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Morpho-molecular traits of Indo-Pacific and Caribbean
Halofolliculina ciliate infections
Simone Montano
1,2
•Davide Maggioni
1,2
•Giulia Liguori
1,2
•Roberto Arrigoni
3,4,5
•
Michael L. Berumen
5
•Davide Seveso
1,2
•Paolo Galli
1,2
•Bert W. Hoeksema
6,7
Received: 1 July 2019 / Accepted: 27 January 2020
ÓSpringer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract Coral diseases are emerging as a major threat to
coral reefs worldwide, and although many of them have
been described, knowledge on their epizootiology is still
limited. This is the case of the Halofolliculina ciliate
infections, recognized as the skeletal eroding band (SEB)
and Caribbean ciliate infection (CCI), two diseases caused
by ciliates belonging to the genus Halofolliculina (Class
Heterotrichea). Despite their similar macroscopic appear-
ance, the two diseases are considered different and their
pathogens have been hypothesized to belong to different
Halofolliculina species. In this work, we analysed the
morphology and genetic diversity of Halofolliculina cili-
ates collected in the Caribbean Sea, Red Sea and Indo-
Pacific Ocean. Our analyses showed a strong macroscopic
similarity of the lesions and similar settlement patterns of
the halofolliculinids from the collection localities. In par-
ticular, the unique erosion patterns typical of the SEB were
observed also in the Caribbean corals. Fine-scale mor-
phological and morphometric examinations revealed a
common phenotype in all analysed ciliates, unequivocally
identified as Halofolliculina corallasia. Phylogenetic
analyses based on nuclear and mitochondrial (COI)
molecular markers consistently found all samples as
monophyletic. However, although the nuclear marker dis-
played an extremely low intra-specific diversity, consistent
with the morphological recognition of a single species, the
analyses based on COI showed a certain level of diver-
gence between samples from different localities. Genetic
distances between localities fall within the intra-specific
range found in other heterotrich ciliates, but they may also
suggest the presence of a H. corallasia species complex. In
conclusion, the presented morpho-molecular characteriza-
tion of Halofolliculina reveals strong similarities between
the pathogens causing SEB and CCI and call for further
detailed studies about the distinction of these two coral
diseases.
Topic Editor Carly Kenkel
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s00338-020-01899-6) contains sup-
plementary material, which is available to authorized users.
&Simone Montano
simone.montano@unimib.it
1
Department of Earth and Environmental Sciences (DISAT),
University of Milan – Bicocca, Piazza della Scienza,
20126 Milan, Italy
2
MaRHE Center (Marine Research and High Education
Center), Magoodhoo Island, Faafu Atoll, Republic of
Maldives
3
Department of Biology and Evolution of Marine Organisms
(BEOM), Stazione Zoologica Anton Dohrn Napoli, Villa
Comunale, 80121 Naples, Italy
4
European Commission, Joint Research Centre (JRC), Ispra,
Italy
5
Division of Biological and Environmental Science and
Engineering, Red Sea Research Center, King Abdullah
University of Science and Technology, Thuwal 23955-6900,
Saudi Arabia
6
Taxonomy and Systematics Group, Naturalis Biodiversity
Center, P.O. Box 9517, 2300 RA Leiden, The Netherlands
7
Groningen Institute for Evolutionary Life Sciences,
University of Groningen, P.O. Box 11103,
9700 CC Groningen, The Netherlands
123
Coral Reefs
https://doi.org/10.1007/s00338-020-01899-6
Keywords Ciliate Protozoan Syndrome
Halofolliculina corallasia Coral reefs Scleractinian
corals
Introduction
Coral reefs are declining worldwide, with an estimated
coral cover loss of 50% in the Indo-Pacific and 80% in the
Caribbean over the last 30 years (Gardener et al. 2003;
Bruno and Selig 2007; Pollock et al. 2011). The causes of
this decline are multiple and complex, with coral diseases
emerging as one of the most affecting threats (Rosenberg
et al. 2007; Bourne et al. 2009; Sutherland et al. 2015).
Currently, it is not clear how many coral diseases exist
globally, and a certain level of confusion emerges from the
incomplete information reported in the literature (Willis
et al. 2004). Indeed, despite their negative impact, the
majority of coral diseases remain a mystery, as a result of
the limited analytic methods, the poor knowledge of the
putative pathogens and the consequent deficiency of epi-
zootiological data (Work and Meteyer 2014; Bourne et al.
2015). One of the most dramatic and recent examples is the
Stony Coral Tissue Loss Disease (SCTLD), which has been
affecting various coral species along the Florida Reef Tract
since 2014 (Aeby et al. 2019). Although its effect is
unprecedented, all the efforts carried out by research have
so far not been successful in discovering its causative
agents (Meyer et al. 2019). The lack of specific and
accurate diagnostic tools coupled with difficulties
encountered in the study of bacteria-based diseases resulted
also in ambiguous classifications of coral diseases that
remain largely open to interpretation (Pollock et al. 2011).
The shortage of multidisciplinary approaches to describe
coral lesions and to identify morphologically and molecu-
larly the pathogens have led to recognize a high number of
possibly different diseases, which often share similar gross
morphology of the lesions, as for the Indo-Pacific White
Syndromes (Bourne et al. 2015). Furthermore, in some
cases, diseases were named differently, even if caused by
the same putative pathogens. This is the case of the Black
Band Disease (BBD) and the Red Band Disease (RBD) of
Palau, in which the filamentous cyanobacteria forming red
and black bands were molecularly identified as belonging
to a single ribotype only after their isolation, culturing and
sequencing (Sussman et al. 2006). Moreover, this scenario
is further complicated by the existence of coral diseases
caused by a consortium of pathogens that may slightly
differ among geographic areas. Therefore, the BBD, rec-
ognized as a worldwide distributed disease caused by a
consortium of microorganism dominated by a cyanobac-
terial component, may differ in its predominant portion
between the Caribbean and Indo-Pacific (Casamatta et al.
2012).
The skeletal eroding band (SEB) is one of the first coral
diseases detected and described from the Indo-Pacific coral
reefs (Antonius 1999). It is caused by the folliculinid ciliate
Halofolliculina corallasia Antonius and Lipscomb (2001)
(Class Heterotrichea; Order Heterotrichida), which not
only attacks the soft tissues of corals but also damages their
skeleton (Antonius and Lipscomb 2001; Riegl and Anto-
nius 2003; Page and Willis 2008). In H. corallasia, the
lorica (sac-like housing) has a rounded posterior and a
cylindrical neck that angles up from the surface at about
45°, and it has an average length and width of 220 lm and
95 lm, respectively. They are settled on the coral skeleton,
usually following the rim of the corallites, and in most
cases, only the neck rises above the coral surface. The
disease manifests as a dark-grey band 1–10 cm thick,
located at the interface between recently exposed skeleton
and apparently healthy coral tissue. The SEB has been
recorded affecting 82 scleractinian species on various Indo-
Pacific and Red Sea coral reefs, with the most affected taxa
being branching species of Acropora and Pocillopora
(Page et al. 2015). According to these data, the SEB shows
the widest host range of any coral disease recorded to date,
reaching the top on the list of harmful coral syndromes
(reviewed by Page and Willis 2008).
In 2004–2005, a similar ciliate infection was reported
from 25 out of about 60 Caribbean coral species (Cro
´quer
et al. 2006a; Page et al. 2015), in which the infection
appeared as a dark band located between healthy tissue and
bare skeleton, showing, on closer inspection, the charac-
teristic spotted appearance of the clustering ciliates (Cro
´-
quer et al. 2006a). The general morphology of the
Caribbean ciliate is very similar to that of Halofolliculina
corallasia from the Indo-Pacific (Cro
´quer et al. 2006b;
Rodrı
´guez et al. 2009). Both ciliates have a free-living
phase that moves towards living tissues, penetrates them
and attaches itself, and a sessile form settled in a lorica,
with the cell body attached at its pointed posterior end,
showing two conspicuous pericytostomial wings bearing
feeding cilia (Antonius 1999; Antonius and Lipscomb
2001; Cro
´quer et al. 2006b).
Despite a similar fine-scale morphology among Halo-
folliculina ciliates affecting Indo-Pacific and Caribbean
corals, the skeletal erosion is often associated with SEB,
but not with the Caribbean ciliate infections to date (Page
et al. 2015). However, no information is present in the
literature about the apparent no-eroding pattern of ciliate
affecting Caribbean corals, leaving space for additional in-
depth studies.
Initially, it was proposed that these ciliates might have
recently invaded the Caribbean from the Indo-Pacific
region (Cro
´quer et al. 2006b), but then the authors stated
Coral Reefs
123
that the Caribbean and Indo-Pacific ciliates are different
species, based on unpublished data (Cro
´quer et al. 2006a).
Despite the Caribbean pathogen is still to be formally
characterized and described at species level, researchers
suggested the name Caribbean ciliate infections (CCI) to
indicate the presumed new disease, due to the apparent
differences in aetiology (Weil and Hooten 2008; Rodrı
´guez
et al. 2009; Weil and Rogers 2011). By contrast, it has also
been reported by Sweet and Se
´re
´(2015) that SEB and CCI
are caused by the same pathogen, despite an absence of
evidence to support this conclusion.
Therefore, the goal of this study is to improve the
knowledge concerning the Halofolliculina ciliate infections
(sensu Page et al. 2015) by investigating the aetiology of
SEB and CCI through a morpho-molecular approach, in
order to assess and confirm possible taxonomic affinities
between the Halofolliculina species.
Materials and methods
Sampling was conducted between June 2017 and October
2019 in three geographic areas, including the Indian Ocean
(Republic of the Maldives), the Red Sea (Saudi Arabia) and
the Caribbean Sea (Curac¸ao and Bonaire) (Fig. 1).
The presence of the Halofolliculina ciliate infection was
qualitatively recorded both by snorkelling and SCUBA
diving through a roving technique (Hoeksema and Koh
2009). A dive of approximatively one hour was carried out
at each sampling locality, starting from a maximum depth
of 10–25 m and moving towards shallower waters. In each
locality, two to four small diseased coral fragments were
taken with a hammer and chisel from colonies showing the
characteristic band. Underwater photographs were taken
using a Canon GX7 Mark II camera in a Fantasea GX7 II
underwater housing. Diseased coral colonies used in the
study were chosen randomly (depending on their abun-
dance) and include Pocillopora spp. and Porites lutea in
the Indo-Pacific and the Red Sea, and Eusmilia fastigiata
and Diploria labyrinthiformis in the Caribbean Sea. After a
preliminary observation, samples were fixed in formalin
6% and ethanol 99%, for further morphological and
molecular analyses, respectively.
Halofolliculinid protozoans were initially observed
in vivo with a Leica EZ4 D stereomicroscope to examine
the protozoan aggregations and to search for possible
macroscopic differences, such as a different colouration
and the shape of the lorica. Then, single individuals were
detached from the coral skeleton using needles, precision
forceps and micropipettes and placed on a glass slide to
observe their morphology at higher magnification under a
Zeiss Axioskop 40 microscope. Subsequently, ten coral
fragments belonging to the four genera (Diploria,Eusmilia,
Pocillopora,Porites) were observed using both the Tescan
Vega TS 5136 XM scanning electron microscope, operat-
ing at beam energies of 20 kV, and the Zeiss Gemini
SEM500 scanning electron microscope operating at beam
energies of 5 kV. About 30 loricae were randomly chosen
for each coral fragment to take measurements of the
diameter, length and width of both the neck and the
ampulla, according to Antonius and Lipscomb (2001) and
Primc-Habdija and Matonickin (2005) (Fig. S1). Mea-
surements were recorded using the Scanning Electron
Microscope measuring software SmartSEM (ZEISS,
Oberkochen, Germany) with maximum resolution of 1 nm.
Additionally, a preliminary characterization of the skeletal
erosion caused by the CCI has also been carried out
through SEM imaging.
The morphometric measurements were tested for nor-
mality distribution with a Shapiro–Wilk test of normality.
One-way analyses of variance (ANOVA) were performed
to test for differences in the neck diameter between
localities, whereas differences in the length of the neck and
diameter of the neck brim were tested using a nonpara-
metric Kruskal–Wallis test, since the data were not nor-
mally distributed (Zar 1999). Ampullae length and width of
Halofolliculina loricae were analysed by descriptive
statistics due the few observations obtained. Statistical
analyses were performed using SPSS ver. 24 (IBM, New
York). All data are presented as mean ±standard error
(SE), unless otherwise stated.
Molecular analyses
The genomic DNA was extracted following a protocol
already successfully used for different taxa (Montano et al.
2015; Beli et al. 2018). Two molecular markers were
amplified, namely a portion of the nuclear ITS (*400 bp)
and the mitochondrial COI gene (*600 bp), following the
protocols described in Fernandes et al. (2016) and Stru
¨der-
Kypke and Lynn (2010), respectively. These two DNA
regions where chosen because they are generally consid-
ered reliable markers to infer ciliate phylogeny (e.g. Sun
et al. 2010; Yi and Song 2011; Fernandes et al. 2016) and
because they show different substitution rates, with the
COI being the more variable marker and having been
already used to assess ciliates intra-specific variability (e.g.
Gentekaki and Lynn 2009; Stru
¨der-Kypke and Lynn 2010).
The amplicons were purified and sequenced in both for-
ward and reverse directions using a DNA Analyser 3730xl
(Applied Biosystems, California, USA). The obtained
sequences were imported, assembled, and visually checked
into Geneious R7. For each molecular marker, a dataset
was assembled adding sequences belonging to halofolli-
culinid relatives downloaded from GenBank. Sequences
from two karyorelictid species (Corlissina maricaensis,
Coral Reefs
123
Loxodes vorax) were also included as outgroups. Sequen-
ces were aligned using the E-INS-i option in MAFFT 7.402
(Katoh and Standley 2013), and were then run through
Gblocks (Castresana 2000; Talavera and Castresana 2007)
to remove low quality and ambiguously aligned positions.
Phylogenetic analyses were performed using Bayesian
Inference (BI) and Maximum Likelihood (ML). Jmod-
elTest2 2.1.6 (Darriba et al. 2012) was run to find the
proper molecular models, and the best-fitting model
selected for both datasets was GTR?G, as suggested by the
Aikaike Information Criterion (AIC). BI analyses were
performed using MrBayes 3.2.6 (Ronquist et al. 2012): four
parallel Markov Chain Monte Carlo runs (MCMC) were
run for 10
7
generations, trees were sampled every 1000th
generation, and burn-in was set to 25%. ML analyses were
performed with RAxML v8.2.10 (Stamatakis 2006,2014)
using 1000 bootstrap replicates. Resulting trees were
displayed and edited using FigTree 1.4.0 (Rambaut 2012)
and CorelDraw X7 (Corel Corporation, Ottawa, Canada).
Genetic distances (uncorrected p-distances, 1000 boot-
strap) among and within heterotrich lineages were obtained
for both molecular markers using MEGA-X (Kumar et al.
2018). The obtained sequences were deposited in GenBank
(accession numbers ITS: MN829871-MN829873; COI:
MN905752-MN905758) with relative sample codes and
sampling sites.
Results
Morphological results
Our ecological surveys reveal the presence of Halofolli-
culina ciliate infections diseases in all three investigated
Fig. 1 Sampling localities in the Caribbean Sea (Curac¸ao and Bonaire), Red Sea (Saudi Arabia), and Indo-Pacific (Republic of the Maldives)
Coral Reefs
123
areas. First examinations revealed clusters of protozoans
placed on coral surface between recently exposed skeleton
and apparently healthy coral tissues, forming dense bands
characterized by a dark green, almost black, colour pattern
in diseased corals from both the Caribbean and the Indo-
Pacific (Fig. 2a, b). In all samples, protozoans matched the
description of Halofolliculina corallasia provided by
Antonius and Lipscomb (2001). The body was covered by
rows of cilia and showed the two characteristic bifurcate
pericystomial wings bearing the oral polykinetids (Fig. 2c,
d).
To better visualize the morphology of the loricae and to
confirm their identification, a total number of 109 loricae
(24 on Porites lutea,29onEusmilia fastigiata,10onDi-
ploria labyrinthiformis,35onPocillopora damicornis, and
11 on Pocillopora verrucosa) were examined using the
SEM. All the loricae had a rounded posterior and a
cylindrical neck with a single sculpture line circumscribing
it (Fig. 3). Furthermore, no significative differences in the
overall distribution, settlement patterns and general size of
the loricae have been observed between the coral genera
and localities investigated. In general, the mean ampulla
length (l) and width (w) were higher in the Caribbean
samples (l= 146.4 ±4.3 lm; w= 83.3 ±6.2 lm) com-
pared to the Maldivian specimens (l= 112.7 ±4.1 lm;
w= 62.1 ±4.7 lm) and the Red Sea specimens
(l= 54.3 lm; w= 51.1 lm). Regarding the length of the
neck, mean values of 40.1 ±1.0 lm and 40.9 ±3.1 lm
were observed in the Maldivian and Red Sea specimens,
respectively, whereas a mean value of 32.2 ±1.1 lm was
observed for the Caribbean ciliates. Furthermore, similar
values were found for the neck diameter, with Maldivian,
Red Sea and Caribbean ciliates showing mean values of
31.25 ±0.9 lm, 32.28 ±0.9 lm and 31.20 ±1.0 lm,
respectively (Fig. 4).
According to the parametric and nonparametric tests
performed, the neck diameters and neck lengths were not
statistically different between the three geographic areas
(neck diameters: ANOVA F
2,90
= 0.406, p= 0.668; neck
length: Kruskal–Wallis H= 6.06 p= 0.051). In contrast, a
significant difference was observed for the neck brim
diameter between the three geographic areas, with the
Maldivian specimens showing a mean value of
42.2 ±1.4 lm, the Red Sea of 45.5 ±1.0 lm and the
Caribbean of 46.9 ±1.3 lm (Kruskal–Wallis H= 8.38
p= 0.015). Maximum and minimum morphometric data of
the loricae from each of the geographic areas are summa-
rized in Table S1.
In addition, peculiar micro-alterations have been iden-
tified on the surface of the skeleton of the Caribbean
scleractinian genera at locations where Halofolliculina
ciliates were present (Fig. 5a). The ciliates appear to
modify the growth pattern of the host coral, apparently
eroding part of the skeleton and producing a round-shaped
trace where the ciliates were located (Fig. 5b, c). This
results in distinct footprints or marks on the coral surface
attributable to the protozoans settlement. The shape of the
footprint appears cylindrical, although generally not
Fig. 2 Halofolliculina Ciliate
Infection. aCCI affecting
Diploria labyrithiformis;bSEB
affecting a coral of Acropora
muricata;cclose-up of
halofolliculinids on a septum of
Eusmilia fastigiata;dclose-up
of halofolliculinids on a colony
of Acropora muricata.LC live
coral; DC dead coral; the
arrowheads indicate the cluster-
like band of protozoans
Coral Reefs
123
regular, with a diameter ranging from about 50 lmin
width to 95 lm in length (Fig. 5d). In some coral colonies,
a less evident eroded pattern, ascribable to the presence of
the loricae on the skeleton, was also observed (Fig. 5e, f).
Molecular results
The total alignments of the ITS and COI datasets after the
Gblocks treatment were 455 and 688 bp long, and con-
sisted in 36 and 40 sequences, respectively. BI and ML
analyses resulted in almost identical phylogenetic trees,
and therefore only the ML topologies are shown in Fig. 4,
with nodal supports indicated as Bayesian posterior prob-
abilities (BPP) and bootstrap supports (BS). The ITS tree
(Fig. 6a) shows an overall moderate to good nodal support
for the Bayesian analysis, whereas the ML analysis resulted
in less supported relationships. All included genera are
monophyletic, but a few species are not. The Halofolli-
culina sequences obtained from different localities form a
monophyletic clade (BPP = 0.88, BS = 89), sister to Fol-
liculina simplex (BPP = 0.99, BS = 84), the only other
folliculinid included in the analysis. The evolutionary
Fig. 3 Various features of
Halofolliculina corallasia and
the peculiar single sculpture
(S) of the lorica (Lor) in the
three investigated areas: a,
bIndo-Pacific, c,dRed Sea, e,
fCaribbean. Scale bars: 20 lm
Coral Reefs
123
relationships represented in the COI tree (Fig. 6b) show
higher support values for both BI and ML analyses at genus
and species level, but deeper nodes are generally less
supported. However, all genera and species included in the
analysis are monophyletic, including the Halofolliculina
clade (BPP = 1, BS = 100). In comparison with the ITS
analyses, the halofolliculinids collected from different
localities show a higher diversification, and three geogra-
phy-related, fully supported lineages can be identified.
Genetic distances calculated between heterotrich species
and genera are generally high, especially for the COI
dataset (Table S2). The average distances between genera
and species for the COI dataset are 39.9% (28.5–58.5%)
and 32% (8.6–59.1%), respectively, with Halofolliculina
showing the highest distances towards all other sequences
(Table S2). Distances between genera and species in the
ITS dataset are lower, being 21.8% (13.1–38.7%) and 18%
(0.4–39.4%), respectively (Table S2). The intra-specific
distances are higher for the COI dataset, ranging from 0 to
14%, whereas they are lower for the ITS dataset
(0.1–7.3%) (Table S2). Regarding the Halofolliculina
sequences, intra-genus distances are moderately high for
the COI (14 ±0.9%), but very low for the ITS (0.5 ±0.4).
The genetic distances between Halofolliculina samples
from the three localities are high, ranging from 18.6 to
21.1% (Table S2).
Discussion
The present work reveals the relationship between Halo-
folliculina ciliate infections known as skeletal eroding band
in the Indo-Pacific and as Caribbean ciliate infection in the
Caribbean, by the application for the first time, of an
integrative, morphological molecular approach. Coral
lesions and protozoans in the three geographic areas
showed the same macroscopic appearance.
In all investigated affected coral species, halofolliculinid
infestations manifested themselves as areas of tissue loss
and bare skeleton covered by loricae. Halofolliculina cili-
ates settle in clusters following the rim of the corallites and
represent dark dots, giving the skeleton a scattered
appearance. When in high densities, they form a thick band
(1–10 cm), usually black or dark green in colour, between
recently exposed skeleton and dead tissue. They can also
form more speckled bands when in low density and be
more light green in colour. Thus, the in vivo observation of
halofolliculinids confirmed the previous information
reported for the skeletal eroding band disease (Antonius
and Lipscomb 2001; Winkler et al. 2004; Page and Willis
2008) and the Caribbean ciliate infection (Cro
´quer et al.
2006a). In particular, the analysis of the main features of
the protozoans’ body and lorica matched with the
description of Halofolliculina corallasia provided by
Antonius and Lipscomb (2001) for all specimens analysed.
Moreover, a detailed analysis of the skeleton revealed an
erosion on Caribbean diseased corals, suggesting for the
first time settlement patterns similar to those of Indo-
Pacific SEB-affected corals. In particular, slightly eroded
marks related to the presence of Halofolliculina loricae
have been observed on the Caribbean skeletons, revealing
that the ciliate may use the same mechanisms as in Indo-
Pacific SEB-causing ciliates. Indeed, the Caribbean Halo-
folliculina ciliates seems to manifest an apparently chem-
ical activity by leaving ‘‘sack-shaped’’ borings or an
‘‘honeycomb’’ pattern while attach their bodies on the coral
skeleton as already reported for the Indo-Pacific counter-
part (Riegl and Antonius 2003). The footprints were con-
sistent with the position and size of the loricae of H.
corallasia, although apparently different erosion degree
Fig. 4 Mean values and SE of
the neck diameter, neck length
and the neck brim diameters of
the Halofolliculina loricae
affecting Indo-Pacific (Maldive
and Red Sea) and Caribbean
scleractinians. Mean values are
expressed in lm
Coral Reefs
123
within the same samples or host has been detected. If this is
related to the time they remain attached to the coral hosts
or if some other unknown factors are involved needs to be
elucidated in future research.
The main character used to distinguish H. corallasia
from its congeners is the presence of a single sculpture line
circumscribing the neck of the lorica (Antonius and Lip-
scomb 2001; Page et al. 2015). The single line was evident
and easily detectable in all examined protozoans from the
Indo-Pacific, Red Sea and the Caribbean. In addition, all
the obtained measures fall within the ranges estimated for
H. corallasia in its first description (Antonius and Lip-
scomb 2001). Statistical analyses revealed no significant
differences in the neck diameter and length, supporting the
fine-scale morphological similarities of halofolliculinids
from the Caribbean and Indo-Pacific. Although a statistical
Fig. 5 Scanning electron
microscopy images of
Caribbean skeletal eroding
pattern. aCluster of
Halofolliculina ciliates on
septae of Diploria
labyrinthiformis;bClose-up of
loricae apparently eroding the
host skeleton; cThe white
dashed line show the eroding
pattern left by Halofolliculina
ciliates on a colony of Diploria
labyrinthiformis;dThe round-
shaped footprint of the loricae
settlement observed on the same
host; eApparently different
eroding patterns associated to
halofolliculinids; fBlack dashed
line show slight footprints
related to the presence of
halofolliculinids. Scale bars:
a1 mm, b0.5 mm, c,d50 lm,
e10 lm, f20 lm)
Coral Reefs
123
difference was found in the neck brim diameters, we
believe this morphological character needs further inves-
tigation since, in a few cases, loricae were distorted mainly
due to the conservation of the sample and to the great
variability in the extensibility of the lorica (Primc-Habdija
and Matonic
ˇkin 2005), and this may have introduced a bias
in the measurements. Therefore, all individuals have mor-
phologically been identified as H. corallasia, and proto-
zoans found in the Caribbean appeared to be identical to
those causing the SEB in the Indo-Pacific and Red Sea.
Overall, the understanding of the genetic relationships
among ciliates is complicated by the incomplete knowl-
edge of their diversity (Stru
¨der-Kypke and Lynn 2010).
Indeed, ciliated protozoans are largely underrepresented in
current biodiversity estimates for many reasons, such as
their small size and the difficulty in their isolation and
culture (Kher et al. 2011). In line with this gap of knowl-
edge, no genetic information on H. corallasia and the
whole genus Halofolliculina has been presented so far in
the literature, and no sequences have been deposited in
public databases. Consequently, the DNA sequences herein
obtained are the first molecular data for the entire Halo-
folliculina genus and represent a starting point for future
genetic evaluations of the species and related taxa.
The molecular results partially diverge from the mor-
phological characterization, finding relevant differences
between protozoans from different localities. Although
both nuclear and mitochondrial molecular markers
revealed the monophyly of all H. corallasia individuals
sequenced in this work, ITS and COI markers showed
variable levels of variation in protozoans from different
localities. The H. corallasia intra-specific genetic distance
based on the ITS dataset was extremely low, whereas that
based on COI was higher. Moreover, genetic distances
between Halofolliculina from the three different localities
were remarkably high. These results agree with previous
works on ciliate genetic diversity, in which COI showed a
much higher diversification than the ITS region (Gentekaki
and Lynn 2009; Fernandes et al. 2016).
According to these molecular results, we may hypoth-
esize two main opposite scenarios. Firstly, we may be
dealing with a single species with a circumtropical distri-
bution. Indeed, the high intra-Halofolliculina genetic dis-
tances observed for the COI fall within the range of intra-
specific distances found for other heterotrich species. The
interspecific divergence is generally much higher in ciliates
than in animals and the genetic distance thresholds used for
species delimitation differ greatly among ciliate taxa, being
for instance around 1% for Tetrahymena spp. and 18% for
Carchesium spp., based on the COI (Gentekaki and Lynn
2009; Kher et al. 2011). These data suggest that evolution
rates can be extremely high in ciliates, and that their intra-
specific genetic diversity can vary largely among taxa and
could be taxon-specific (Gentekaki and Lynn 2009; Stru
¨-
der-Kypke and Lynn 2010). Researchers have suggested
that a high ciliate genetic diversity can depend on several
factors, such as a strong gene flow and the ability of the
dispersal phase of these microorganisms to reach large
distances (Gentekaki and Lynn 2009). It is also assumed
that in population of ciliates highly isolated from each
other, a positive correlation exists between their genetic
divergence and the geographic distance, such as in the case
of Carchesium polypinum (Zhang et al. 2006). However,
there still is no universal consensus about the possible
biogeographic diversification of ciliates. Even if rarely, in
some cases ciliates population have been demonstrated to
have a genetic structure related to their biogeography
(Miao et al. 2004; Katz et al. 2005), and this may also the
case of H. corallasia.
A second scenario would be the presence of multiple
cryptic species with a similar morphology, as already found
also in other ciliates (e.g. Stru
¨der-Kypke and Lynn 2010;
McManus et al. 2010; Katz et al. 2011; Park et al. 2019). In
this latter case, the ITS region would result as inappropriate
to distinguish between closely related species, whereas the
COI divergence could be explained by the presence of
different species living in the three localities. Indeed, the
COI genetic distances within H. corallasia are comparable
or higher than the interspecific divergences within the other
genera included in the analyses and show, for instance,
patterns similar to those of some Blepharisma species,
which are well-resolved with COI but not with ITS
sequences. Moreover, Maldivian samples are more similar
to Caribbean ciliates rather than the Red Sea ones. This
seems to contradict the scenario of a circumtropical species
with a biogeography-related genetic structure and further
support the species complex hypothesis.
Therefore, the morphological and molecular data
obtained in this work seem to support more the latter
scenario, with the identification of a H. corallasia species
complex as the pathogen associated with both the Car-
ibbean ciliate infection and skeletal eroding band. Even
though it cannot be excluded that the SEB may be sym-
patric with the CCI in some localities, this would represent
an unlikely scenario. The most cautious approach when
describing coral diseases would be to proceed with the
classification of different syndromes only when clear evi-
dence is presented (Bourne et al. 2015). Since we found an
approximately identical morphology at micro- and macro-
scale in ciliates and lesions from different localities, and
since H. corallasia may actually be a complex of multiple
cryptic species, we believe that in order to reduce further
confusion supplementary studies that will clarify if CCI
and SEB should be synonymized are strongly required.
Coral Reefs
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Coral Reefs
123
Acknowledgements SM is grateful to Naturalis Biodiversity Center
for providing Martin Fellowships, which supported fieldwork in
Curac¸ao (2017) and Bonaire (2019). The fieldwork in Bonaire was
also supported by a grant from the WWF-Netherlands Biodiversity
Fund to BWH. The staff of CARMABI Marine Research Center at
Curac¸ao is thanked for logistical support. We are grateful to STI-
NAPA and DCNA at Bonaire for assistance in the submission of the
research proposal and the research permit.
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