Molecular evidence that the genus Cadenatella Dollfus, 1946

(Digenea: Plagiorchiida) belongs in the superfamily

Haploporoidea Nicoll, 1914

Rodney A. Bray • Thomas H. Cribb •

Andrea Waeschenbach • D. Timothy J. Littlewood

Received: 17 April 2014 / Accepted: 9 June 2014

� Springer Science+Business Media Dordrecht 2014

Abstract A re-examination of published lsrDNA

sequence data of haploporid digeneans has shown that

the genus Cadenatella Dollfus, 1946, hitherto consid-

ered a lepocreadioid, is correctly placed within the

superfamily Haploporoidea Nicoll, 1914, although its

relationships within the superfamily are not resolved.

The morphological similarities and differences

between Cadenatella and other haploporoids are

discussed, and the subfamily Cadenatellinae Gibson

& Bray, 1982 is considered the best repository for

Cadenatella spp. at present.

Introduction

Molecular phylogenetics has produced some surpris-

ing results, placing species in situations where their

morphology does not, on first examination, seem to fit.

In the Digenea, for example, the genus Cableia

Sogandares-Bernal, 1959 was originally considered a

lepocreadiid (Sogandares-Bernal, 1959), later an op-

ecoelid (Yamaguti, 1971), then an enenterid (Gibson

& Bray, 1982) and finally an acanthocolpid (Bray

et al., 1996). Molecular studies, based on small subunit

nuclear ribosomal RNA gene (ssrDNA) (=18S rDNA)

and partial large subunit nuclear ribosomal RNA gene

(lsrDNA) (=28S rDNA) sequences, place this genus as

an early divergent monorchiid (Cribb et al., 2001;

Olson et al., 2003; Bray et al., 2005; unpublished). In

the study reported here, we present molecular evi-

dence that another puzzling genus, Cadenatella Doll-

fus, 1946, is not an enenterid, as has been considered

since its discovery, but a member of the Haploporoi-

dea Nicoll, 1914.

Materials and methods

Taxon choice

In this study, we have made use of the considerable

amount of published lsrDNA data available on Gen-

Bank (as at January 2013) to conduct a trematode-wide

survey of this gene. Included sequences were those of

[500 base pair length; duplicate sequences which

showed\1% sequence divergencewere excluded, thus

leaving us with information for discrete species only.

The final dataset consisted of 556 trematode sequences

representing 24 superfamilies and 97 families and

included two Cadenatella spp. This molecular phy-

logeny was utilised, but not published in full, by

Littlewood et al. (2015) in their survey of the biodi-

versity of helminth endoparasites. It indicated, with

strong support, that theCadenatella spp. were included

within the superfamily Haploporoidea. Here, a further

R. A. Bray (&) � A. Waeschenbach � D. T. J. Littlewood

Department of Life Sciences, Natural History Museum,

Cromwell Road, London SW7 5BD, UK

e-mail: rab@nhm.ac.uk

T. H. Cribb

School of Biological Sciences, The University of

Queensland, Brisbane, QLD 4072, Australia

123

Syst Parasitol (2014) 89:15–21

DOI 10.1007/s11230-014-9504-5

Table 1 Provenance data on the sequences used

Superfamily Family Parasite species Host Locality GenBank No. Reference

Opisthorchioidea Heterophyidae Cryptocotyle lingua (Creplin,

1825)

Littorina littorea Off Germany AY222228 Olson et al. (2003)

Lepocreadioidea Enenteridae Enenterum aureum Linton, 1910 Kyphosus vaigiensis Off Moorea AY222232 Olson et al. (2003)

Haploporoidea Atractotrematidae Atractotrema sigani Durio &

Manter, 1969

Siganus lineatus Off Lizard Island AY222267 Olson et al. (2003)

Haploporoidea Atractotrematidae Pseudomegasolena ishigakiensis

Machida & Kamiya, 1976

Scarus rivulatus Off Heron Island AY222266 Olson et al. (2003)

Haploporoidea Haploporidae Hapladena nasonis Yamaguti,

1970

Naso unicornis Off Lizard Island AY222265 Olson et al. (2003)

Haploporoidea [Cadenatellinae] Cadenatella pacifica (Yamaguti,

1970)

Kyphosus vaigiensis Off Heron Island FJ788498 Bray et al. (2009)

Haploporoidea [Cadenatellinae] Cadenatella isuzumi Machida,

1993

Kyphosus vaigiensis Off Heron Island FJ788497 Bray et al. (2009)

Haploporoidea Haploporidae Saccocoelioides sp. Poecilidae indet. Nicaragua EF032696 Curran et al. (2006)

Haploporoidea Haploporidae Intromugil mugilicolus (Shireman,

1964)

Mugil cephalus Mississippi KC430096 Pulis et al. (2013)

Haploporoidea Haploporidae Intromugil alachuaensis Pulis,

Fayton, Curran & Overstreet,

2013

Mugil cephalus Florida KC430095 Pulis et al. (2013)

Haploporoidea Haploporidae Forticulcita gibsoni Blasco-Costa,

Montero, Balbuena, Raga &

Kostadinova, 2009

Mugil cephalus Off Spain FJ211239 Blasco-Costa et al. (2009)

Haploporoidea Haploporidae Spiritestis herveyensis Pulis &

Overstreet, 2013

Moolgarda seheli Off Queensland KC206500 Pulis & Overstreet (2013)

Haploporoidea Haploporidae Capitimitta sp. Selenotoca multifasciata Off Queensland KC206499 Pulis & Overstreet (2013)

Haploporoidea Haploporidae Capitimitta darwinensis Pulis &

Overstreet, 2013

Selenotoca multifasciata Off Darwin KC206498 Pulis & Overstreet (2013)

Haploporoidea Haploporidae Capitimitta costata Pulis &

Overstreet, 2013

Selenotoca multifasciata Off Queensland KC206497 Pulis & Overstreet (2013)

Haploporoidea Haploporidae Saccocoelium brayi Blasco-Costa,

Balbuena, Raga, Kostadinova &

Olson, 2010

Liza saliens Off Spain FJ211234 Blasco-Costa et al. (2009)

Haploporoidea Haploporidae Saccocoelium tensum Looss, 1902 Liza aurata Off Spain FJ211258 Blasco-Costa et al. (2009)

Haploporoidea Haploporidae Saccocoelium cephali Blasco-

Costa, Montero, Gibson,

Balbuena, Raga & Kostadinova,

2009

Mugil cephalus Off Spain FJ211233 Blasco-Costa et al. (2009)

16

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Parasito

l(2014)89:15–21

123

study of the status of Cadenatella was undertaken

utilising available haploporoid lsrDNA sequences

(Table 1). The ingroup is composed of 19 haploporoids

and two representatives of Cadenatella, the latter

having been shown to group separately from other

enenterids in a previous molecular phylogeny (Bray

et al., 2009). The outgroup choice was informed by the

above-mentioned phylogenetic analyses encompass-

ing the whole of the Digenea (unpublished) and is

composed of Cryptocotyle lingua (Creplin, 1825)

(Opisthorchioidea Looss, 1899:Heterophyidae Leiper,

1909) and Enenterum aureum Linton, 1910 (Lepo-

creadioidea Odhner, 1905: Enenteridae Yamaguti,

1958). The sister group to the Haploporoidea in the

trematode-wide tree was found, with very low support,

to be the Opisthorchioidea and the sister to this joint

‘clade’ was found to be, with similar levels of support,

the Lepocreadioidea. GenBank accession numbers are

given in Fig. 1 and Table 1.

Phylogenetic analysis

Sequences were aligned using MAFFT version 6.611b

(Katoh et al., 2005) with 1,000 cycles of iterative

refinement and the genafpair algorithm. An alignment

mask, excluding sites of uncertain positional homol-

ogy, was generated using ZORRO (Wu et al., 2012).

ZORRO uses a pair Hidden Markov Model and a

weighted sum of pairs scheme (guided by a guide tree)

that sums up the probability that a given alignment

column appears over the total alignment landscape,

thus providing an objective estimate whether positions

consist of correctly aligned, homologous residues.

Default settings were used, except for the invocation

of the sample option, which calculates the alignment

columns posterior probabilities based on a random

sampling of pairs of sequences, rather than exhaus-

tively sampling each pair, thus speeding up the

running time; positions with confidence scores\0.4

were excluded from subsequent analyses. Modeltest

version 3.7macX (Posada & Crandall, 1998) was used

to select models of evolution using the Akaike

Information Criterion. Phylogenetic trees were con-

structed using Bayesian inference (BI) with MrBayes,

version 3.1.2 (Huelsenbeck & Ronquist, 2001). Like-

lihood settings were set to nst = 6, rates = in-

vgamma, equivalent to the GTR?I?G model of

sequence evolution. Four chains (temp = 0.2) were

run for 6,000,000 generations and sampled every

1,000 generations; 2,500,000 generations wereTable

1continued

Superfamily

Fam

ily

Parasitespecies

Host

Locality

GenBankNo.

Reference

Haploporoidea

Haploporidae

LecithobotrysputrescensLooss,

1902

Lizasaliens

OffSpain

FJ211236

Blasco-Costaet

al.(2009)

Haploporoidea

Haploporidae

Haploporusbenedenii(Stossich,

1887)

Lizaramada

OffSpain

FJ211237

Blasco-Costaet

al.(2009)

Haploporoidea

Haploporidae

Ragaia

lizaeBlasco-Costa,

Montero,Gibson,Balbuena&

Kostadinova,

2009

Lizaaurata

OffSpain

FJ211235

Blasco-Costaet

al.( 2009)

Haploporoidea

Haploporidae

Dicrogastercontracta

Looss,1902

Lizaaurata

OffSpain

FJ211261

Blasco-Costaet

al.( 2009)

Haploporoidea

Haploporidae

DicrogasterperpusillaLooss,

1902

Lizaramada

OffSpain

FJ211238

Blasco-Costaet

al.(2009)

Syst Parasitol (2014) 89:15–21 17

123

discarded as burn-in, at which point the average

standard deviation of split frequencies were\0.01.

Results

The lsrDNA tree (Fig. 1) shows the superfamily

Haploporoidea Nicoll, 1914, including the families

Atractotrematidae Yamaguti, 1939 and Haploporidae

Nicoll, 1914 and the Cadenatella spp. as a well-

supported monophyletic group; the topology is iden-

tical to that obtained in the lsrDNA analysis by

Littlewood et al. (2015), apart from the addition of the

recently published sequences of the haploporids

Intromugil mugilicolus (Shireman, 1964), I. alachua-

ensis Pulis, Fayton, Curran & Overstreet, 2013,

Spiritestis herveyensis Pulis & Overstreet, 2013, an

unnamed species of Capitimitta Pulis & Overstreet,

2013, C. darwinensis Pulis & Overstreet, 2013 and

C. costata Pulis & Overstreet, 2013, which had not

been included by Littlewood et al. (2015). The

Atractotrematidae, represented here by just two spe-

cies, is also well supported as monophyletic. The two

species of Cadenatella are monophyletic, forming a

group for which the subfamily name Cadenatellinae

Gibson & Bray, 1982 is used here, it being in our

opinion premature to decide on the hierarchical status

of this clade. These results strongly indicate that

Cadenatella is not an enenterid, or indeed a lepoc-

readioid, as previously thought.

The Haploporidae is less well supported in that

Hapladena Linton, 1910 is not clustered with the

remainder of the Haploporidae. Within the Haplopo-

ridae, there are two well-supported groups: a sister

Fig. 1 Bayesian inference analysis of published haploporoid lsrDNA sequences, constructed using MrBayes under the GTR?I?G

model. Analyses were run for 6,000,000 generations with 2,500,000 generations discarded as burn-in. The branch-length scale-bar

indicates the number of substitutions per site

18 Syst Parasitol (2014) 89:15–21

123

group relationship between Saccocoelioides Szidat,

1954 and IntromugilOverstreet & Curran, 2005, and a

clade in which species of Saccocoelium Looss, 1902

form the sister group to an unresolved assemblage of

(Lecithobotrys Looss, 1902, Haploporus Looss,

1902), Ragaia lizae Blasco-Costa, Montero, Gibson,

Balbuena & Kostadinova, 2009 and species of

Dicrogaster Looss, 1902. However, the interrelation-

ships between those two lineages, Forticulcita gibsoni

Blasco-Costa, Montero, Balbuena, Raga & Kostadi-

nova, 2009, Spiritestis herveyensis and Capitimitta

spp. are unresolved.

Discussion

The Haploporoidea

The monophyly of the Haploporoidea, including

Cadenatella, is well supported, thus supplying strong

evidence of the superfamilial position of Cadenatella.

The haploporid subfamily Haploporinae is well sup-

ported (including the genera Saccocoelium, Lecitho-

botrys, Haploporus, Ragaia Blasco-Costa, Montero,

Gibson, Balbuena & Kostadinova, 2009 and Dicrog-

aster), but the subfamily status of other haploporids is

less clear. Blasco-Costa et al. (2009) erected the

subfamily Forticulcitinae Blasco-Costa, Balbuena,

Kostadinova & Olson, 2009 for Forticulcita Over-

street, 1982 and retained Saccocoelioides within the

Chalcinotrematinae Overstreet & Curran 2005 fol-

lowing Overstreet & Curran (2005). Pulis & Over-

street (2013) produced sequences of Spiritestis

Nagaty, 1948 and Capitimitta, which we have

included in our analysis, but their relationships within

the family are not resolved. The sequences of Introm-

ugil spp. produced by Pulis et al. (2013) cluster with

Saccocoelioides sp. adding weight to their statement

that Intromugil ‘‘will be more closely allied with the

chalcinotrematines’’ although no molecular data are

yet available for Chalcinotrema Freitas, 1947.

Cadenatella

Dollfus (1946) originally erected Cadenatella as a

subgenus of Enenterum Linton, 1910 and in the same

paper erected another subgenus, Jeancadenatia Doll-

fus, 1946. Manter (1947) considered that both sub-

genera probably warranted generic rank and Nagaty

(1948) used both names at the generic level. Nahhas &

Cable (1964) synonymised Jeancadenatia with Ca-

denatella, a situation followed by many subsequent

workers. Recent revisions have all considered Caden-

atella an enenterid because of the general morpho-

logical similarity and especially, superficially, of the

oral sucker lobation and the restriction of Cadenatella

to hosts of the genus Kyphosus which is the most

important host taxon for enenterids (Brooks et al.,

2000; Bray & Cribb, 2001, 2002). Molecular evidence

in Bray et al. (2009) showed that Cadenatella was not

a lepocreadioid, and, therefore, not an enenterid. The

sample size used in their analysis did not produce

convincing evidence of the status of Cadenatella.

Jones (2005) and Overstreet & Curran (2005)

considered that members of the Haploporoidea are

characterised by the presence of a sac enclosing the

terminal genitalia, considered a ‘hermaphroditic sac’.

Cadenatella lacks a hermaphroditic sac, but the exact

condition of the terminal genitalia is variably

described. Dollfus (1946) described the male terminal

genitalia of the type-species C. cadenatiDollfus, 1946

as ‘‘La poche de cirre occupe l’espace entre la

bifurcation intestinale et bord anterieur de l’acetabu-

lum, il y a une vesicule seminale interne et des cellules

prostatiques; il ne semble exist de vesicule seminale

externe.’’ The other species described by Dollfus

(1946), C. brumpti (Dollfus, 1946), originally placed

in the subgenus Jeancadenatia, has ‘‘La poche de

cirre, extremement petit et mal distincte, est situee au

contact du bord anterieur gauche de l’acetabulum’’.

Manter (1949) described the terminal genitalia of

C. americana Manter, 1949 as ‘‘Cirrus sac thin-

walled, small and very inconspicuous’’ and described

an external seminal vesicle. Nahhas & Cable (1964)

stated that ‘‘reexamination of the holotype reveals that

the pars prostatica probably was misinterpreted as a

cirrus sac’’. Overstreet (1969) examined further spec-

imens and stated that a ‘‘sectioned specimen does not

have a cirrus sac, confirming what Nahhas and Cable

(1964: 192) believed. A thin membrane, however,

appears to surround the vesicle in some whole-

mounts’’. Winter (1957) described a large cirrus-sac

in C. dohenyi (Winter, 1957), and presented a micro-

photograph which shows a large, sperm-filled sac (?

seminal vesicle) in the posterior forebody. Yamaguti

(1970) stated that C. pacifica (Yamaguti, 1970) had

‘‘no cirrus-sac’’ a situation confirmed by Machida

(1993) and Bray & Cribb (2001). According to

Syst Parasitol (2014) 89:15–21 19

123

Hafeezullah (1980), C. dollfusi (Hafeezullah, 1980)

has a ‘‘short cirrus sac … enclosing short tubular

internal seminal vesicle’’. Machida (1993) apparently

found the cirrus-sac absent in C. isuzumi Machida,

1993 and Bray & Cribb (2001) confirmed this finding.

Brooks et al. (2000) re-examined specimens of

C. americana, C. dohenyi, C. kyphosi Nahhas &

Cable, 1964, C. floridae Overstreet, 1969, C. isuzumi

and C. pacifica and found the cirrus-sac missing in all

but C. dohenyi, which they considered along with

C. brumpti and C. dollfusi, to have a cirrus-sac

‘‘partially enclosing the seminal vesicle’’. In no case

was a hermaphroditic sac described. Gibson & Bray

(1982) erected the enenterid subfamily Cadenatelli-

nae, to contain Cadenatella (and its now recognised

synonym Jeancadenatia Dollfus, 1946), based, inter

alia, on the lack of a cirrus-sac.

Nahhas & Cable (1964) commented ‘‘it may be

noted that the genera Enenterum, Cadenatella, and

Jeancadenatia which generally are placed in the

Lepocreadiidae, have many features in common with

those of families included in the Haploporoidea and

ultimately may be transferred to that superfamily’’.

The major characteristic features of Cadenatella are

the elaborately lobed oral sucker, the uroproct and the

single testis. Comparison with the superfamily diag-

nosis of the Haploporoidea in Jones (2005) reinforces

the view that most features of Cadenatella are shared

with members of this superfamily. We know that

complex lobation of the oral sucker occurs in the

Haploporoidea, e.g. in Waretrema Srivastava, 1937,

Spiritestis Nagaty, 1948, Capitimitta Pulis & Over-

street, 2013. Pulis & Overstreet (2013) illustrated

examples from these genera, both with microphoto-

graphs and line drawings. An uroproct is shared with

the genusMyoderaMontgomery, 1957 (see Overstreet

& Curran, 2005), whose species are also reported in

kyphosids (Montgomery, 1957; Sogandares-Bernal,

1959). A single testis is also the usual condition in the

Haploporidae. In fact, Overstreet & Curran (2005)

considered it possible that the three genera whose

members exhibit two testes, Vitellibaculum Mont-

gomery, 1957, Metamegasolena Yamaguti, 1970 and

Megasolena Linton, 1910, ‘‘do not even belong in the

family Haploporidae’’. All of the haploporoid species

used in the current study have a single testis.

The evidence from lsrDNA is, therefore, strong that

Cadenatella has been misplaced by previous workers.

The topology of the tree (Fig. 1) suggests that the

terminal genitalia of Cadenatella are derived from the

‘hermaphroditic sac’ by loss of the wall. The lobation

of the oral sucker is not homologous with that of

species of Enenterum and, in detail, the lobation

appears rather distinct (see Bray & Cribb, 2001,

figures 2, 7 vs 14, 15, 18, 19). The current placement

of Cadenatella in the haploporoid tree is poorly

resolved. Convincing relationships within the Hap-

loporoidea await the molecular study of many more

haploporoids, but it is probable that the subfamily

Cadenatellinae will be recognised at the family level.

In retrospect, the confusion relating to the position of

Cadenatella can be seen as having arisen from a case

of convergent evolution driven, apparently, by the

nature of the habitat provided by the kyphosid gut. We

note too that identity as a haploporoid, which implies a

likely two-host life cycle in which the cercariae encyst

in the open (Bartoli & Gibson, 2007), is entirely

consistent with the herbivorous diet of species of

Kyphosus.

Acknowledgements We would like to thank Peter Foster

(NHM) for assistance in compiling the original data set drawn

from GenBank.

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