWP) is at its greatest in the Coral Triangle, the archipelagos of Indonesia, the Philippines and Papua New Guinea. Marine richness declines in every direction from the Coral Triangle. For example, richness of shore fishes declines from 1693 species in the ‘centre of the centre’, the Philippines [1], to as few as 767 species in the Society Islands (French Polynesia) and just 259 species in the ⁎ Corresponding author. Tel.: +61 7 3365 2581; fax: +61 7 3365 4699. E-mail addresses:
[email protected] (T.H. Cribb),
[email protected] (R.D. Adlard),
[email protected] (R.A. Bray),
[email protected] (P. Sasal),
[email protected] (S.C. Cutmore). 1383-5769/$ – see front matter © 2013 Published by Elsevier Ireland Ltd. http://dx.doi.org/10.1016/j.parint.2013.11.009
Gambier Archipelago (French Polynesia) [2]. Thus, whereas the GBR has eight species of Siganidae, French Polynesia has only two [3]. In some cases the decline in richness means that significant higher taxa are absent entirely. Hawaii lacks Siganidae entirely and has no naturally occurring shallow water Lutjanus species [4]. Similarly, French Polynesia has no Nemipteridae [5]. The absence of widespread species is, however, sometimes accompanied by the appearance of significant numbers of endemic species, especially in peripheral areas [6]; Hawaiian fishes have an endemicity of 23% [7] and those of the Marquesas (French Polynesia, still being explored) have an endemicity of 11.6% [8]. Similar overall patterns, especially in the reduction of richness away from the Coral Triangle, have been reported for other groups of free-living animals such as strombid gastropods [9] and hermatypic corals [10].
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Whereas the patterns of richness of many free-living animal groups in the TIWP are relatively well understood, the biogeography of marine parasites in the region remains poorly characterised. This is doubtless because, overall, so much work remains to be done to describe the parasite fauna [11]. Although there are huge numbers of records of parasites of fishes from the TIWP, relatively few studies have reported systematically the presence or absence of parasites in the same or related hosts over broad ranges. We know that many digenean species are apparently widespread (at least on the basis of morphological identifications) and a few studies [e.g. 12–14] have used molecular data to demonstrate distributions encompassing both the Indian and Pacific Oceans. However, we have little sense of broad biogeographical patterns in this region. Just a handful of studies have established some key patterns. Hayward [15] explored the richness of ectoparasites of sillaginids and found evidence for relatively distinct Asian and Australian faunas. Rigby et al. [16] explored the internal helminth richness of the serranid Epinephelus merra from sites between the GBR and French Polynesia and reported a dramatic reduction in richness from west to east. Rigby et al. [17] concluded that overall trematode richness was lower in French Polynesia than on the GBR. Downie et al. [18] and Diaz et al. [19] found that two species of each of the trematode genera Symmetrovesicula and Paradiscogaster were absent from French Polynesia despite the presence of suitable hosts. McNamara et al. [20] demonstrated that Moorea (French Polynesia) has the lowest richness of monorchiid trematodes in chaetodontid fishes (6 species) of any of six well-studied TIWP sites (the remaining having 7–10 species); again in several cases known fish hosts for monorchiid species were present at Moorea but the parasites were absent. In contrast, studies of the richness of dactylogyrid monogeneans of chaetodontid fishes [21,22] found, counter to the general TIWP paradigm, that the greatest richness was in French Polynesia. However, these studies did not report sample sizes so the significance of the reported absences is not certain. Overall, the extent to which parasite taxa are broadly distributed in the TIWP appears patchy, but reliable data, including the reporting of evidence of absence, remains inadequate. A recent series of studies [23–27] has shown that the Transversotrematidae, trematode parasites that live beneath the scales of fishes, is rich and abundant on a wide range of fishes in several areas of the TIWP (northern and southern GBR, Ningaloo Reef, New Caledonia and Palau). These parasites are probably under-reported because they have rarely been sought specifically. In total, these papers reported evidence of 24 species (some only distinguished by molecular data) of which 18 occur on the GBR where the family has been studied most intensively. Here we present evidence regarding the richness of the family on fishes of French Polynesia in direct comparison with previously recorded richness and prevalence data for the GBR. Three categories of outcomes are possible — species found in French Polynesia that occur elsewhere, species found in French Polynesia that are endemic to the region, and species known elsewhere that are lacking in French Polynesia. 2. Materials and methods
examined under a stereo microscope. Trematodes collected from the supernatant were fixed by pipetting into near boiling saline, and preserved in 70% ethanol to allow for either morphological or molecular analysis. 2.2. Morphological analysis Specimens for morphological analysis were stained in Mayer's haematoxylin, destained in dilute HCl, neutralised in dilute ammonia solution, dehydrated in a graded series of ethanol, cleared in methyl salicylate and mounted in Canada balsam. Drawings were completed using an Olympus BH-2 compound microscope and drawing tube. Measurements were recorded on an Olympus BH-2 microscope using a Nikon Digital Sight DS-LI digital camera (Nikon Corporation, Japan) and are in micrometres as the range with the mean in parentheses. All voucher specimens are paragenophores [sensu 29] and are lodged in the Queensland Museum, Brisbane, Australia. 2.3. Molecular analysis Total genomic DNA was extracted using standard phenol/ chloroform extraction techniques [30]. The ITS2 nuclear ribosomal DNA region was amplified using the forward primer 3S (5′-GGT ACC GGT GGA TCA CGT GGC TAG TG-3′) [31] and the reverse primer ITS2.2 (5′-CCT GGT TAG TTT CTT TTC CTC CGC-3′) [32]. PCR was performed with a total volume of 20 μl consisting of approximately 10 ng of DNA, 5 μl of 5× MyTaq Reaction Buffer (Bioline), 0.75 μl of each primer (10 pmol) and 0.25 μl of Taq DNA polymerase (Bioline MyTaq™ DNA Polymerase), made up to 20 μl with Invitrogen™ ultraPURE™ distilled water. Amplification was carried out on a MJ Research PTC-150 thermocycler using the following profile: an initial single cycle of 95 °C denaturation for 3 min, 45 °C annealing for 2 min, 72 °C extension for 90 s, followed by 4 cycles of 95 °C denaturation for 45 s, 50 °C annealing for 45 s, 72 °C extension for 90 s, followed by 30 cycles of 95 °C denaturation for 20 s, 52 °C annealing for 20 s, 72 °C extension for 90 s, and followed by a final 72 °C extension for 5 min. Amplified DNA was purified using a Bioline ISOLATE II PCR and Gel Kit, according to the manufacturer's protocol. Cycle sequencing of purified DNA was carried out using ABI BigDye™ v.3.1 chemistry following the manufacturer's recommendations, using the amplification primers and the additional forward primer GA1 (5′-AGA ACA TCG ACA TCT TGA AC-3′) [33]. Cycle sequencing was carried out at the Australian Genome Research Facility using an AB3730xl capillary sequencer. Sequencher™ version 4.5 (Gene Codes Corp.) was used to assemble and edit contiguous sequences. GenBank accession numbers and number of replicates for transversotrematids sequenced in this study are shown in Table 1.
Table 1 Collection data and GenBank accession numbers for Transversotrema polynesiae n. sp. ITS2 rDNA data generated during this study. Host
Collection locality
# of replicates
GenBank accession #
Toau Atoll, Tuamotu Archipelago, French Polynesia (15°49′S, 146°09′W) Toau Atoll, Tuamotu Archipelago, French Polynesia (15°49′S, 146°09′W)
1
KF765503
1
KF765502
2
KF765501
2.1. Specimen collection and preparation Fish were collected from four sites in French Polynesia over several years, Moorea (Society Islands, 17°29′S 149°52′W), Gambier Archipelago (southern Tuamotus, based around 23°07′S 134°56′W), the Marquesas Archipelago (north-east of Tahiti, based around 9°00′S, 140°00′W) and the Tuamotus Archipelago (east of Tahiti, based around the atolls of Toau (15°49′S, 146°09′W) and Fakarava (16°30′S, 145°29′ W). The fish were collected mainly by spearing but sometimes at anaesthetic stations. After being killed by neural pithing, fish for examination for transversotrematids were soaked in saline solution (approximately 0.9% NaCl) for a minimum of 30 min as described by Cribb and Bray [28]. The supernatant was then decanted and the resulting sediment
Chaetodontidae Chaetodon auriga
Chaetodon ephippium
Lutjanidae Lutjanus gibbus
Fakarava Atoll, Tuamotu Archipelago, French Polynesia (16°30′S, 145°29′W); Toau Atoll, Tuamotu Archipelago, French Polynesia (15°49′S, 146°09′W)
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2.4. Phylogenetic analysis The ITS2 rDNA sequences generated during this study were aligned with ITS2 rDNA transversotrematid sequences available from GenBank (Table 2) using MUSCLE version 3.7 [34] with ClustalW sequence weighting and UPGMA clustering for iterations 1 and 2. The resultant alignment was refined by eye using MESQUITE [35] and the ends of each fragment were trimmed to match the shortest sequence in the alignment. Bayesian inference analysis was conducted to explore relationships between the new taxa and all Transversotrema Witenberg, 1944 species available on GenBank. Neighbour-joining analysis was also performed and was restricted to only closely related Transversotrema taxa (“T. borboleta complex species”). Bayesian inference analysis was performed using MrBayes version 3.2.2 [36] run on the CIPRES portal [37]. The software jModelTest version 0.1.1 [38] was used to estimate the best nucleotide substitution model for the dataset. Bayesian inference analysis was conducted using the GTR + I + G model predicted as the best estimators by the Akaike Information Criterion (AIC) in jModelTest. Bayesian inference analysis was run over 10,000,000 generations (ngen = 10,000,000) with two runs each containing four simultaneous Markov Chain Monte Carlo chains (nchains = 4) and every 1000th tree saved (samplefreq = 1000). Bayesian analyses used the following parameters: nst = 6, rates =gamma, ngammacat = 4, and Table 2 ITS2 rDNA sequences from GenBank analysed in this study. Species Family Transversotrematidae Transversotrema atkinsoni Transversotrema borboleta G1 Transversotrema borboleta G1 Transversotrema borboleta G1 Transversotrema borboleta G1 Transversotrema borboleta G1 Transversotrema borboleta G1 Transversotrema borboleta G1 Transversotrema borboleta G1 Transversotrema borboleta G1 Transversotrema borboleta G1 Transversotrema borboleta G2 Transversotrema borboleta G2 Transversotrema borboleta G2 Transversotrema borboleta G2 Transversotrema borboleta G2 Transversotrema borboleta G2 Transversotrema borboleta G3 Transversotrema borboleta G3 Transversotrema borboleta G3 Transversotrema cabrarum Transversotrema cardinalis Transversotrema carmenae Transversotrema chevrarum Transversotrema cutmorei Transversotrema damsella Transversotrema elegans Transversotrema espanola Transversotrema fusilieri Transversotrema gigantica Transversotrema gigantica Transversotrema lacerta Transversotrema licinum Transversotrema manteri Transversotrema tragorum Transversotrema witenbergi Transversotrema sp. A Transversotrema sp. C Transversotrema sp. Transversotrema sp. Transversotrema sp. Outgroup taxa Crusziella formosa Prototransversotrema steeri
Host species
GenBank accession #
Scolopsis bilineatus Chaetodon auriga Chaetodon citrinellus Chaetodon ephippium Chaetodon rafflesi Chaetodon ulietensis Chaetodon vagabundus Lutjanus bohar Lutjanus carponotatus Lutjanus monostigma Macolor niger Chaetodon auriga Lutjanus fulviflamma Lutjanus fulvus Lutjanus kasmira Lutjanus quinquelineatus Lutjanus russellii Lutjanus adetii Lutjanus sebae Pterocaesio marri Parupeneus spilurus Lutjanus carponotatus Scolopsis bilineatus Parupeneus multifasciatus Upeneus tragula Crenimugil crenilabis Choerodon graphicus Lutjanus carponotatus Caesio cuning Scarus ghobban Scarus schlegeli Diagramma labiosum Liza argentea Caesio cuning Parupeneus multifasciatus Caesio cuning Sillago ciliata Chaetodon lunula Lutjanus bohar Parupeneus barberinus Parupeneus ciliatus
GU174456 HQ201706 HQ201682 GU174468 HQ201683 JF412536 HQ201684 HQ201686 HQ201685 JQ041373 JF412526 JF412524 GU174470 HQ201707 HQ201710 HQ201708 HQ201711 GU174472 HQ201687 GU174471 GQ162866 HQ201689 HQ201697 GQ162849 JN375553 HQ201700 HM748982 HQ201701 HQ201703 HM748989 HM748984 GU174437 GU174446 JF412523 GQ162858 HQ201704 GU174464 JF412534 GU174450 GQ162828 GQ162837
Crenimugil crenilabis Aldrichetta forsteri
GU174461 GU174458
287
the priors parameters of the combined dataset were set to ratepr = variable. Samples of substitution model parameters, and tree and branch lengths were summarised using the parameters ‘sump burnin = 3000’ and ‘sumt burnin = 3000’. These ‘burnin’ parameters were chosen because the log likelihood scores ‘stabilised’ well before 3,000,000 replicates in the Bayesian inference analyses. Species of the transversotrematid genera Crusziella Cribb, Bray & Barker, 1992 and Prototransversotrema Angel, 1969 were designated as functional outgroups. Distance Analysis using the complete deletion option within MEGA5 [39] was used to determine base pair differences relative to the present form. A separate alignment of the “T. borboleta complex” only was explored by Distance Analysis within MEGA5 [39] to generate a NJ tree and a table of genetic distances. 2.5. Species accumulation analysis To explore discovery of transversotrematids at different localities we constructed sets of Heron Island and Lizard Island fishes to be compared with matching sets from French Polynesia. Each data set included as many individuals of any given taxa as were found at the less well studied site. As a result, the data sets had as few as one or as many as 22 individuals of specific fish taxa. We chose fish for inclusion in the order in which they had been examined. Records of transversotrematids from the GBR were consistent with the three recent papers on the subject [24–26] but with minor modification for collections and identifications made subsequent to those studies. In a few cases, where a fish had been examined and shown to be infected but the Transversotrema species was unknown, it was replaced with a later collected fish where the Transversotrema identity was known. No such substitution affected the number of species of Transversotrema included in the data set. Different fish families were treated depending on our understanding of the host-specificity of the transversotrematids that infect them. Thus, for the taxonomic levels of caesionine lutjanids, Lethrinus (Lethrinidae), Abudefduf (Pomacentridae), Parupeneus and Mulloidichthys (Mullidae; genera considered separately) and Scaridae, there is little evidence of host-specificity beneath that level so that fish were not matched below these taxonomic levels. In contrast, there is evidence of distinctions in the transversotrematids that occur in different species of Chaetodon (Chaetodontidae) and Lutjanus (Lutjanidae) so the samples for these important genera were matched species by species. Species accumulation curves were generated with 10,000 randomisations and the default settings in the software EstimateS [40]. Presence or absence of species of fish in French Polynesia was determined by reference to Randall [5]. 3. Results 3.1. General results Table 3 summarises the fish examined for transversotrematids at French Polynesia, subdivided by locality. From the 304 individuals of 53 species of seven families of scaled fishes known to be susceptible elsewhere, transversotrematid infections were found on specimens of two species of Chaetodontidae (Chaetodon auriga Forsskål and C. ephippium Cuvier) and on one species of Lutjanidae (Lutjanus gibbus (Forsskål)). 3.2. Morphological results We detected no differences between samples from the three host species from French Polynesia or between them and the three genotypes of T. borboleta reported from the GBR by Hunter and Cribb [25]. However, in view of the molecular differences reported below, we propose T. polynesiae n. sp. for this form.
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Table 3 Scaled fishes examined for Transversotrematidae in French Polynesia. 1. Gambiers Archipelago; 2. Marquesas Archipelago; 3. Moorea, Society Islands; 4. Tuamotus Archipelago. Family
Fish Genus
Species
1
2
Chaetodontidae
Chaetodon
auriga bennetti citrinellus ephippium lineolatus lunula lunulatus ornatissimus pelewensis quadrimaculatus reticulatus trichrous trifascialis ulietensis unimaculatus vagabundus flavissimus chrysostomus monoceros undulatus insidiator hardwicke lutescens aureolineatus atkinsoni olivaceous virescens tile bohar fulvus gibbus kasmira monostigma marri flavolineatus mimicus cyclostomus insularis multifasciatus vitta septemfasciatus sexfasciatus nigricans microrhinos sordidus altipinnis forsteni frenatus ghobban globiceps koputea rubriviolaceus sp.
6
3
1
2
2 1
5
Forcipiger Heniochus Labridae
Cheilinus Epibulus Thalassoma
Lethrinidae
Gnathodentex Lethrinus
Lutjanidae
Mullidae
Aprion Caesio Lutjanus
Pterocaesio Mulloidichthys Parupeneus
Pomacentridae
Upeneus Abudefduf
Scaridae
Stegastes Chlorurus Scarus
Totals
3
2
1
1 2 4
1 1
4
Total
8 2 12 9 1 9 7 4 8 6 9
17 2 14 12 1 17 10 9 8 7 10 2 7 10 3 4 17 11 5 1 4 1 1 4 1 1 1 1 8 6 11 30 7 6 1 2 10 5 7 1 3 5 2 1 5 1 1 1 1 1 5 2 1 304
2 1 2 3
6 8 4 10 2
1
6 9 5
1 4 1 1 4 1 1 1
1
1 7 3 6 30 1 6
1 2 5 6 1
1 2
2 9 4 4
2
1 1 1 1 5 2
1 2
3 1 1 1 1 1 5 2
30
94
1 56
124
Species found infected in bold.
3.3. Molecular results Four identical ITS2 rDNA sequences were generated from French Polynesian samples, one from each of Chaetodon auriga and C. ephippium and two from Lutjanus gibbus. The sequences, when trimmed at the primers, were 419 bp including 123 bp of flanking 5.8S DNA, 247 bp of ITS2 and 49 bp of flanking 28S rDNA. Bayesian inference analysis of Transversotrema polynesiae n. sp. together with all other transversotrematids for which ITS2 rDNA sequences are available (Fig. 1) resolved all species of Transversotrema as monophyletic relative to the transversotrematid genera Prototransversotrema and Crusziella, and recognised the same three major clades within Transversotrema as recognised by Hunter et al. [27] — Clade A associated exclusively with mullids, Clade
B with haemulids, labrids, lethrinids and scarids, and Clade C which incorporates T. polynesiae n. sp. and 19 other species from a wide range of families. Clade C has two recognisable subclades (Clades C1 and C2) which each have only modest support. Of these, T. polynesiae n. sp. falls in Clade C2 which is otherwise comprised of T. witenbergi, the three genotypes of T. borboleta (to which T. polynesiae n. sp. was the sister taxon but with low support), T. sp. C from a chaetodontid from Palau, T. sp. A from a sillaginid from Moreton Bay and T. licinum. In Distance Analysis (all taxa), T. polynesiae n. sp. differed from Clade A species by 39–45 bp, from Clade B species by 43–50 bp, and from Clade C1 species by 15–38 bp. In an alignment of Clade C2 taxa only, T. polynesiae n. sp. differed from other taxa by 4–11 bp. Within this clade, T. witenbergi, T. licinum and T. sp. A were phylogenetically distinct from T. borboleta, T. polynesiae n. sp., and T. sp. C ex Chaetodon from Palau and differed from T. polynesiae n. sp. by 8–11 bp. The T. borboleta genotypes, T. polynesiae n. sp. and T. sp. C ex Chaetodon formed a poorly supported clade and, in an alignment of just these species, differed from each other by 2–8 bp; T. polynesiae n. sp. differed from other taxa by 4–8 bp (Table 4). The single available sequence of Transversotrema sp. D of Hunter and Cribb [25] from Chaetodon flavirostris from New Caledonia is similar to these sequences but is here excluded from analysis because of the shortness of the available sequence (297 bp). These taxa are here considered to form the T. borboleta complex. Fig. 2 shows the relationships of these taxa in a NJ tree. Transversotrema polynesiae n. sp. (Fig. 3) Type-host: Chaetodon auriga (Forsskål), Threadfin butterflyfish (Perciformes: Chaetodontidae). Other hosts: Chaetodon ephippium (Cuvier), Saddle butterflyfish (Perciformes: Chaetodontidae); Lutjanus gibbus (Forsskål), Humpback red snapper (Perciformes: Lutjanidae). Type-locality: Toau, Tuamotus Archipelago, French Polynesia (15°49′S, 146°09′W). Other locality: Fakarava, Tuamotus Archipelago, French Polynesia (16°30′S, 145°29′W). Site: Beneath the scales. Material examined: 13 ex C. auriga; 1 ex C. ephippium; 25 ex L. gibbus. Molecular sequence data: ITS2 rDNA, four identical replicates, three submitted to GenBank (Table 1). Deposited specimens: Holotype QM G234305; Paratypes QM G234306-19. Etymology: The name is derived from the type locality, French Polynesia. Description: measurements of 20 mature specimens given in Table 5. Body transversely elongated, lancet shaped. Tegumental spines prominent. Eyespots prominent, paired, central in anterior half of body, level with anterior margin of pharynx; no pigment evident other than in eyespots. Ventral sucker well posterior to eyespots. Mouth mid-ventral, inconspicuous. Pharynx well developed. Oesophagus indistinct, almost entirely ventral to pharynx. Intestinal bifurcation dorsal to ventral sucker. Caeca form cyclocoel enclosing testes, ovary and some vitelline follicles. Testes deeply lobed. Seminal vesicle distinctly bipartite, composed of enclosed and extracaecal portions; enclosed portion saccular, distinctly lobed, anterodextral to right testis, constricted distally to form narrow duct which passes ventral to cyclocoel and leads to extracaecal portion; extracaecal portion tubular, winding, long, passes laterally along line of cyclocoel towards midline of body, turning anteriorly and proceeding between eyespots, dextral to pharynx, forms naked ejaculatory duct distally. Common genital pore precisely in midline on anterior margin of worm. Ovary deeply lobed, sinistral to but not contiguous with left testis. Oviduct passes medioposteriorly from ovary. Seminal receptacle uterine, proximal to genital pore. Laurer's canal unites with oviduct posterior to ovary, passes posteriorly to open dorsally. Vitelline reservoir immediately anterior to left testis. Uterus passes medially between anterior half of cyclocoel and testes then between right testis and saccular portion of seminal
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Fig. 1. Relationships between Transversotrema polynesiae n. sp. and other transversotrematid taxa based on Bayesian inference analysis of the ITS2 rDNA dataset. Posterior probabilities are shown at the nodes; values b50 not shown.
vesicle. Proximal portions of uterus act as seminal receptacle. Vitelline follicles scattered in extracaecal and enclosed areas of body. Extracaecal vitelline follicles most dense lateral and posterior to cyclocoel, loosely assembled anterior to cyclocoel to midway from lateral margin to pharynx. Enclosed follicles in one mass at each lateral extremity, absent along posterior margin of cyclocoel posterior to testes. Eggs wide, tanned, no operculum observed, apparently unembryonated. Excretory vesicle opens posteriorly at small notch in middle of posterior margin, extends anteriorly initially as narrow tube which then expands into large sac which passes ventrally to cyclocoel anterior to which it becomes laterally directed. 3.4. Species accumulation curves Separate randomised species accumulation curves for French Polynesia (all localities pooled) relative to Heron Island (southern GBR) and Lizard Island (northern GBR) are shown in Fig. 4. Table 6 lists the species and numbers of fishes incorporated in the two comparisons. In the case of the Heron Island comparison, 103 individual fish were compared, four species of Transversotrema were detected at Heron Island, and one species in French Polynesia. Transversotrematids Table 4 Pairwise distances between ITS2 rDNA sequences for taxa in the Transversotrema borboleta complex. Base pair differences below the diagonal, percent differences above the parallel.
1 2 3 4 5
T. borboleta G1 T. borboleta G2 T. borboleta G3 T. sp. C (Palau) T. polynesiae n. sp.
n
1
2
3
4
5
10 6 3 1 4
x 4 4 8 8
0.95 x 2 4 4
0.95 0.48 x 6 6
1.91 0.95 1.43 x 4
1.91 0.95 1.43 0.95 x
from mullids at Heron Island have not yet been characterised so all infections were treated as belonging to a single species. Three further species of Transversotrematidae known from Heron Island (Crusziella formosa Cribb, Bray & Barker, 1992, Transversotrema elegans Hunter, Ingram, Adlard, Bray & Cribb, 2010 and Transversotrema espanola Hunter & Cribb, 2012) were not detected in the comparison set. In the case of the Lizard Island comparison, 146 individual fishes were compared; 10 species of Transversotrema were detected at Lizard Island and one species was detected in French Polynesia. The comparative data set included all the species of Transversotrematidae known at Lizard Island but the two genotypes of T. borboleta present at Lizard Island were pooled as a single species because so many samples had not been distinguished between the two genotypes and their relative specific status remains unresolved. 4. Discussion 4.1. Taxonomy Neither morphological analysis nor ITS2 rDNA sequences showed any variation between samples of Transversotrema from French Polynesian chaetodontids and lutjanids and all the specimens are thus interpreted as relating to a single species. Although such low specificity is unusual in trematodes of coral reef fishes [41], it has been reported previously for other transversotrematids [25]. The proposal of a new species for the French Polynesian specimens requires careful justification. Parasites are typically distinguished on the basis of combinations of four kinds of data — morphological, molecular, host distribution and geographic distribution. The most clearly distinct species differ in all four areas, the most similar may overlap in several of these respects. In the present case we have been unable
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Fig. 2. NJ tree based on ITS2 rDNA sequences of samples of T. borboleta complex.
to distinguish the French Polynesian material on the basis of morphology or host-specificity. Host distribution, morphology and molecular analysis all indicated that the present specimens are similar to the three genotypes of T. borboleta (G1, G2 and G3) from the GBR, T. sp. C from chaetodontids from Palau and T. sp. D from Chaetodon flavirostris from New Caledonia [25]. These taxa, together with T. polynesiae n. sp., are here considered as forming the T. borboleta complex and as comprising six operational taxonomic units (OTU's). These forms account for all the transversotrematids that have been reported from chaetodontids. Numerous other species are known from lutjanids (Transversotrema cardinalis Hunter & Cribb, 2012, Transversotrema espanola, Transversotrema fusilieri Hunter & Cribb, 2012, Transversotrema licinum Manter, 1970, Transversotrema manteri Hunter & Cribb, 2012 and T. witenbergi Hunter & Cribb, 2012), but all are clearly morphologically and genetically distinct from the T. borboleta complex. The host range of the present specimens, lutjanids and chaetodontids, is consistent with the remainder of the T. borboleta complex which in two cases are also from chaetodontids and lutjanids, in one case from lutjanids only, and in the other two (each based on
limited sampling) from chaetodontids only. Notably, the species of Chaetodon reported infected here, C. auriga and C. ephippium, belong to Chaetodon Clade 4 sensu Bellwood et al. [42] as do the great majority of records of T. borboleta (two genotypes) that are from chaetodontids and all the records of T. sp. C and T. sp. D. Examination of the morphology of the present specimens revealed no reliable distinctions relative to the original descriptions of three genotypes of T. borboleta or the limited material available of T. sp. C and T. sp. D. In describing T. borboleta, Hunter and Cribb [25] commented that it is “probably a complex of at least three closely related species but these are not yet sufficiently well delineated to allow separate descriptions”. The key difficulties were that the genotypes occurred broadly sympatrically (although two genotypes had been found only at Lizard Island on the northern GBR and one only at Heron Island on the southern GBR), that the hosts were broadly overlapping (although again there were some suggestions of possible subtle distinctions) and, that the three forms were essentially morphologically indistinguishable. The key evidence that we invoke here is thus the molecular data. Of the five OTU's with which the French Polynesian specimens
Fig. 3. Transversotrema polynesiae n. sp., ventral view. Scale bar: 200 μm.
T.H. Cribb et al. / Parasitology International 63 (2014) 285–294 Table 5 Measurements of Transversotrema polynesiae n. sp. Chaetodon ephippium
Chaetodon auriga
Lutjanus gibbus
n=1
n=9
n = 10
Body length Body width Length/width Pharynx to ant. margin Cyclocoel to post. margin Eye-spots apart % body width Ventral sucker length Ventral sucker width Pharynx length Pharynx width Left testes area μm2
521 1668 3.20 190
347–480 (416) 1062–1624 (1364) 2.92–3.72 (3.26) 129–188 (155)
331–538 (462) 1153–1708 (1476) 2.81–3.68 (3.22) 116–212 (175)
86
53–74 (63)
44–86 (67)
137 5.16 98 97 79 88 34408
Right testes area μm2
25038
Ovary area μm2 Egg length Egg width
8857
76–128 (106) 4.00–5.42 (4.64) 87–100 (95) 79–100 (92) 48–82 (64) 53–86 (74) 14518–32069 (22999) 10901–27651 (19470) 2993–8416 (5961) 84–130 (101) 37–55 (45)
79–134 (107) 3.69–5.39 (4.52) 64–114 (95) 70–118 (97) 60–83 (75) 63–97 (83) 17959–38203 (27086) 14694–34644 (23490) 3255–8428 (5596) 83–111 (99) 44–70 (54)
are clearly aligned, only T. borboleta has been formally named. Although three genotypes were noted at the time of the description of the species, the name was unambiguously associated with Genotype 1 which is known from chaetodontids and lutjanids at Lizard Island (northern GBR) by way of the holotype which was collected from Chaetodon lunula. The French Polynesian material differs consistently from T. borboleta G1 at eight base positions. This level of variation exceeds that between several combinations of sympatrically occurring and clearly morphologically distinct species of Transversotrema. For
Fig. 4. Species accumulation curves (randomised observed) for species of Transversotrematidae based on matched sets of specimens. A. Heron Island (southern Great Barrier Reef) and French Polynesia. B. Lizard Island (northern Great Barrier Reef) and French Polynesia.
291
Table 6 Matched fish taxa numbers for analysis of transversotrematid richness in French Polynesia (FP) relative to the Great Barrier Reef at Lizard Island (LI) and Heron Island (HI). Family
Genus
Species
FP/HI
FP/LI
Chaetodontidae
Chaetodon
auriga citrinellus ephippium lineolatus lunula lunulatus ulietensis vagabundus
Lethrinidae Lutjanidae
(pooled)
14 7 7 1 3 10 10 4 2
17 14 12 1 2 10 10 4 2
(pooled) bohar fulvus gibbus kasmira monostigma (pooled) (pooled) (pooled)
7 4
7 8 6 5 1 3 3 22 6 13
Caesioninae Lutjanus
Mullidae Pomacentridae Scaridae
Mulloidichthys Parupeneus Abudefduf (pooled)
1 11 3 19
example, Transversotrema gigantica Hunter, Ingram, Adlard, Bray & Cribb, 2010 and T. elegans, which have subtle morphological differences and are restricted to scarids and labrids respectively at Heron Island, differ by just four base pairs. Similarly, Transversotrema damsella Hunter & Cribb, 2012 and T. espanola differ at just six base pairs, but can be easily distinguished morphologically and on the basis of the separate families of fishes that they infect. In the light of these levels of molecular variation between clearly distinct pairs of species being lower than that seen here, we conclude that the French Polynesian form should be recognised as a distinct, cryptic species. The geographical separation of T. polynesiae n. sp. from T. borboleta informs the decision to recognise it as a distinct species in that, practically, it makes it possible to identify specimens from these hosts at this locality without the need to sequence every sample. This is in contrast to the three genotypes of T. borboleta which all occur on the GBR, including two at the same locality. Recognition of T. polynesiae n. sp. raises questions as to the status of the other four OTU's in this group which do not have formal names. The levels of molecular distinction between some of these combinations are as low as two base pairs of ITS2 rDNA. Such low levels of distinction sharpen the need for corroborative evidence before new names might be proposed. In the cases of T. sp. C and T. sp. D, there is a clear need for more specimens for both morphological and molecular analysis. In the cases of T. borboleta Genotypes 2 and 3, the problems outlined by Hunter and Cribb [25] remain; further exploration of host-specificity, perhaps exploration of independent genetic markers, and further search for morphological distinctions are all desirable before these OTU's can be reliably characterised as separate species. Some other combinations of fish trematodes have been recognised partly on the basis of very low levels of molecular difference where the biological corroboration has been convincing. For example, Nolan and Cribb [43] found that highly replicated and consistent molecular distinction of three base pairs of ITS2 rDNA between the aporocotylids Phthinomita jonesi Nolan & Cribb, 2006 and Phthinomita hallae Nolan & Cribb, 2006 was corroborated by their consistent infection of separate, sympatric siganids and subtle morphological distinctions. Ultimately we suspect that this species complex should comprise six named species and we think it at least possible, perhaps likely, that further species will be found at more localities if they are sought. A major proliferation of species names for this group might be considered practically problematic and biologically meaningless if the parasites are parasitising the same hosts and have indistinguishable (or nearly so) morphology. The issues relating to the naming of cryptic species and the value of DNA data in doing so have been dealt with in many analyses [e.g. 44–46]. In our view it is best to treat problems of apparently cryptic
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species on a case by case basis. In the present system we note that the characteristics of this apparent complex of species is in contrast both with other transversotrematids, in that so far only one species has been found in French Polynesia, and with other trematodes that retain their genetic integrity over wide ranges. Several trematodes of coral reef fishes have been reported as having identical ITS2 sequences for specimens from the GBR and French Polynesia [12,47]. Thus, there is no prospect that every trematode morpho-species will need recognition as separate species at multiple TIWP localities. The extent to which the T. borboleta complex has radiated appears to be exceptional and, in this context, the recognition of separate species for its components is an appropriate reflection of the nature of the system. 4.2. Biogeography and richness We conclude from the comparative analysis of transversotrematid species discovery on the GBR and in French Polynesia that transversotrematids are exceptionally depauperate in French Polynesia. French Polynesia covers a vast geographical area (2.5 million km2) and incorporates a wide variety of habitats of which we have sampled only a few. It would therefore be foolhardy to state that there are no more transversotrematid species to be detected there. In addition, we do not contend that we have demonstrated that none of the species found on the GBR occur in French Polynesia. However, we think it is clear from our comparative analysis of the pattern of discovery that transversotrematids are far less rich there than on the GBR, despite the presence of many suitable fish host families. In terms of the possible biogeographical patterns hypothesised above, we have found a single species endemic to French Polynesia and no evidence at all for widespread species (species shared between the GBR and French Polynesia). Thus, the evidence is that transversotrematids follow the typical pattern of decline in richness from the centre to more peripheral regions of the TIWP as well as the appearance of some endemicity that is seen for free-living animals. However, the decline, from 18 species known on the GBR to just one known in French Polynesia, is unusually steep. The paucity of the French Polynesian transversotrematid fauna has two distinct elements. First, hosts for several species that occur on the GBR are simply not present in French Polynesia. Such absences probably preclude at least five species from occurring in French Polynesia. Transversotrema atkinsoni Hunter & Cribb, 2012, Transversotrema carmenae Hunter & Cribb, 2012 and Transversotrema nova Hunter & Cribb, 2012 are all known only from nemipterids, a family which does not occur in French Polynesia. Transversotrema elegans is known overwhelmingly from species of the labrid genus Choerodon which is entirely absent from French Polynesia. A single individual of this species was recorded from the labrid Gomphosus varius from the GBR, a species present in French Polynesia but not examined by us there. Given the evident reliance of T. elegans on species of Choerodon we conclude that the species is probably absent from French Polynesia. Transversotrema espanola is primarily a parasite of Lutjanus carponotatus on which it is abundant, especially on the southern GBR where 38 of 49 individuals examined were infected; it has been found rarely on other lutjanids and occasionally on haemulids (the latter being entirely absent from the French Polynesian sites we sampled). Lutjanus carponotatus does not occur in French Polynesia and we suspect this absence excludes T. espanola from the region. The second element of absence relates to species for which suitable hosts are present in French Polynesia but for which samples have so far produced no infections. Such host groups include the Lethrinidae, Lutjanidae, Mullidae, Pomacentridae (Abudefduf) and Scaridae. Although one species is now known from a lutjanid in French Polynesia, a total of seven are known in Australian waters, including three on caesionines and four on lutjanines. Between them, these five families harbour at least eight species of transversotrematids on the GBR that have not yet been detected in French Polynesia, despite the examination of varying numbers of suitable hosts. The plateauing shapes of the
accumulation curves for the two GBR sites relative to those for French Polynesia make it clear that the overall lack of richness identified in French Polynesia is not an artefact of undersampling. Other groups of helminth parasites of fishes that have been studied in French Polynesia show varying evidence of richness, species shared with other regions, species loss and local endemicity. For dactylogyrid monogeneans of chaetodontid fishes there have been more species reported from French Polynesia (11) than at either of two sites on the GBR (6 and 8) [21,22]. Although the robustness of these figures is weakened by the lack of published sample sizes for the fish hosts, it is clear that dactylogyrids are abundant in French Polynesia in a way that contrasts dramatically with the paucity of transversotrematids. For trematodes, studies comparing the GBR and French Polynesia based on systematic collecting or molecular analyses have revealed a range of effects. Lo et al. [47] used molecular analyses (ITS2 rDNA sequence data) to demonstrate that three species (Schistorchis zancli Hanson, 1953, Preptetos laguncula Bray & Cribb, 1996, Neohypocreadium dorsoporum Machida & Uchida, 1987) were identical (or at least partly so) between French Polynesia and the GBR. Similarly, Chambers and Cribb [12] demonstrated that the lecithasterid Quadrifoliovarium pritchardae Yamaguti, 1965 had identical ITS2 rDNA sequences from Ningaloo Reef in Western Australia, the GBR and French Polynesia and Miller and Cribb [14] used combined molecular and morphological evidence to report an even larger distribution for the cryptogonimid Retrovarium brooksi Miller & Cribb, 2007 from the Maldives (Indian Ocean), the GBR and French Polynesia. These three studies established that trematode species of TIWP fishes may have very wide distributions and remain genetically identical (at least for ITS2 rDNA sequences) over these ranges. In contrast, Downie et al. [18] and Diaz et al. [19] inferred that two species each of Symmetrovesicula (Fellodistomidae) and Paradiscogaster (Faustulidae) are absent from French Polynesia despite the presence of suitable chaetodontid hosts there. Morphological analysis of Hurleytrematoides (Monorchiidae) of chaetodontid fishes [20,48] found 10 species in GBR fishes but only six species in French Polynesia. Of these, just four were in common; two found in French Polynesia were not found on the GBR despite the presence of suitable hosts, two found on the GBR were not found in French Polynesia despite the presence of suitable hosts, and two found on the GBR lacked suitable hosts in French Polynesia. Overall, of the French Polynesian fish helminth groups studied in any detail, the fauna of the Transversotrematidae appears to be the most depauperate relative to that known elsewhere and the only fauna composed only of endemic species. Why the transversotrematids of French Polynesia are so depauperate is not immediately obvious. Certainly the family is not absent because of the absence of suitable definitive hosts but it is possible that some intermediate hosts are absent. This is difficult to analyse because life cycles of marine transversotrematids are entirely unknown. Freshwater species use gastropods of the families Thiaridae [49,50] and Hydrobiidae [51], which gives little guide as to the likely identity of the hosts of marine species beyond an expectation that they will be gastropods. The mollusc fauna of French Polynesia is known to be rich; Tröndlé and Boutet [52] reported 2373 species for the region. In addition, recent dramatic findings regarding molluscan richness in the TIWP [e.g. 53,54] suggest that the richness and biogeography of molluscs of the TIWP remain poorly known. Subject to definitive information regarding the identity of the marine hosts of transversotrematids, we think it unlikely that molluscan intermediate host availability is more limiting for Transversotrematidae than for other families of trematodes. We predict that the explanation for the absence of transversotrematids in French Polynesia lies in the nature of the life cycle. Only freshwater life cycles are known for the family but we can assume that marine cycles are broadly comparable given that there is no general pattern of distinction between marine and freshwater cycles of species from the same trematode family. Freshwater transversotrematids have two-host cycles in which large, short-lived cercariae emerge from gastropods and infect their definitive hosts directly. Cribb [51] reported
T.H. Cribb et al. / Parasitology International 63 (2014) 285–294
that the cercaria of Prototransversotrema steeri Angel, 1969 survives for only about 18 h. The other free-living stage, the miracidium which emerges from the egg to infect the gastropod intermediate host, was shown by Bundy [55] to survive for no more than 8 h for Transversotrema patialense (Soparkar, 1924). The adults of transversotrematids require well-developed scales on fishes and the molluscan infection presumably requires development of the gastropod host well beyond that seen in pelagic larval stages. However, most adult coral reef fishes and gastropods have low vagility so that there is thus no stage obviously capable of dispersing these parasites over long distances. Unlike many trematodes, transversotrematids lack a second intermediate host in which a metacercaria develops. The trematodes of the TIWP with the largest known ranges demonstrated by genetic studies, species of Schistorchis (Apocreadiidae), Neohypocreadium and Preptetos (Lepocreadiidae), and Retrovarium (Cryptogonimidae) (reviewed above) belong to families that use second intermediate hosts; thus the use of second intermediate hosts may be important in enabling the broad distributions seen for these species. That transversotrematids have low vagility is implied by the significant differences in the species composition previously demonstrated between the southern and northern GBR [24–26] and by the fact that the single species found in French Polynesia is distinct from (although clearly closely related to) species known on the GBR and elsewhere. Thus, we infer that the nature of the transversotrematid fauna in French Polynesia relates to the combination of large short-lived cercariae, short-lived miracidia, the absence of second intermediate hosts, and dependence on definitive and intermediate hosts that have limited vagility. This conclusion parallels that of Meyer [56] for cypraeid gastropods — ‘shorter larval duration results in smaller ranges, higher speciation rates, but also higher turnover’. Two predictions arise from this study. First we predict that further sampling will demonstrate that the richness of the family Transversotrematidae decreases progressively eastwards across the Pacific from the GBR. We suspect that the family does not occur at all in Hawaii which is already known for the absence of many marine animal taxa and where two major trematode workers, Yamaguti and Manter, both aware of the existence of transversotrematids, did extensive work. Perhaps a gradient of richness also occurs westwards across the Indian Ocean. However, it is already known that at least two species of transversotrematids occur in the Red Sea [57–59]. The second prediction is that further examples of transversotrematid endemicity will be detected, although they may well require molecular analysis for their recognition. Acknowledgements Collecting in French Polynesia was supported by The University of Queensland, BioCode, the Coral Spot programme (financially supported by the French Polynesian Territory and the French Government) and Pakaihi ite Moana in Marquesas (financially supported by AAMP, by the French Polynesian Territory and the French Government). We are also grateful for the generous support of the Khaled bin Sultan Living Oceans Foundation. Collecting on the Great Barrier Reef was supported by the Australian Research Council and the Australian Biological Resources Study. We thank the many colleagues and past students who helped collect specimens on the Great Barrier Reef and elsewhere. We especially thank Serge Planes, Geoff Williams and Erwan DelrieuTrottin for their assistance with collection and identification of fish in French Polynesia. References [1] Carpenter KE, Springer VG. The center of the center of marine shore fish biodiversity: the Philippine Islands. Environ Biol Fish 2005;72:467–80. [2] Kulbicki M. Biogeography of reef fishes of the French territories in the South Pacific. Cybium 2007;31:275–88.
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