DOI: 10.2478/s11686-007-0019-y
© 2007 W. Stefañski Institute of Parasitology, PAS
Acta Parasitologica, 2007, 52(2), 104–113; ISSN 1230-2821
Two new cryptogonimid genera (Digenea, Cryptogonimidae)
from Lutjanus bohar (Perciformes, Lutjanidae):
Stefañski analyses of ribosomal DNA reveals wide geographic distribution
and presence of cryptic species
Terrence L. Miller1* and Thomas H. Cribb1,2
1School
of Molecular and Microbial Sciences and 2Centre for Marine Studies, The University of Queensland, Brisbane,
Queensland 4072, Australia
Abstract
We describe three new species of Cryptogonimidae belonging to two new genera, Caulanus gen. nov. and Latuterus gen. nov.,
from the large piscivorous reef fish Lutjanus bohar ForsskDl, 1775, recovered from Heron and Lizard Islands off the Great
Barrier Reef and Rasdhoo Atoll, Maldives. To support our morphologically based taxonomic approach, three nuclear ribosomal
DNA regions (28S, ITS1 and ITS2) were sequenced and analysed to explore the geographic distribution and integrity of the
putative species recovered from these widespread localities. Sequencing of the rDNA regions included multiple replicates and
revealed three distinct genotypes. Two of the observed genotypes were associated with phenotypically similar specimens of
Latuterus, but were each restricted to a single locality, Lizard Island, GBR or Rasdhoo Atoll, Maldives. A posteriori analysis
of the associated morphotypes revealed distinct morphological differences and these consistent differences, in combination with
the consistent genetic differences led to the recognition of two distinct species in the system. Caulanus is distinguished by having oral spines, caeca which open via ani at the posterior end of the body, tandem testes and uterus that extends from the posterior end of the body to the pharynx. Latuterus is distinguished by lacking oral spines, having multiple/follicular testes, a uterus
that is extensive in both fore- and hindbody and vitelline follicles which are confined to the region from the pharynx to oral sucker. Caulanus thomasi sp. nov. had identical sequences for all of the rDNA regions examined from specimens recovered from
all three localities, indicating that this species has a wide Indo-Pacific distribution. The species reported here are evidently
restricted to Lutjanus bohar because they were never found in large numbers of other lutjanid species sampled at the same localities.
Key words
28S, ITS1, ITS2, cryptic species, internal transcribed spacers, biogeography, Cryptogonimidae, Lutjanidae
Introduction
Lutjanus bohar ForsskDl, 1775 is a large predatory reef fish
Skóra
distributed widely throughout the tropical Indo-Pacific
Ocean
from East Africa to the Marquesas and Line Islands, north to
the Ryuku Islands and south to Australia (Allen 1985). This
species is relatively long-lived and comprises an important
fishery for many island nations, although it is banned from
sale in some countries because of its reported implication in
ciguatera poisoning (Marriott and Mapstone 2006). Despite
L. bohar being common on reefs throughout its range, the intestinal parasite fauna infecting this species has not been thoroughly characterised; only two trematodes have been identified to species, the acanthocolpid Stephanostomum casum
*Corresponding
Linton, 1910 by Arthur and Lumanlan-Mayo (1997) and the
opeceolid Hamacreadium mutabile Linton, 1910 by Bray and
Cribb (1989) and Rigby et al. (1999). The only other report in
the literature is of an unidentified species of Cryptogonimidae
from L. bohar off French Polynesia by Rigby et al. (1999).
A survey of the intestinal trematode fauna infecting species of Indo-West Pacific Lutjanidae revealed the presence of
undescribed species of Cryptogonimidae infecting L. bohar
from three localities. Here we describe three new species in
two new genera from specimens of L. bohar collected from
sites on the Great Barrier Reef (GBR) and in the Maldives. In
addition to morphological comparison, we augment our taxonomic approach with analysis of molecular data from three
ribosomal DNA regions, the 28S and the internal transcribed
author: [email protected]
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105
Œl¹ski
spacers 1 and 2 (ITS1 and ITS2), to explore the geographic
distribution and integrity of these species. This combined taxonomic approach utilizing comparative analyses of ribosomal
DNA sequences to support traditional morphologically based
studies is becoming more frequently used to characterise digenean systems because of the ability to reveal richness or phylogenetic affinities that morphology alone cannot distinguish.
Presently, the internal transcribed spacers are the most frequently used regions to distinguish digenean species, with
data (ITS1, ITS2 or both) reported from at least 24 families
(Nolan and Cribb 2005). These regions have been used in
trematode systems to separate combinations of species with
little phenotypic variation (cryptic species), which may not
have been recognized as distinct without the accompanying
molecular data (Despres et al. 1995, Iwagami et al. 2000,
Jousson and Bartoli 2000, Jousson et al. 2000).
Analyses of the three ribosomal DNA regions for the
species described herein are compared with those reported
recently for other cryptogonimid genera by Miller and Cribb
(in press-a) to examine levels of intergeneric and interspecific variation between these taxa, which all infect species of
tropical Indo-West Pacific Lutjanidae.
Materials and methods
Host and parasite collection
Fishes were collected using baited line or spear from the following localities: Lizard Island (14°40´S, 145°27´E) in the
northern GBR, Heron Island (23°26´S, 151°54´E) in the
southern GBR and Rasdhoo Atoll (4°16´N, 72°58´E), Maldives. Fish were euthanased by neural pithing and the intestine
immediately removed, washed in vertebrate saline (0.85%),
and examined for the presence of endohelminths. Trematodes
were washed in saline and killed by pipetting them into nearly boiling saline. Specimens for morphological analysis were
then stored in 10% formalin and specimens for DNA extraction and analysis were stored in 95–100% ethanol at –20°C.
Morphological samples
Preserved specimens for morphological analysis were washed
in fresh water and placed in Mayer’s haematoxylin for staining. The specimens were overstained and then destained by
placing them in a solution of 1.0% HCl and subsequently neutralized in a 0.5–1.0% ammonium hydroxide solution. Stained
specimens were then dehydrated through a graded series of
ethanol for at least half an hour at each dehydration step,
cleared in methyl salicylate and mounted in Canada balsam.
Drawings were made with the aid of a drawing tube. All measurements were made using an ocular micrometer and are in
micrometers with the mean followed by the range in parentheses. Type-specimens were deposited in the Queensland
Museum, Brisbane, Australia.
Multivariate statistical analyses [principal components
analysis (PCA) and discriminant analysis] of normalised mor-
phometric data were performed between all individuals of
morphologically similar species using the software PAST
(Hammer et al. 2001). Scatterplots were drawn to indicate the
position of individual specimens on the axes (Principal component axis 1 vs 2).
Molecular samples
Total genomic DNA from species of Caulanus gen. nov. and
Latuterus gen. nov. was isolated by a standard proteinase K
and phenol:chloroform extraction procedure (Sambrook et
al. 2001). Amplification of the 28S rDNA region was performed with the primers LSU5 (5’-TAGGTCGACCCGCTGAAYTTAAGCA-3’ Littlewood et al. 2000) and ECD2 (5’CCTTGGTCCGTGTTTCAAGACGGG-3’ Littlewood et al.
2000), the ITS1 region with the primers BD1 (5’-GTCGTAACAAGGTTTCCGTA-3’ Bowles and McManus 1993)
and 4S (5’-TCTAGATGCGTTCGAARTGTCGATG-3’ Bowles and McManus 1993) and the ITS2 region with the primers
3S (5’-GGTACCGGTGGATCACGTGGCTAGTG-3’Anderson and Barker 1993) and ITS2.2 (5’-CCTGGTTAGTTTCTTTTCCTCCGC-3’ Anderson and Barker 1993). PCR was
conducted for all three rDNA regions with a total volume of
20 µl consisting of approximately 10 ng of template DNA,
0.75 µl of each primer (10 pmols), 1.6 µl MgCL2, 2 µl of
10 × reaction buffer, 0.8 µl deoxyribonucleotide triphosphate (dNTP) (each 2.5 mM), and 0.25 µl of Taq DNA polymerase. Amplifications of the 28S, ITS1 and ITS2 rDNA regions
were carried out on a MJ Research PTC-150 thermocycler
(Waltham, MA). The following profile was used to amplify
the 28S and ITS2 rDNA regions: an initial 96°C denaturation
for 5 min, followed by 25 cycles of 96°C denaturation for
1 min, 54°C annealing for 15 s, 72°C extension for 30 s, and
a final 72°C extension for 4 min. The following profile was
used to amplify the ITS1 region: an initial 95°C denaturation
for 5 min, followed by 30 cycles of 95°C denaturation for
30 s, 55°C annealing for 30 s, 72°C extension for 1 min, and a
final 72°C extension for 10 min. Amplified DNA was purified
using QIAGEN® QIAquick™ PCR purification kit according
to manufacturer’s protocol. Cycle sequencing was conducted
using the same primers utilized for PCR amplification with
ABI Big Dye™ v.3.1 chemistry following manufacturer’s recommendations. Precipitation with 3 M sodium acetate (pH
approximately 5) and alcohol was done to remove dye terminators, and the pellets were then dried for 30–60 min at 39°C
and sequenced using an ABI 3730xl automated sequencer.
The resulting sequences were edited and contigs constructed
using Sequencher™ version 4.5 (GeneCodes Corp.). GenBank accession numbers for all taxa sequenced in this study are
provided in Table I. The consensus sequences for each taxon
utilized in this study were constructed from multiple replicates
(each replicate being both a forward and reverse sequence from
a single individual from different infections when possible)
from different host/parasite/location combinations whenever
possible. The total number of replicates for each rDNA region
sequenced in this study are shown in Table I.
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Terrence L. Miller and Thomas H. Cribb
Stanis³a
Table I. Total number of molecular sequence replicates for each of the rDNA regions
produced during this study followed by the corresponding GenBank accession numbers
for species of Caulanus gen. nov. and Latuterus gen. nov.
Species
Number of replicates and GenBank Accession No.
28S
Caulanus thomasi sp. nov.
Latuterus tkachi sp. nov.
Latuterus maldivensis sp. nov.
7
3
2
EF428144
EF428145
EF428146
ITS1 and ITS2
9
4
2
EF428141
EF428142
EF428143
Comparative DNA analyses
The 28S, ITS1 and ITS2 rDNA regions from taxa sequenced
in this study were initially aligned using ClustalX version 1.83
(Thompson et al. 1997) under the following parameters: pairwise alignment parameters = gap opening 10.00, gap extension 0.10, DNA weight matrix International Union of Biochemistry (IUB); multiple alignment parameters = gap opening 10.00, gap extension 0.20, delay divergent sequences 30%,
DNA weight matrix IUB. The resulting sequence alignments
were exported from ClustalX in FASTA and NEXUS formats,
and refined by eye using MacClade version 4.08 (Maddison
and Maddison 2005). After alignments of all three rDNA regions were edited, the ends of each fragment were trimmed to
match the shortest sequence in the alignment. Distance matrices for each of the three rDNA regions were constructed with
the absolute pairwise character difference and the percentage
of pairwise character differences. Pairwise comparisons of
absolute sequence divergence for all taxa were calculated with
gaps treated as missing data.
The three rDNA regions for species of Caulanus and Latuterus were also aligned with those reported for the species
Retrovarium amplorificium, R. exiguiformosum, R. formosum,
R. gardneri, R. sablae, R. snyderi, Neometadena ovata and
an undescribed species of Siphoderina (previously Neoparacryptogonimus ovatus and Paracryptogonimus sp., see Miller
and Cribb in press-b) by Miller and Cribb (in press-a) for comparative purposes. The three rDNA regions were then combined and assigned partitions in a single NEXUS file. Minimum evolution (ME) analyses of each of the three rDNA
regions independently and of the combined dataset of these
taxa was performed using PAUP* version 4.0b10 (Swofford
2003) and estimated by total distance, with heuristic searches employing tree bisection-reconnection (TBR) swapping.
Nodal support for ME analyses was conducted using 10,000
bootstrap replicates.
and 15 at Rasdhoo Atoll, Maldives). Putative trematode species were separated into two morphotypes, genomic DNA was
extracted from individuals representing each of these morphotypes, and the 28S, ITS1 and ITS2 rDNA regions were
sequenced from as many different host/parasite/location combinations as possible. Sequencing of the rDNA revealed three
unique genotypes for each of the three rDNA regions examined. Multiple replicates obtained for the three rDNA regions
from the three sequence types revealed no variation within the
sequences, which corresponded with the morphological and
geographic distribution diversity seen in the morphotypes.
The combination of morphological, molecular and biogeographic distribution seen in this system forms the basis of our
recognition of species here. One genotype, associated with the
species described here as Caulanus thomasi sp. nov., had identical rDNA sequences (all three regions) from Heron Island,
Lizard Island and the Maldives. The other two genotypes, differed consistently in all three rDNA regions between Lizard
Island and the Maldives; neither was found at Heron Island.
Analysis of the phenotypically similar morphotypes associated with the two remaining genotypes revealed that the specimens from the Maldives were distinctly smaller (fully mature
specimens approximately only half of the size) than the specimens recovered from Lizard Island, and we choose to recognize the two distinct morpho- and genotypes as separate
species here for reasons explained in the Discussion section.
Results
ITS rDNA
Sequencing of the ITS1, 5.8S and ITS2 rDNA yielded fragments that ranged from approximately 1100 bp in Caulanus
thomasi to approximately 1050 bp in Latuterus tkachi and
L. maldivensis. Over this entire region, C. thomasi differed by
52 bp (5.0%) from L. tkachi and by 54 bp (5.2%) from L. maldivensis. Latuterus tkachi and L. maldivensis differed from
each other by 6 bp (0.6%).
Molecular data
Species level taxonomy in light of molecular and morphological data
A total of 33 specimens of Lutjanus bohar were collected
from the three localities (7 at Heron Island, 11 at Lizard Island
28S rDNA
Sequencing of the 28S rDNA yielded an average of approximately 900 base pairs (bp) for all taxa. The aligned and trimmed sequences incorporated a total of 893 characters (base
pairs and gaps) for analysis. Caulanus thomasi differed by 36
bp (4.0%) from Latuterus tkachi sp. nov. and by 34 bp (3.8%)
from L. maldivensis sp. nov. Latuterus tkachi and L. maldivensis differed by 3 bp (0.4%) from each other.
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Two new cryptogonimid genera from Lutjanus bohar
Roborzyñski
rosbœŸæv
107
fjad kadsææ¿æ
The ITS1 region ranged in size from 576 bp in C. thomasi
to 525 bp in L. tkachi and L. maldivensis. Caulanus thomasi
differed by 33 bp (6.3%) from L. tkachi and by 35 bp (6.7%)
from L. maldivensis. Latuterus tkachi and L. maldivensis differed from each other by 2 bp (0.4%).
The 5.8S region was 160 bp in length and was identical for
all three taxa.
The ITS2 region was 289 bp in length in all three species.
Caulanus thomasi differed by 19 bp (6.6%) from both
L. tkachi and L. maldivensis. Latuterus tkachi and L. maldivensis differed from each other by 4 bp (1.4%).
Intergeneric variation
Alignment of the 28S regions of Caulanus thomasi, species of
Latuterus, Neometadena, Retrovarium and Siphoderina found
C. thomasi differed from Neometadena ovata by 49 bp (5.7%),
from species of Retrovarium by 56–63 bp (6.5–7.3%) and
from the undescribed species of Siphoderina by 37 bp (4.3%).
Species of Latuterus differed from N. ovata by 61–63 bp (7.0–
7.3%), from species of Retrovarium by 60–65 bp (6.9–7.5%)
and from the undescribed species of Siphoderina by 47–49 bp
(5.4–5.7%).
Variation in the 5’ half of the ITS1 made alignment between species of Caulanus, Latuterus, Neometadena, Retrovarium and Siphoderina in this region impossible, so only the
3’ half of the ITS1 were included as in Miller and Cribb (in
press-a), because this region was alignable. In the 3’ half of the
ITS1, C. thomasi differed from N. ovata by 41 bp (11.2%),
from species of Retrovarium by 24–37 bp (6.4–9.7%) and
from the undescribed species of Siphoderina by 24 bp (6.2%).
Species of Latuterus differed from Neometadena ovata in the
3’ half of the ITS1 by 46–47 bp (12.6–12.9%), from species of
Retrovarium by 28–41 bp (7.5–10.8%) and from the undescribed species of Siphoderina by 25–26 bp (6.6–6.8%).
Alignment of the ITS2 region revealed that Caulanus
thomasi differed from N. ovata by 29 bp (8.7%), from species
of Retrovarium by 33–39 bp (9.9–11.6%) and from the undescribed species of Siphoderina by 23 bp (6.8%). Species of
Latuterus differed from N. ovata by 34 bp (10.2%), from
species of Retrovarium by 35–39 bp (10.5–11.8%) and from
the undescribed species of Siphoderina by 29 bp (8.6%).
Minimum evolution analysis (Fig. 1, ME score = 419.99)
of the combined dataset supported the results seen with each
of the three rDNA regions when analysed independently. The
combined dataset revealed intergeneric relationships similar
to those observed by Miller and Cribb (in press-a) and resulted in the undescribed species of Siphoderina forming a well
supported clade with species of Caulanus and Latuterus.
Morphological data
Family Cryptogonimidae Ward, 1917
Caulanus gen. nov.
Diagnosis: Body elongate; length/width ratio c. 2.9–3.7. Tegument with small to minute spines. Oral sucker wider than
Fig. 1. Phylogram of relationships between species of Caulanus gen.
nov., Latuterus gen. nov., Neometadena, Retrovarium and an undescribed species of Siphoderina inferred from minimum evolution
analysis of total genetic distance for the combined (28S, ITS1 and
ITS2) rDNA datasets. Bootstrap values are indicated at the nodes,
with values <50 not shown
long, with enlarged oral spines, opens terminally. Ventral
sucker unspecialised, embedded in ventrogenital sac. Ratio
oral/ventral sucker width c. 1.3–1.7. Forebody occupies c. 1/3
body length. Prepharynx long, often longer than oesophagus.
Oesophagus short. Intestinal bifurcation midway between
ventral sucker and pharynx. Caeca open via ani at posterior
end of body. Testes two, entire, tandem, in mid-hindbody. Seminal vesicle saccular, at level of or slightly posterior to ventral
sucker. Common genital pore immediately anterior to ventral
sucker. Gonotyl absent. Ovary trilobed or with few lobes,
immediately anterior and adjacent to testes in mid-hindbody.
Laurer’s canal present. Seminal receptacle tubulosaccular,
immediately dorsal or anterior to ovary. Vitelline follicles in
two lateral groups ventrally, confluent dorsally, extend from
ovary to pharynx. Uterine coils extensive in fore- and hindbody, extend from posterior end of body to pharynx. Excretory vesicle Y-shaped; arms reach to level of pharynx. In marine fishes (Lutjanidae); tropical Indo-West Pacific.
Type-species: Caulanus thomasi sp. nov.
Etymology: The name Caulanus is derived from the Latin
caula meaning opening, hole or passage and the Latin anus
meaning intestine or rectum. It is proposed in recognition of
the caeca, which open via ani at the posterior end of the body
in these species. It is a noun to be treated as masculine.
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Caulanus thomasi sp. nov. (Fig. 2)
Description: Based on 10 specimens. Body elongate, longer
than wide, 1221 (790–1443) long by 373 (260–429) wide;
length/width ratio 3.27 (2.93–3.61). Oral sucker 113 (69–133)
long by 184 (139–211) wide. Oral spines 47 (41–52), length
15 (11–20). Ventral sucker 119 (93–138) long by 126 (99–
149) wide. Ratio oral/ventral sucker width 1.46 (1.37–1.64).
Forebody occupying 32 (31–33)% body length. Prepharynx
80 (58–107) long. Pharynx 88 (69–106) long by 85 (67–104)
wide. Ventral sucker/pharynx width ratio 1.49 (1.38–1.74).
Genital pore immediately anterior to ventral sucker. Oesophagus 24 (16–35) long. Intestinal bifurcation immediately
anterior to ventral sucker. Intestinal caeca open via ani at posterior end of body 937 (579–1131) long. Testes two, tandem,
immediately posterior to ovary, anterior testis 127 (90–147)
long by 150 (109–179) wide, posterior testis 128 (80–152)
long by 160 (118–192) wide. Seminal vesicle saccular, at level
of ventral sucker or slightly posterior to it. Ovary trilobed or
with few lobes, at midbody immediately anterior and adjacent
to testes, 95 (69–122) long by 125 (104–147) wide. Laurer’s
canal present. Seminal receptacle tubulosaccular, immediately dorsal or anterior to ovary. Vitelline follicles extend from
level of ovary to pharynx; confluent dorsally. Uterine coils
extensive in fore- and hindbody, extend from posterior end of
body to level of pharynx. Eggs small, darkly tanned, 18 (15–
20) long by 8 (7–9) wide. Excretory vesicle Y-shaped, bifurcates dorsal to ovary; arms extend to level of pharynx, 969
(631–1157) long.
Type host: Lutjanus bohar (ForsskDl, 1775), Perciformes,
Lutjanidae, red bass.
Type locality: Lizard Island, Great Barrier Reef (14°40´S,
145°27´E), Queensland, Australia.
Other localities: Heron Island, Great Barrier Reef (23°26´S,
151°54´E), Queensland, Australia; Rasdhoo Atoll (4°16´N,
72°58´E), Maldives.
Site: Rectum.
Prevalence: 10 of 33 (30%); 5 of 11 (45%) at Lizard Island; 2 of 7 (29%) at Heron Island; 3 of 15 (20%) at Rasdhoo
Atoll.
Deposited specimens: Holotype G227607, paratypes
G227608-G227616.
Etymology: The species epithet thomasi is in honour of Mr
Thomas Miller, the father of the senior author, in recognition
of his support and encouragement of this work.
Latuterus gen. nov.
Fig. 2. Caulanus thomasi sp. nov. from the rectum of Lutjanus bohar
from Lizard Island, Great Barrier Reef, Australia. Scale bar = 200 µm
Diagnosis: Body squat, slightly longer than wide; length/
width ratio c. 1–1.1. Tegument with small to minute spines.
Oral sucker nearly round in outline, without enlarged oral
spines, opens slightly subterminally to almost terminally. Ventral sucker unspecialised, embedded in ventrogenital sac. Ratio oral/ventral sucker width c. 1.7–2. Forebody occupies c.
1/3–2/5 body length. Prepharynx very short. Oesophagus very
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short. Intestinal bifurcation immediately posterior to pharynx.
Caeca blind, terminate close to posterior end of body. Testes
multiple/follicular, 9 in number, near midbody. Seminal vesicle bipartite, at level of ventral sucker. Common genital pore
immediately anterior to ventral sucker. Gonotyl absent. Ovary
deeply lobed, slightly posterior to midbody at level of testes.
Laurer’s canal present. Seminal receptacle saccular, dorsal to
ventral sucker. Vitelline follicles restricted entirely to forebody,
confluent dorsally and ventrally, extend from pharynx to oral
sucker. Uterine coils extensive in fore- and hindbody, extend
from slightly posterior to testes to anterior end of pharynx.
Excretory vesicle Y-shaped; arms reach to level of pharynx. In marine fishes (Lutjanidae); tropical Indo-West Pacific.
Type-species: Latuterus tkachi sp. nov.
Etymology: The name Latuterus is derived from the Latin
latus meaning broad or wide and the Latin uterus meaning
uterus or womb. It is proposed in recognition of the uterus,
which is extensive throughout the fore- and hindbody in these
species. It is a noun to be treated as masculine.
Latuterus tkachi sp. nov. (Fig. 3)
Description: Based on 4 specimens. Body squat, slightly
longer than wide, 1294 (1168–1384) long by 1234 (1168–
1304) wide; length/width ratio 1.05 (1–1.07). Oral sucker 226
(202–241) long by 288 (280–302) wide. Ventral sucker 145
109
(133–156) long by 157 (153–159) wide. Ratio oral/ventral
sucker width 1.84 (1.76–1.94). Forebody occupying 39 (34–
43)% body length. Prepharynx effectively absent. Pharynx
wider than ventral sucker, 210 (205–221) long by 210
(198–218) wide. Ventral sucker/pharynx width ratio 0.75
(0.73–0.77). Genital pore immediately anterior to ventral
sucker. Oesophagus effectively absent. Intestinal bifurcation
at or immediately posterior to pharynx. Intestinal caeca blind,
960 (806–1053) long, terminate close to posterior end of body.
Testes multiple/follicular, 9 in number, near midbody, individual follicles 185 (143–234) long by 182 (127–228) wide.
Seminal vesicle saccular, at level of ventral sucker. Ovary
deeply lobed 189 (150–231) long by 392 (338–442) wide,
near midbody at level of testes. Laurer’s canal present. Seminal receptacle saccular, dorsal to ventral sucker. Vitelline
follicles confined entirely to forebody, confluent dorsally and
ventrally, extend from pharynx to oral sucker. Uterine coils
extensive in fore- and hindbody, extend from level of testes
or slightly posterior to testes to level of pharynx or oral sucker. Eggs small, darkly tanned, 16 (15–19) long by 8 (7–8)
wide. Excretory vesicle Y-shaped, bifurcates dorsal to ovary;
arms extend to level of pharynx, 856 (793–995) long.
Type host: Lutjanus bohar (ForsskDl, 1775), Perciformes,
Lutjanidae, red bass.
Type locality: Lizard Island, Great Barrier Reef (14°40´S,
145°27´E), Queensland, Australia.
Figs 3 and 4. Latuterus gen. nov. from the intestine of Lutjanus
bohar (ventral view). 3. L. tkachi sp. nov. from Lizard Island, Great
Barrier Reef, Australia. 4. L. maldivensis sp. nov. from Rasdhoo
Atoll, Maldives. Scale bars for both figures = 500 µm
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Site: Intestine and pyloric caeca.
Prevalence: 3 of 11 (27%) at Lizard Island.
Deposited specimens: Holotype G227617, paratypes G227618-G227620.
Etymology: The species epithet tkachi is in honour of Dr
Vasyl Tkach in recognition of his contributions to platyhelminth systematics and taxonomy.
Latuterus maldivensis sp. nov. (Fig. 4)
Description: Based on 2 specimens. Body squat, slightly
longer than wide, 788 (736–840) long by 752 (736–768) wide;
length/width ratio 1.05 (1–1.09). Oral sucker 128 (127–130)
long by 187 (182–192) wide. Ventral sucker 101 long by 102
(94–111) wide. Ratio oral/ventral sucker width 1.86 (1.67–
2.04). Forebody occupying 38 (36–39%) body length. Prepharynx effectively absent. Pharynx wider than ventral sucker, 133 (130–137) long by 132 (130–133) wide. Ventral sucker/pharynx width ratio 0.78 (0.71–0.85). Genital pore immediately anterior to ventral sucker. Oesophagus effectively absent. Intestinal bifurcation at or immediately posterior to pharynx. Intestinal caeca blind, 546 (494–611) long, terminate
close to posterior end of body. Testes multiple/follicular, 9 in
number, near midbody, individual follicles 100 (94–114) long
by 105 (94–124) wide. Seminal vesicle tubulosaccular, at level of ventral sucker. Ovary deeply lobed 146 (124–169) long
by 216 (215–218) wide, near midbody posterior to ventral
sucker. Laurer’s canal present. Seminal receptacle saccular,
dorsal to ventral sucker. Vitelline follicles confined entirely
to forebody, confluent dorsally and ventrally, extend from
pharynx to oral sucker. Uterine coils extensive in fore- and
hindbody, extend from level of testes or slightly posterior to
testes to level of pharynx or oral sucker. Eggs small, darkly
tanned, 16 (15–17) long by 8 (7–8) wide. Excretory vesicle Yshaped, bifurcates dorsal to ovary; arms extend to level of
pharynx, 471 (403–540) long.
Type host: Lutjanus bohar (ForsskDl, 1775), Perciformes,
Lutjanidae, red bass.
Type locality: Rasdhoo Atoll (4°16´N, 72°58´E), Maldives.
Site: Intestine and pyloric caeca.
Prevalence: 4 of 15 (27%) at Rasdhoo Atoll.
Deposited specimens: Holotype G227621, paratype G227622.
Etymology: The species epithet maldivensis refers to the
locality where this species was recovered.
Discussion
Caulanus gen. nov.
Caulanus gen. nov. is distinguished from all other cryptogonimid genera by the combination of an elongate body, oral
spines, caeca that open via ani at the posterior end of the body,
tandem testes, uterine loops that are extensive in the fore- and
hindbody and vitelline follicles that extend from the ovary to
the pharynx.
Terrence L. Miller and Thomas H. Cribb
This genus most closely resembles Pseudallacanthochasmus Velasquez, 1961, also from lutjanids, but differs from it
primarily in having caeca that open via ani at the posterior
end of the body and a uterus which is extensive in the foreand hindbody. Six other cryptogonimid genera (Acanthostomum Looss, 1899, Caimanicola Teixeira de Freitas et Lent,
1938, Novemtestis Yamaguti, 1942, Orientodiploproctodaeum
Bhutta et Khan, 1970, Proctocaecum Baugh, 1957 and Timoniella Rebecq, 1960) also have caeca that open via ani at the
posterior end of the body, but all are easily distinguished from
Caulanus. Species of Acanthostomum, Caimanicola, Proctocaecum and Timoniella all have entire ovaries, the uterus does
not enter the forebody and the testes are located at the posterior end of the body. Orientodiploproctodaeum is distinguished from Caulanus by lacking oral spines and in the distinct ‘collar’ surrounding the oral sucker, and Novemtestis differs from Caulanus in having multiple/follicular testes.
The preferred site of infection for C. thomasi was the rectum of Lutjanus bohar. Interestingly, Anoiktostoma coronatum (Wagener, 1852) Stossich, 1899, a parasite of the sciaenid
Sciaena umbra Linnaeus, 1758, also only inhabits the rectum
of its host and is similar to C. thomasi in having a uterus that
loops well into the forebody, oral spines and a trilobed or few
lobed ovary (although some specimens may have an entire
ovary (Bartoli and Gibson 1995)). This species can be distinguished from C. thomasi by lacking caeca that open via ani at
the posterior end of the body, a prepharynx, having opposite to
slightly oblique testes and in its restriction to Mediterranean
sciaenids.
Latuterus gen. nov.
Latuterus gen. nov. can be distinguished from all other cryptogonimid genera by the combination of a squat body, lack of
oral spines, presence of multiple/follicular testes, a uterus that
is extensive in the fore- and hindbody and vitelline follicles
that are restricted entirely to the forebody. Three cryptogonimid genera (Iheringtrema Travassos, 1947, Polyorchitrema
Srivastava, 1939 and Siphodera Linton, 1910) resemble Latuterus in lacking oral spines and in having multiple/follicular
testes, but differ fundamentally from this genus in that the
uterus is restricted entirely to the hindbody. Siphodera is the
genus most morphologically similar to Latuterus, and some of
its species also infect species of Lutjanidae. The type-species,
S. vinaledwardsii Linton, 1910 is distinguished from L. tkachi
by having a more elongate body, a distinct prepharynx and
oesophagus, and vitelline follicles and uterus that are restricted to the hindbody. Siphodera aegyptensis Hassanine et Gibson, 2005, and S. gurukun Machida, 1986, both parasites of
lutjanids, have vitelline follicles that are extensive in the forebody, but differ from L. tkachi in having elongate bodies and
a distinct prepharynx and oesophagus. Polyorchitrema, a genus restricted primarily to freshwater siluriforms (although a
species has been reported from a marine sparid), is distinguished from Latuterus by having a ventral sucker that is
approximately the same size as or larger than the oral sucker
and numerous testicular follicles (8–50) located at the poste-
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Two new cryptogonimid genera from Lutjanus bohar
rior end of the body. The monotypic Iheringtrema is restricted to freshwater siluriforms and differs from Latuterus in having an elongate body, testes located at the posterior end of the
body and the vitelline follicles extending from the posterior
end of the body to the pharynx.
Latuterus tkachi resembles L. maldivensis closely, differing morphologically by having a distinctly larger body and
genetically by 9 base positions (3 in the 28S, 4 in the ITS2 and
2 in the ITS1) over the three ribosomal DNA regions examined here. Principal components analysis conducted using all
measured morphometric variables (component axis 1, 76%,
axis 2, 8%) resulted in the unambiguous separation of L. tkachi and L. maldivensis (Fig. 5), although only six specimens
were available. Discriminant analysis between the two morphometric datasets also resulted in statistically significant separation of the two species along the discriminant axis.
This level of interspecific variation in rDNA sequences
was reported recently for species of Retrovarium (Cryptogonimidae) by Miller and Cribb (in press-a). Morphologically,
genetically and ecologically (host specific) distinct species of
Retrovarium were observed to differ by as few as 4 bp over the
entire ITS2 region, 5 bp over the 3’ half of the ITS1 and 7 bp
over the same region of the 28S rDNA examined here. Two
species, R. exiguiformosum and R. formosum, which were
each only found at one of two widely separated localities
(R. exiguiformosum only at Heron Island and R. formosum
only at Lizard Island) sampled on the Great Barrier Reef from
the same lutjanid fish, Symphorus nematophorus, differed
consistently from each other by 2 bp in all three rDNA regions
examined. Although similar, these two species differed consistently from each other morphologically in body size and
oral spine number and this combination of consistent genetic, geographic and morphological differences led to the conclusion that two distinct species were present in the system.
Also, the observed base differences between the species reported by Miller and Cribb (in press-a) and between L. tkachi
and L. maldivensis reported here agree with the consistent
genetic variation of as few as one to a handful of bases observed in the ITS that has been interpreted as evidence of distinct species in other digenean systems (Morgan and Blair
1995; Anderson and Barker 1998; Bell et al. 2001; Snyder and
Tkach 2001; Nolan and Cribb 2006a, b). For example, similar interspecific variation to that observed for cryptogonimids
has been observed in the Didymozoidae (0.5% over the ITS2
region) by Andersen and Barker (1998), the Echinostomatidae
(0.8% over the entire ITS region) by Sorensen et al. (1998),
the Haematoloechidae (0.5% over the entire ITS region) by
Snyder and Tkach (2001), the Strigeidae (0.3% over the ITS2)
by Bell et al. (2001) and the Sanguinicolidae (0.3% over the
ITS2 region) by Nolan and Cribb (2006a).
The relatively low intergeneric variation of 3.8–4.0% in the
28S, 6.3–6.7% in the ITS1 and 6.6% in the ITS2 between these
two morphologically distinct genera also suggests that a small
amount of interspecific variation represents considerable difference. In addition, the distinct morphological and genetic
homogeneity of Caulanus thomasi over the same wide geo-
111
Fig. 5. Scatterplot indicating the position of each individual specimen of Latuterus tkachi sp. nov. and L. maldivensis sp. nov. plotted
on the principal component axes. Crosses represent individuals of
L. maldivensis from Rasdhoo Atoll, Maldives and squares represent
individuals of L. tkachi from Lizard Island, Great Barrier Reef,
Australia
graphic range as L. tkachi and L. maldivensis suggests that the
integrity of species parasitising Lutjanus bohar over large geographic distances can be maintained. Consequently, we consider the consistent morphological, genetic and geographic differences to warrant the recognition of two distinct species in the
system, Latuterus tkachi and L. maldivensis, from localities
separated by over 9600 kilometres (Lizard Island and the
Maldives).
Geographic distribution
The consistent morphological and molecular data (identical
rDNA sequences from all localities) for Caulanus thomasi
suggest that this species has a wide geographic distribution of
over 9600 kilometres (direct water route). The identical rDNA
sequences reported here from widely separated localities is
consistent with that reported for two species of cryptogonimids (Miller and Cribb, in press-a) and at least 16 other species representing 9 families of trematodes which have displayed identical ITS (either the ITS1 or ITS2 independently or the
entire ITS region) sequences from wide geographic ranges
(Nolan and Cribb 2005).
Nuclear rDNA sequence data has also revealed wide distributions from a number of additional digenean families infecting marine fish in the Indo-Pacific. Identical ITS2 sequences between Lizard and Heron Islands on the GBR (separated by over 1100 kilometres) were reported by Cribb et al.
(1998) for two species of bivesiculids. Lo et al. (2001) reported identical ITS2 sequences between the GBR and French
Polynesia (separated by over 6000 kilometres) for two lepocreadiids and an apocreadiid. Recently, Chambers and Cribb
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112
Terrence L. Miller and Thomas H. Cribb
Table II. Numbers and localities of individuals of Lutjanidae collected and examined for cryptogonimids. The caesionines listed here
are considered members of the Lutjanidae rather than Caesionidae
(Miller and Cribb, in press-c)
Lutjanidae
HI
Caesioninae
Caesio caerulaurea
Caesio cuning
Pterocaesio marri
Pterocaesio pisang
Etelinae
Aprion virescens
Lutjaninae
Lutjanus adetii
Lutjanus argentimaculatus
Lutjanus bohar
Lutjanus carponotatus
Lutjanus erythropterus
Lutjanus fulviflamma
Lutjanus fulvus
Lutjanus gibbus
Lutjanus kasmira
Lutjanus malabaricus
Lutjanus monostigma
Lutjanus quinquelineatus
Lutjanus russelli
Lutjanus sebae
Lutjanus vitta
Macolor niger
Paradicichthyinae
Symphorichthys spilurus
Symphorus nematophorus
61
21
Locality
LI
MA
2
18
3
3
63
4
7
144
24
13
2
3
3
15
3
2
5
11
60
1
36
7
1
1
3
10
17
1
11
1
25
15
Host specificity
Despite many lutjanid species being collected from the same
localities as Lutjanus bohar during this survey (Table II), all
three species reported here were recovered only from L.
bohar. In particular a number of L. gibbus were sampled from
Lizard Island and the Maldives and these were not infected
with species of Caulanus or Latuterus. Lutjanus gibbus is sister taxon to L. bohar (Miller and Cribb, in press-c) and the
most likely candidate for infection due to its phylogenetic
relatedness and ecological and dietary preferences (Allen
1985), but was not infected with either of these genera.
Although a large number of individuals and species of
Lutjanidae were sampled from different Indo-West Pacific
localities, many more species remain unexamined and may be
found to harbour species of Caulanus or Latuterus.
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5
3
5
3
3
HI – Heron Island, GBR; LI – Lizard Island, GBR; MA – Rasdhoo
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