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Diversity, host specificity and biogeography in the Cladocorynidae
(Hydrozoa, Capitata), with description of a new genus
Davide Maggioni
a,b
*, Agust
ın Garese
c
, Danwei Huang
d
, Bert W. Hoeksema
e,f
,
Roberto Arrigoni
g
, Davide Seveso
a,b
, Paolo Galli
a,b
, Michael L. Berumen
h
,
Enrico Montalbetti
a,b
, Daniela Pica
i,j
, Fabrizio Torsani
k
and
Simone Montano
a,b
a
Department of Earth and Environmental Sciences (DISAT), University of Milano-Bicocca, Piazza della Scienza, Milano, 20126, Italy;
b
Marine
Research and High Education (MaRHE) Center, University of Milano-Bicocca, Faafu Magoodhoo Island, 12030, Republic of Maldives;
c
Instituto
de Investigaciones Marinas y Costeras (IIMyC), Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata (UNMDP) –
Consejo Nacional de Investigaciones Cient
ıficas y T
ecnicas (CONICET), Mar del Plata, 7600, Argentina;
d
Department of Biological Sciences,
Tropical Marine Science Institute and Centre for Nature-based Climate Solutions, National University of Singapore, Singapore, 117558, Singapore;
e
Taxonomy, Systematics and Geodiversity Group, Naturalis Biodiversity Center, Leiden, 2300 RA, The Netherlands;
f
Groningen Institute for
Evolutionary Life Sciences, University of Groningen, Groningen, 9700 CC, The Netherlands;
g
Department of Biology and Evolution of Marine
Organisms (BEOM), Stazione Zoologica Anton Dohrn, Villa Comunale, Napoli, 80121, Italy;
h
Red Sea Research Center, Division of Biological
and Environmental Science and Engineering, King Abdullah University of Science and Technology (KAUST), Thuwal, 23955-6900, Saudi Arabia;
i
Department of Biological and Environmental Sciences and Technologies, University of Salento, Lecce, 73100, Italy;
j
CoNISMa –Consorzio
Nazionale Interuniversitario per le Scienze del Mare, Roma, 00196, Italy;
k
Department of Life and Environmental Sciences, Polytechnic University
of Marche, Ancona, 60131, Italy
Accepted 17 June 2021
Abstract
The hydrozoan family Cladocorynidae inhabits tropical to temperate waters and comprises the two genera Pteroclava and
Cladocoryne.Pteroclava lives in association with some octocorals and hydrozoans, whereas Cladocoryne is more generalist in
terms of substrate choice. This work provides a thorough morpho-molecular reassessment of the Cladocorynidae by presenting
the first well-supported phylogeny of the family based on the analyses of three mitochondrial and four nuclear markers. Nota-
bly, the two nominal genera were confirmed to be monophyletic and both morphological and genetic data led to the formal
description of a new genus exclusively associated with octocorals, Pseudozanclea gen. nov. Maggioni & Montano. Accordingly,
the diagnosis of the family was updated. The ancestral state reconstruction of selected characters revealed that the symbiosis
with octocorals likely appeared in the most recent common ancestor of Pteroclava and Pseudozanclea. Additionally, the presence
of euryteles aggregation in the polyp stage and the exumbrellar nematocyst pouches with euryteles represent synapomorphies of
all cladocorynid taxa and probably emerged in their most recent common ancestor. The analysis of several Pteroclava krempfi
colonies from Indo-Pacific and Caribbean localities associated with several host octocorals revealed a high intra-specific genetic
variability. Single- and multi-locus species delimitations resulted in three to five species hypotheses, but the statistical analysis of
morphometric data showed only limited distinction among the clades of P. krempfi. However, P. krempfi clades showed differ-
ences in both host specificity, mostly at the octocoral family level, and geographic distribution, with one clade found exclusively
in the Caribbean Sea and the others found in the Indo-Pacific.
©2021 The Authors. Cladistics published by John Wiley & Sons Ltd on behalf of Willi Hennig Society.
*Corresponding author:
E-mail address: davide.maggioni@unimib.it
Cladistics
Cladistics (2021) 1–25
10.1111/cla.12480
©2021 The Authors. Cladistics published by John Wiley & Sons Ltd on behalf of Willi Hennig Society
This is an open access article under the terms of the Creative Commons Attribution License, which permits use,
distribution and reproduction in any medium, provided the original work is properly cited.
Introduction
Coral reefs are well known to host a great diversity
of symbiotic relations, with scleractinian corals being
associated with numerous other organisms, spanning
all domains of life (Gates and Ainsworth, 2011; Stella
et al., 2011; Hoeksema et al., 2012, 2017). Coral asso-
ciates may interact with each other and the host,
resulting in positive outcomes for the whole coral sym-
biome (Ainsworth et al., 2020), but in many cases
these interactions are still poorly characterized, in par-
ticular for their taxonomic composition and ecological
relevance (Gates and Ainsworth, 2011). Other benthic
coral-reef invertebrates also provide habitats for a
plethora of organisms (e.g., Antokhina and Britayev,
2012; Neo et al., 2015; Sch€
onberg et al., 2015; Garc
ıa-
Hern
andez et al., 2019), even if these associations are
rarely well characterized (Montano, 2020).
Octocorals represent a prominent group among non-
scleractinian reef dwellers (Fabricius and Alderslade,
2001), and, due to their topological complexity and
abundance, they host several associated organisms
(Goh et al., 1999; Hoeksema et al., 2015). For
instance, Maggioni et al., (2020a) recently reported the
octocoral Bebryce cf. grandicalyx to recurrently host
the hydroid Zanclea timida Puce, Di Camillo and
Bavestrello, 2008 together with a suberitid sponge in
the Maldives, and many other invertebrates were
observed to dwell on the octocoral or sponge surface.
This octocoral-hydrozoan symbiosis is not an excep-
tion, as many hydrozoan and octocoral species are
reported to live in intimate associations (Bo et al.,
2011). Some of these hydroids appear to be obligate
symbionts of octocorals and have never been reported
growing on other substrates (Puce et al., 2008a, b).
The aforementioned Z. timida, and the cladocorynid
Pteroclava krempfi (Billard, 1919) are two examples of
obligate alcyonancean-associated hydrozoan species
(Puce et al., 2008b). Both species are known to grow
on a variety of hosts: Z.timida is associated with the
alcyonacean genera Paratelesto Utinomi, 1958 and
Bebryce Philippi, 1841 (Puce et al., 2008b; Maggioni
et al., 2020a), whereas P. krempfi associates with at
least nine alcyonacean genera (Billard, 1919; Varela
and Cabrales Caballero, 2010; Seveso et al., 2016,
2020; Maggioni et al., 2016; Montano et al., 2017).
Genetic data of P. krempfi have revealed the presence
of a complex of multiple cryptic species with a possible
host specificity at the octocoral genus or family level
(Maggioni et al., 2016; Montano et al., 2017). Specifi-
cally, a Maldivian clade was associated with Paraplex-
aura K€
ukenthal, 1909 (family Plexauridae Gray, 1859),
a second Maldivian clade was found exclusively on the
Alcyoniidae Lamouroux, 1812 genera Sinularia May,
1898, Sarcophyton Lesson, 1834, and Lobophytum
Marenzeller, 1886, whereas a third Caribbean clade
was associated with Antillogorgia Bayer, 1951 (family
Gorgoniidae Lamouroux, 1812). Despite the host pref-
erences and genetic divergence, no morphological dif-
ferences were detected among the three P. krempfi
clades. This, along with (i) the lack of suitable genetic
material of P. krempfi from the type locality (in Viet-
nam), and (ii) the type’s host Cladiella krempfi (Hick-
son, 1919) and (iii) the scant information on the
congeneric and hydrozoan-associated species Ptero-
clava crassa (Pictet, 1893), did not allow the authors
to name the species.
Taxonomic confusion is also evident in the other
cladocoryinid genus, Cladocoryne Rotch, 1871. All
Cladocoryne species are characterized by the unique
aboral branched tentacles (Bouillon et al., 2006), and
the number and arrangement of aboral tentacles are
key distinguishing characters for the species of this
genus (Schuchert, 2003). This taxon currently contains
five species, three of which [Cladocoryne travancorensis
(Mammen, 1963), Cladocoryne littoralis (Mammen,
1963), Cladocoryne minuta Watson, 2005] have never
been observed again after their first description (Mam-
men, 1963; Watson, 2005). The other two well-
established species, namely Cladocoryne floccosa
Rotch, 1871 and Cladocoryne haddoni Kirkpatrick,
1890, can be easily distinguished by the number of
aboral branched tentacles and the cnidome (Bouillon
et al., 1987). The presence of unnamed Cladocoryne
species has also been hypothesized by Schuchert (2003)
while discussing Indonesian colonies described by Ste-
chow and M€
uller (1923) and Vervoort (1941), yet no
molecular analyses have been performed to assess the
precise diversity of the genus. Moreover, the type
material of C. floccosa,C. travancorensis,andC. lit-
toralis cannot currently be located, and is presumably
lost.
In contrast to Pteroclava Weill, 1931, the genus
Cladocoryne is a generalist in terms of substrate
choice, and has been reported on a variety of sub-
strates, including rocks, algae, seagrasses, sponges,
octocorals, bryozoans, polychaete tubes, and other
hydroids (Bouillon et al., 1987; Gravili et al., 2015).
Additional differences are found in the general mor-
phology and reproductive structures, since Pteroclava
has moniliform tentacles and produces free-swimming
medusae, whereas Cladocoryne has oral capitate and
aboral branched capitate tentacles and produces cryp-
tomedusoids that do not detach from the parental
polyp (Bouillon et al., 2006). However, both genera
share the presence of euryteles grouped in rounded
clusters in the polyp stage, a feature considered to be
a synapomorphy of taxa ascribed to Cladocorynidae
Allman, 1872 (Petersen, 1990). Other unique features
of the family are (i) the presence of euryteles in the
exumbrellar cnidocyst pouches, occurring in the free-
swimming medusa of P. krempfi (Boero et al., 1995),
2D. Maggioni et al. / Cladistics 0 (2021) 1–25
and (ii) the branched aboral tentacles in all Cladoco-
ryne species (Bouillon et al., 2006).
With this work we aimed at performing a thorough
assessment of the family Cladocorynidae, including
detailed morphological characterisations, phylogenetic
analyses, single and multi-locus DNA-based species
delimitations, statistics of morphological measure-
ments, character evolution, and host-specificity assess-
ment. Additionally, the phylogenetic position of the
octocoral-associated species Zanclea timida was
assessed based on new morphological information (i.e.,
the medusa stage) and genetic data.
Material and methods
Sampling and morphological assessment
Sampling was carried out by snorkelling (0–5 m deep) and diving
(5–30 m deep) between November 2013 and November 2018 in sev-
eral localities across the Indo-Pacific, Red Sea, Caribbean Sea, and
Mediterranean Sea (Fig. 1, Table S1). All species were collected in
this depth range, with the only exception of Zanclea timida colonies
at 40–50 m depth. When the presence of hydrozoan polyps was
recorded in situ, small fragments of the substrate and associated
hydroids were collected. Animals were anesthetized with menthol
crystals, detached from their hosts using precision forceps, hypoder-
mic syringe needles, and micropipettes and subsequently fixed in
both 99% ethanol for molecular analyses and 10% formalin for
morphological analyses. When possible, live animals were reared in
constantly oxygenated bowls filled with seawater, at room tempera-
ture, under artificial light, and fed with Artemia nauplii until medusa
release occurred.
Hydrozoans were identified to the species level following Boero
et al., (1995), Bouillon et al., (1987), Schuchert (2006), and Puce
et al., (2008). Cladocorynid specimens were observed and pho-
tographed under a Leica EZ4 D stereo microscope to assess their
general morphology, and analyzed under a Zeiss Axioskop 40 com-
pound microscope to study the fine-scale morphology of polyps,
medusae, and cnidocysts. All measurements were taken using ImageJ
1.52p software. For the identification of host octocorals, small por-
tions of tissue were immersed in a 10% sodium hypochlorite solution
for 6 h to obtain sclerites. Octocorals were identified to genus level
or, when possible, to the species level by examining the general mor-
phology of the colony and the sclerites following Fabricius and
Alderslade (2001) and Williams and Chen (2012).
New material for each investigated species was deposited at the
Museo Civico di Storia Naturale di Milano (Italy) and the Aus-
tralian Museum (Sydney, Australia). The present work is registered
in ZooBank under: http://zoobank.org/urn:lsid:zoobank.org:pub:
EED5AABF-43CC-4793-A84E-3C49857D33B8.
DNA extraction and sequencing
Total genomic DNA was extracted from ethanol-fixed hydroids
using the protocol described in Maggioni et al., (2020b). Additional
DNA samples of Cladocoryne floccosa colonies from the Caribbean
and Mediterranean Sea were obtained from Peter Schuchert
(Mus
eum d’Histoire Naturelle of Geneva, Switzerland, Table S1).
AU
SG
MV
SA
EG,IL
EUX
MZ
PA GP
ES FR
ES
GR
Pteroclava krempfi Cladocoryne floccosa Cladocoryne haddoni
Pseudozanclea timida Sampling localities Type localities
Fig. 1. Map of the sampling sites. Triangles show type localities of the investigated species, whereas pins show sampling localities, coloured by
species, as shown in the legend. Caribbean Sea: GP, Guadeloupe; PA, Panama; EUX, St. Eustatius. Mediterranean Sea: ES, Spain; FR, France;
GR, Greece. Red Sea: EG, Egypt; IL, Israel; SA, Saudi Arabia. Indo-West Pacific: AU, Australia; MV, Maldives; MZ, Mozambique; SG, Singa-
pore.
D. Maggioni et al. / Cladistics 0 (2021) 1–25 3
Seven gene regions were amplified using the primers and protocols
described in Maggioni et al., (2020c), namely portions of the mito-
chondrial large ribosomal RNA (16S rRNA), cytochrome oxidase
subunit I (COX1), cytochrome oxidase subunit III (COX3), and
nuclear small ribosomal RNA (18S rRNA), large ribosomal RNA
(28S rRNA), internal transcribed spacer (ITS; including partial
ITS1, 5.8S, and partial ITS2 regions), and histone H3 (H3)
(Table 1). PCR products were checked through 1.5% agarose elec-
trophoretic runs, purified with Illustra ExoStar (GE Healthcare:
Amersham, UK) and sequenced in both directions with ABI 3730xl
DNA Analyzer (Applied Biosystems: Carlsbad, CA, USA). Geneious
6.1.6 was used to visually check, correct, and assemble the obtained
chromatograms and to check for the presence of open reading
frames in protein-coding genes. The consensus sequences obtained in
this study were deposited in GenBank with the accession numbers
listed in Table S1.
Sequences of each DNA region were aligned using MAFFT 7.110
(Katoh and Standley, 2013) with the E-INS-i option, after adding
outgroup sequences following Maggioni et al., (2017, 2018) (Tables
S1). The ITS alignment was further run through Gblocks (Castre-
sana, 2000; Talavera and Castresana, 2007) using the default ‘less
stringent’ settings to remove ambiguously aligned regions. All align-
ments were also concatenated using Mesquite 3.2 (Maddison and
Maddison, 2006).
Phylogenetic analyses
Descriptive statistics and variability of each DNA region were cal-
culated with DnaSP 6 (Rozas et al., 2017) (Table 1). Substitution
models were determined using jModelTest 2 (Darriba et al., 2012)
for single-locus datasets, whereas partition schemes and models were
determined with PartitionFinder 1.1.1 (Lanfear et al., 2012) for the
multi-locus dataset (Table S2), using the Akaike Information Crite-
rion (AIC).
Phylogenetic inference was performed using maximum parsimony
(MP), maximum likelihood (ML), and Bayesian inference (BI) using
both single- and multi-locus datasets and running the analyses on
the CIPRES server (Miller et al., 2010). MP analyses were performed
using PAUP 4.0b10 (Swofford, 2003) with heuristic searches stepwise
addition and tree-bisection-reconnection branch swapping. Node
support was assessed using 1000 bootstrap replicates with randomly
added taxa. For ML analyses, the substitution model GTR+G was
selected for each DNA region and partition, and analyses were run
using RAxML 8.2.12 (Stamatakis, 2014) with 1000 non-parametric
bootstrap replicates. BI analyses were performed using MrBayes
3.2.6 (Ronquist et al., 2012) using the substitution models and parti-
tions listed in Table S2. Two independent runs for four Markov
chains were conducted for 50 million generations, with trees sampled
every 5000th generation. For all Bayesian analyses, parameter esti-
mates and convergence were checked using Tracer 1.6 (Rambaut
et al., 2014), where burn-in was set at 25%.
MEGA X (Kumar et al., 2018) was used to calculate genetic dis-
tances within and among the obtained clades. Genetic distances were
calculated as % uncorrected p-distances with 1000 non-parametric
bootstrap replicates.
A species tree was obtained using *BEAST (Heled and Drum-
mond, 2009) in BEAST 2.2.0 (Bouckaert et al., 2014). Specimens
were assigned to different species according to the species delimita-
tion analyses described below, and mitochondrial loci were consid-
ered as a single partition, whereas nuclear loci were kept separate.
Yule process prior, together with a linear and constant-root
population-size model, were used and each analysis was run for 10
8
generations, sampling every 10000th generation, and setting a 25%
burn-in. The convergence of the analysis was checked using Tracer
1.6 (Rambaut et al., 2014), as done for the gene trees.
To better visualize the presence of host-related and geography-
related genetic structure within Pteroclava clades, median-joining
haplotype networks were built using the software PopART 1.7
(Leigh and Bryant, 2015). The most complete mitochondrial single-
locus dataset (16S rRNA) was used, with haplotypes coloured
according to the genus of the host and the geographic provenance
(Indian Ocean, Pacific Ocean, Red Sea).
Finally, a set of characters was mapped onto a reduced multi-
locus phylogenetic reconstruction of the Zancleida Russel, 1953 (in-
cluding one specimen for each nominal species) obtained with
BEAST 1.8.2 (Drummond et al., 2012) using the same parameters
described for the *BEAST analysis. The mapped characters were: (i)
the organisation of euryteles in the polyp stage (not organized, orga-
nized in clusters or a ring); (ii) the exumbrellar cnidocyst pouches in
the medusa stage (absent, containing euryteles, containing stenote-
les); (iii) the substrate choice of the polyp stage (generalist, symbi-
otic); (iv) the host of the polyp stage (none, octocorals, sponges,
scleractinians, bryozoans). Stochastic mapping (Huelsenbeck et al.,
2003) was used to map probable realizations of the evolution of the
considered characters on the Zancleida tree. The analyses were run
in the R environment (R Core Team, 2020), using the ‘make.sim-
map’ function in the package ‘Phytools’ (Revell, 2012). Reconstruc-
tions were performed under the ‘equal rates’ (ER) and the ‘all rates
different’ (ARD) models, comparing the fit of the models with a like-
lihood ratio test using the function ‘pchisq’. The ER model was
selected for the characters ‘organization of euryteles’ and ‘substrate
choice’, whereas the ARD model for the characters ‘cnidocyst
pouches’ and ‘host’. 10000 stochastic mapping replicates were con-
ducted for each analysis and the results were summarized with pie
charts representing the posterior probability of each internal node
being in each state.
DNA-based species discovery and validation
Distance- and tree-based species delimitation approaches were
used to investigate the presence of multiple species hypotheses in
octocoral-associated Pteroclava. All analyses were run separately on
Table 1
Molecular statistics and substitution models for each molecular markers
Region Primers S Sv (%) SPI (%) H Hd Nd Substitution models
16S rRNA SHB-SHA 625 157 (25.1) 123 (19.7) 55 0.973 0.008 0.100 0.007 HKY +I+G
COX1 LCO1490-HCO2198 677 187 (27.6) 167 (24.7) 52 0.976 0.009 0.131 0.006 HKY +I+G
COX3 CO3F-CO3R 668 264 (39.5) 233 (34.9) 65 0.990 0.004 0.150 0.008 GTR +I+G
18S rRNA 18SA-18SB 1712 185 (10.8) 109 (6.4) 29 0.910 0.016 0.012 0.002 GTR +I+G
28S rRNA 28SHF-R2077 1684 250 (14.8) 165 (9.8) 25 0.831 0.030 0.022 0.002 GTR +I+G
ITS (Gblocks) HITSF-HITSR 662 191 (28.9) 133 (20.1) 21 0.809 0.031 0.074 0.011 GTR +I+G
H3 H3F-H3R 352 68 (19.3) 56 (15.9) 22 0.873 0.023 0.091 0.009 GTR +G
H, number of unique haplotypes; Hd, haplotype diversity; Nd, nucleotide diversity; S, number of sites; SPI, number of parsimony-
informative sites; Sv, number of variable sites.
4D. Maggioni et al. / Cladistics 0 (2021) 1–25
the single-locus datasets, keeping only Pteroclava sequences and col-
lapsing the alignments into haplotypes by removing identical
sequences, using FaBox (Villesen, 2007). Sequences with different
length but identical nucleotides were considered the same haplotype.
The distance-based Automatic Barcode Gap Discovery (ABGD;
Puillandre et al., 2012) and Assemble Species by Automatic Parti-
tioning (ASAP; Puillandre et al., 2020) methods were performed
using matrices of genetic distances (Kimura 2-Parameter) as inputs.
The ABGD delimitations were run on the website ‘abgd web’
(https://www.abi.snv.jussieu.fr/public/abgd/abgdweb.html), setting
parameters as follows: P
min
=0.001, P
max
=0.1, Steps =100,
X=1.5, Nb bins =100. The ASAP delimitations were run on the
website ‘asap web’ (https://bioinfo.mnhn.fr/abi/public/asap/) consid-
ering only the partitions showing the lowest asap-score.
For tree-based methods, single-locus ML trees were obtained as
described above and ultrametric Bayesian trees were obtained with
BEAST 1.8.2 (Drummond et al., 2012), setting a coalescent tree
prior and an uncorrelated lognormal relaxed clock. Three replicate
analyses were run for 10
8
million generations, with trees sampled
every 10000th generation, and were combined using LogCombiner
1.8.2 (Drummond et al., 2012) with a burn-in set to 25%. Maximum
clade credibility trees were obtained using TreeAnnotator 1.8.2
(Drummond et al., 2012). The obtained trees were used as inputs for
the Poisson Tree Process (PTP) and Generalized Mixed Yule Coales-
cence (GMYC) methods. Multiple-threshold PTP analyses (mtPTP:
Kapli et al., 2017) were performed on the website ‘mPTP Webser-
vice’ (https://mptp.h-its.org) using ML trees as input, whereas
GMYC analyses were run in the R environment (R Core Team,
2020), using ultrametric trees as input. Specifically, single-threshold
GMYC (stGMYC; Pons et al., 2006) and bGMYC (Reid and Car-
stens, 2012) analyses were run using the packages ‘Splits’ (Ezard
et al., 2009), ‘Ape’ (Paradis et al., 2004), and ‘bGMYC’ (Reid and
Carstens, 2012). bGMYC analyses were performed using a subset of
100 trees retrieved from the 10000 posterior trees obtained with each
BI analysis. Single-threshold PTP and multiple-threshold GMYC
methods were previously shown to be outperformed by the other
implementations of PTP and GMYC used in this work (Fujisawa
and Barraclough, 2013; Kapli et al., 2017) and were therefore not
included in the analyses.
To validate the species hypotheses proposed by the single-locus
analyses, the multi-locus dataset was analyzed under the multispecies
coalescent model using Bayesian Phylogenetics and Phylogeography
(BPP) 3.4 (Yang and Rannala, 2010). The algorithm A11 (joint spe-
cies delimitation and species tree inference) was used and different
values of the root age (s) and prior distributions of the ancestral
population size (h) were tested, due to the lack of knowledge about
these parameters in Pteroclava and to test their possible influence on
the number of delimited species (Leach
e and Fujita, 2010). We tested
for: (i) large ancestral population size and deep divergence [h~IG
(3, 0.04), s~IG (3, 0.02)]; (ii) small ancestral population size and
shallow divergence [h~IG (3, 0.004), s~IG (3, 0.002)]; (iii) small
ancestral population size and deep divergence [h~IG (3, 0.004), s~
IG (3, 0.02)]; (iv) large ancestral population size and shallow diver-
gence [h~IG (3, 0.04), s~IG (3, 0.002)]. Each analysis was run
using both the reversible-jump MCMC algorithm 0 (ɛ= 1) and
algorithm 1 (a=1.5, m =1.5) to assess the impact of the fine-tuning
parameters of reversible-jump algorithms on the speciation probabili-
ties, and was repeated two times, to confirm the stability of results
across runs. For each run, 50000 samples were collected with a sam-
pling frequency of 10 iterations after a burn-in of the first 5000
cycles.
Finally, DNA diagnostic characters were searched for each
P. krempfi clade, using the package QUIDDICH (K€
uhn and Haase,
2020) in the R environment (R Core Team, 2020). Specifically, char-
acter attributes (i.e., single nucleotides) present in all members of a
defined clade but absent in members of other clades were searched,
and the analyses were run for each molecular marker, considering
different species hypotheses.
Alignments and raw phylogenetic trees are provided as supple-
mentary files.
Pteroclava cnidocyst size analyses
To explore differences in the size of cnidocysts among P. krempfi
clades, we selected 15 colonies, five for each of the clades Ia, IIa,
and III, whereas measurements were not performed for clades Ib
and IIb (Fig. 2), since insufficient material was available. The ana-
lyzed colonies were associated with nine different hosts and collected
from five localities. For each colony, ten polyps were selected and,
for each polyp, up to 15 capsules per type were measured. The
descriptive statistical parameters of all cnidocyst types [length, width
(mean sd), range (maximum-minimum)] were calculated for each
clade in the R environment (R Core Team, 2020) using the ‘Rcmdr’
package (Fox and Bouchet-Valat, 2020). Kernel density plots
(Sheather, 2004) were produced for the length of each cnidocyst type
and clade. Stratified box plots for each cnidocyst type, clade, locality
and host were also obtained. All graphics were made using the pack-
age ‘ggplot2’ (Wickham, 2016).
To study the variation of cnidocyst sizes in the P. krempfi clades,
linear mixed models (LMM) or generalized linear mixed models
(GLMM) were fitted for each of the three cnidocyst types (eurytele,
large stenotele, small stenotele). Only the length data were used,
since width data are in general less variable (Garese et al., 2016).
The selection of LMM or GLMM was performed assessing the nor-
mality or not of the residuals of the models (following Garese et al.,
2016). The models were performed using the R package ‘lme4’ (Bates
et al., 2015). The general form of the models was: y
i
=b
i
x+z
i
µ
i
+e
i
, where yis the response variable, b
i
is the fixed effect parame-
ter, µ
i
is the random effect parameter, xis the matrix of fixed
parameters, zis the matrix of random effects and e
i
is the matrix of
errors. The global model form included ‘cnidocyst size’ as the depen-
dent variable and ‘clade’ as fixed effect. Since datasets were assem-
bled by measuring polyps from different colonies, localities, and/or
hosts, these variables were at first considered as random effects and
then their significance evaluated. By consequence, the global model
form was: Cnidocyst sizes ~Clade +(1|Polyp) +(1|Colony) +(1|
Locality) +(1|Host) +e.
To find the best model, the significance of each random effect was
tested by deleting them one by one, using ‘ranova’ function of the R
package ‘lmerTest’ (Kuznetsova et al., 2017), and to rank LMMs or
GLMMs the Akaike Information Criterion (AIC) was used. Once
the best model was determined, the normality of residuals of the
LMM were tested by Shapiro–Wilks test and, graphically, in QQ-
plots, and the homoscedasticity of variance was checked in fitted vs
residual graphs. When the assumption of normality was not met, a
GLMM was fitted by maximum likelihood (Laplace Approximation)
with Gamma distribution for errors and inverse link function. Then,
the best models were compared through ANOVA with a null model
(Cnidocyst sizes ~Clade) to test the significance of the LMM or
GLMM. Finally, the model estimates for each clade, the confidence
intervals, and the standard deviation for random effects, were
extracted and analyzed. The R scripts used for the LMM and
GLMM are shown in File S1.
Results
A total of 75 colonies were collected from the stud-
ied localities and identified as Cladocoryne floccosa,
C. haddoni,Pteroclava krempfi and Zanclea timida.
D. Maggioni et al. / Cladistics 0 (2021) 1–25 5
0.02
F 292B (SA) Melithaea
M 182A (MV)
F 250B (SA) Melithaea
M 243A (MV) Bebryce
A 010U (AU) Sarcophyton
K 056A (SA) Paralemnalia
F 477B (SA) Melithaea
D 044A (EG) Rhytisma
G 008R (GR)
M 293A (MV)
M 223A (MV)
K 046A (SA) Sinularia
M 307A (MV) Bebryce
DNA660 (GP)
G 003R (GR)
E 009I (IL) Sarcophyton
K 044A (SA) Rhytisma
AU008 (AU) Sarcophyton
F116B(SA) Sinularia
M 364A (MV)
K 041A (SA) Rhytisma
M 186A (MV) Lobophytum
K 021A (SA) Sarcophyton
F 252B (SA) Melithaea
DNAN062 (ES)
S 136N (SG) Ctenocella
F 308B (SA) Astrogorgia
M 302A (MV) Bebryce
K 095A (SA) Rhytisma
F 317B (SA) Astrogorgia
S 013E (EUX) Antillogorgia
S 138N (SG) Ctenocella
M 188A (MV) Lobophytum
F 040B (SA) Sinularia
F 227B (SA) Melithaea
M 234A (MV) Paraplexaura
P 045O (MZ) Melithaea
M 306A (MV) Bebryce
G 007R (GR)
F 277B (SA) Sinularia
MA189 (MV) Paraplexaura
F 294B (SA) Astrogorgia
P 095O (MZ) Melithaea
M 279A (MV)
M 185A (MV) Sarcophyton
M 190A (MV) Paraplexaura
K 050A (SA) Rhytisma
S 140N (SG) Ctenocella
M 299A (MV) Bebryce
M 200A (MV)
M 183A (MV) Sinularia
F 305B (SA) Melithaea
S 012E (EUX) Antillogorgia
K 052A (SA) Sinularia
F 140B (SA) Sinularia
S 134N (SG) Ctenocella
M 214A (MV) Bebryce
M 224A (MV)
D043A
(EG)
Rhytisma
K 042A (SA) Rhytisma
F 327B (SA) Melithaea
M 244A (MV) Paraplexaura
A 006U (AU) Acanthogorgia
DNA981 (FR)
F 027B (SA) Sinularia
F 298B (SA) Melithaea
F 047B (SA) Sinularia
P 074O (MZ) Dendronephthya
M 187A (MV) Lobophytum
M 181A (MV) Sinularia
B 006T (PA) Antillogorgia
K 075A (SA) Sinularia
F 231B (SA) Melithaea
DNA679 (ES)
K 054A (SA) Sarcophyton
M 130A (MV) Lobophytum
M 366A (MV)
M 184A (MV) Sarcophyton
K 058A (MV) Sinularia
Pt. krempfi II
Pt. krempfi III
*
*
*
*
*
*
*
*
*
*
*
*
Pt. krempfiI
Pteroclava
Pseudozanclea
Cladocoryne
C. floccosa
C. haddoni
Ps. timida
91/71/1
71/99/1
98/100/1
72/95/1
Corynida
Pennariidae
Moerisiidae
Sphaerocorynidae
Asyncorynidae
Milleporidae
Solanderiidae
Zancleidae
Cladocorynidae
*
*
-/80/1
83/92/1
-/88/1
-/85/1
94/100/1
71/99/1
MV
MZ
AU
SG
EG
IL
SA
EUX
GP
PA
Maldives
Mozambique
Australia
Singapore
Egypt
Israel
Saudi Arabia
Sint Eustatius
Guadeloupe
Panama
Indian Ocean
Pacific Ocean
Red Sea
Caribbean Sea
LOCALITIES
ES
FR
GR
Spain
France
Greece
Mediterranean Sea
(a) (b)
(c)
Ia
Ib
IIa
IIb
Sinularia
Pt. krempfi Ia
Pt. krempfi IIb
Pt. krempfi III
Ps. timida
C. floccosa
C. haddoni
Pt. krempfi IIa
Pt. krempfi Ib
1
1
0.98
0.88
0.69
0.94
1
MP
ML
BI
16S
COX1
COX3
18S
ITS
28S
H3
≥ 75 (MP, ML) or ≥ 0.9 (BI)
< 75 (MP, ML) or < 0.9 (BI)
node absent
Nodal support in single-locus analyses:
Nodal support in multi-locus analyses:
*: MP = 100, ML = 100, BI = 1
MP / ML / BI
6D. Maggioni et al. / Cladistics 0 (2021) 1–25
The morphology of all investigated species corre-
sponded to the original descriptions, with the excep-
tion of Z. timida, as reported in the ‘Taxonomic
account’ section. Indeed, the morphological analysis of
the medusa stage of Z. timida, conducted herein for
the first time, highlighted strong similarities with the
medusa of P. krempfi, specifically the presence of eury-
teles in the exumbrellar cnidocyst pouches. On the
other hand, the polyp stage was not ascribable to the
genus Pteroclava, being more similar to typical Zan-
clea polyps (i.e., with oral and aboral capitate tenta-
cles). For these reasons, and in accordance with
phylogenetic results, the new cladocorynid genus Pseu-
dozanclea gen. nov. was established to accommodate
Z. timida, resulting in the new combination Pseudozan-
clea timida comb. nov.
In addition, specimens from Mozambique, Singa-
pore, and Australia represent new geographic records
for P. krempfi. The host range of the Pteroclava-
octocoral association was also widened with the inclu-
sion of the alcyonacean genera Melithaea Milne
Edwards, 1857 (family Melithaeidae Gray, 1870),
Ctenocella Valenciennes, 1855 (Ellisellidae Gray, 1859),
Acanthogorgia Gray, 1857 (Acanthogorgiidae Gray,
1959), Dendronephthya K€
ukenthal, 1905 and Paralem-
nalia K€
ukenthal, 1913 (Nephtheidae Gray, 1862).
Therefore, P. krempfi is currently known to live in
association with 14 octocoral genera belonging to
seven families (Table 2).
Molecular phylogenetics and species delimitation
The length of single-locus alignments was 625 bp for
the 16S rRNA, 677 bp for COX1, 668 bp for COX3,
1712 bp for 18S rRNA, 1684 bp for 28S rRNA,
662 bp for ITS (927 bp before the Gblocks treatment),
and 352 bp for H3 (Table 1), resulting in a total of
6380 bp for the concatenated alignment. Overall, the
molecular markers were amplified and sequenced with
high success (Table 3) and approximately 80% of the
terminals in the concatenated dataset were represented
by all markers. When this was not the case, one to
three markers were missing (Table S1). The single- and
multi-locus phylogeny reconstructions were broadly
concordant (Fig. 2a, b; Fig. S1; Table S3). Although
the relationships among clades varied across the
single-locus analyses, each of the analyzed colonies
belonged to the same molecular clade in almost all
trees and phylogeny reconstruction criteria (Table S3).
Multi-locus analyses resulted in the same ingroup
topology with high nodal support (Fig. 2a, b), with
only minor variation in the relationships among out-
groups (Fig. 2a; Fig. S1). Here we considered the node
support as maximal when bootstrap values (BS) for
MP and ML analyses were =100 and Bayesian poste-
rior probabilities (BPP) =1, high when BS ≥75 and
BPP ≥0.9, and low when BS <75 and BPP <0.9. The
family Cladocorynidae was monophyletic in all multi-
locus analyses, with maximal support in ML and BI
analyses, and lower support in the MP analysis
(BS =71), and its phylogenetic position as sister group
of the families Asyncorynidae Kramp, 1949, Millepori-
dae Fleming, 1828, Solanderiidae Marshall, 1892, and
Zancleidae Russel, 1953 was confirmed (Fig. 2a,
Fig. S1). Both cladocorynid genera, Cladocoryne and
Pteroclava, and all the analyzed species, were consis-
tently recovered as monophyletic with high or maximal
support values (Fig. 2b). All the specimens belonging
to Pseudozanclea timida, and collected in the Maldives,
formed a monophyletic clade that was sister to Ptero-
clava in all multi-locus analyses with maximal nodal
support. The phylogenetic placement of this species
within the Cladocorynidae was therefore demon-
strated, supporting the transfer from the genus Zan-
clea and family Zancleidae to the new genus
Pseudozanclea. In all analyses, Pteroclava krempfi was
characterized by a strong intra-specific genetic varia-
tion, spanning in most cases five well-supported clades
(Fig. 2b; Fig. S1; Table S3): Pteroclava krempfi Ia, Ib,
IIa, IIb, and III. The clades P. krempfi I, II, and III
were recovered in all analyses, whereas sub-clades Ia,
Ib, IIa, and IIb were recovered in most analyses, with
the exception of single-locus ITS, 28S, and H3 analy-
ses (Table S3).
The genetic distances among the nominal species
were high for all DNA regions, with nuclear 18S and
28S rRNA showing the lowest values, whereas intra-
specific distances were low for all species but
P. krempfi and C. floccosa (Table 4; Table S4). These
two species showed high intra-specific distances espe-
cially in mitochondrial regions, with values of 4.7%
for P. krempfi and 3.7% for C. floccosa for the 16S
rRNA dataset (Table S4). Regarding P. krempfi, the
inter-clade genetic distances were also generally high,
Fig. 2. Phylogenetic hypotheses of the Cladocorynidae. (a) Cladogram showing the phylogenetic placement of the Cladocorynidae in the super-
family Zancleida and (b) ML tree showing the phylogenetic relationships among the cladocorynid species, based on the concatenated multi-locus
dataset. (c) Species tree of the cladocorynid species. Numbers at nodes in (a) and (b) represent bootstrap values of MP and ML analyses, and
Bayesian posterior probabilities of BI analyses, respectively, for the multi-locus analyses, with asterisks indicating maximal nodal support for all
analyses; nodal supports obtained with single-locus analyses are shown in panels, coloured as coded in the legend. In (c) numbers only represent
Bayesian posterior probabilities. In (b) clades are highlighted with different colours and alphanumeric codes; sampling localities, as coded in the
legend, and hosts are also reported for each terminal taxon.
D. Maggioni et al. / Cladistics 0 (2021) 1–25 7
and a reasonable decrease in the intra-clade distances
was observed by grouping sequences in the five clades
obtained with phylogenetic analyses, with no overlap
with intra-clade distances (Table 4; Table S4).
Single-locus DNA-based species delimitation analyses
of P. krempfi showed variable results, according to the
method used and DNA region analyzed (Fig. 3a), and
resulted in five possible scenarios, spanning one to five
species hypotheses (Fig. 3b). According to phylogenetic
and genetic distance analyses, the delimitation based on
mitochondrial regions revealed the highest number of
species hypotheses, whereas those based on nuclear
regions mostly retrieved three species hypotheses
(Fig. 3a). The BPP multi-locus delimitation analyses
consistently inferred a five-species model with high pos-
terior support (always >0.99), regardless of the
algorithm and sets of priors used. Consistently, the pos-
terior probabilities for each of the five delimited species
hypotheses were always high (>0.99) (Table S5).
Diagnostic characters were searched in P. krempfi
clades considering the three-species model and the five-
species model (Table S6). In the three-species model,
diagnostic characters were detected for all clades and
molecular markers, with highest numbers in the mito-
chondrial COX1 and COX3 genes and the ITS. On
the other hand, in the five-species model diagnostic
characters were found for all clades only in the 16S
rRNA and COX3 genes. Overall, clade III consistently
showed the highest number of diagnostic characters in
all DNA regions.
A species tree of the Cladocorynidae family was pro-
duced considering P. krempfi as composed of five
Table 2
Host range of Pteroclava krempfi
Host Distribution Clade
fam. Alcyoniidae
Cladiella Vietnam*unknown*
Lobophytum Maldives Ia
Rhytisma Red Sea (Egypt, Saudi Arabia) Ia
Sinularia Maldives, Red Sea (Saudi Arabia) Ia, Ib
Sarcophyton Australia, Maldives, Red Sea (Israel, Saudi Arabia) Ia, Ib
fam. Acanthogorgiidae
Acanthogorgia Australia IIb
fam. Ellisellidae
Ctenocella Singapore IIa
fam. Gorgoniidae
Antillogorgia Caribbean (Panama, Sint Eustatius) III
fam. Melithaeidae
Melithaea Mozambique, Red Sea (Saudi Arabia) IIa
fam. Nephtheidae
Dendronephthya Mozambique IIa
Paralemnalia Red Sea (Saudi Arabia) Ia
fam. Plexauridae
Astrogorgia Red Sea (Saudi Arabia), Indonesia*IIa, unknown*
Paraplexaura Maldives IIa
Plexaurella Caribbean (Cuba)*unknown*
*Data from literature.
Table 3
Summary of sequencing results for each species/clade, showing the % of successfully sequenced specimens for each DNA region
Species/Clade (No. of specimens) 16S COX1 COX3 18S 28S ITS H3 Conc
Cladocoryne floccosa (n=7) 100 0 100 100 100 43 86 0
Cladocoryne haddoni (n=7) 100 100 100 100 100 100 86 86
Pseudozanclea timida (n=6) 100 100 100 100 100 100 100 100
Pteroclava krempfi Ia (n=28) 100 100 100 100 100 100 100 100
Pteroclava krempfi Ib (n=4) 100 100 100 100 100 100 100 100
Pteroclava krempfi IIa (n=23) 100 100 100 100 100 100 100 100
Pteroclava krempfi IIb (n=1) 100 0 100 0 100 100 100 0
Pteroclava krempfi III (n=3) 100 67 67 100 100 100 33 0
Outgroups (n=12) 100 83 92 100 100 92 75 58
Total (n=91) 100 88 95 99 100 95 92 78
The column ’Conc’ refers to the % of terminals with all markers sequenced in the concatenated dataset
8D. Maggioni et al. / Cladistics 0 (2021) 1–25
species (Fig. 2c). The topology of the species tree was
identical to that of the concatenated multi-locus trees,
although an overall lower support was obtained
(Fig. 2b, c). Specifically, the relationships among Pte-
roclava clades did not vary, Pseudozanclea timida was
confirmed as the sister group of Pteroclava, and Clado-
coryne as the sister group of Pteroclava +Pseudozan-
clea.
According to the ancestral state reconstructions, the
organisation of euryteles in clusters or rings in the
polyp stage (Fig. 4a) and the presence of exumbrellar
cnidocyst pouches with euryteles in the medusa
(Fig. 4b) resulted to likely be synapomorphies of the
Cladocorynidae, being appeared in the most recent
common ancestor (MRCA) of the extant family mem-
bers. The establishment of symbiotic relationships
between the polyp stage and other benthic organisms
used as substrates emerged multiple times in the super-
family Zancleida (Fig. 4c), but the association with
octocorals probably emerged in the MRCA of the Pte-
roclava +Pseudozanclea clade (Fig. 4d).
Distribution, host specificity, and geographic structure
of Pteroclava clades
Both Pteroclava krempfi clades I and II were found
across the Indo-Pacific and Red Sea, whereas clade III
was exclusively observed in Caribbean localities and
associated with Antillogorgia. Clade I was associated
with members of the octocoral family Alcyoniidae
(Sinularia,Sarcophyton,Lobophytum,Rhytisma), with
the only exception of a colony associated with Par-
alemnalia (family Nephtheidae) (Table 2). Clade Ib
included four colonies collected in the Red Sea and
Australia, associated with Sarcophyton and Sinularia
octocorals, whereas the remaining specimens belonged
to clade Ia. Within clade I, no clear host-related
genetic structure was observed (Fig 4a), with two hap-
lotypes associated with multiple hosts, whereas popu-
lations from the Indian Ocean and Red Sea appeared
to be moderately differentiated, with the exception of
a Red Sea haplotype more related to Indian Ocean
haplotypes (Fig. 5b). Clade II was composed of clade
IIa, associated with the genera Astrogorgia and Para-
plexaura (Plexauridae), Melithaea (Melithaeidae),
Ctenocella (Ellisellidae) and Dendronephthya (Neph-
theidae), and clade IIb, represented by a single colony
associated with Acanthogorgia (Acanthogorgiidae)
(Table 2). In clade II no host-related genetic structure
was also clearly detectable, and the most common
haplotype was associated with two different octocoral
genera (Fig. 5c). Clade II showed a lower number of
haplotypes compared to clade I and in this case differ-
ent localities did not share the same haplotypes, even
if a clear geographic pattern was not observed
(Fig. 5d).
Table 4
16S rRNA genetic distances shown as % uncorrected p-distance (standard deviation) among and within Pteroclava krempfi clades
Pt. krempfi Ia 1.0 (0.2)
Pt. krempfi Ib 4.4 (0.7) 1.0 (0.3)
Pt. krempfi IIa 6.9 (0.9) 6.8 (0.9) 1.1 (0.2)
Pt. krempfi IIb 7.5 (1.0) 7.2 (1.0) 4.3 (0.7) n.c.
Pt. krempfi III 8.8 (1.3) 9.3 (1.3) 9.3 (1.3) 9.0 (1.3) 1.8 (0.5)
1sp
Ia
Ib
I aI
I bI
III
2sp
4sp
5sp
3sp
(b)
16S
COX1*
COX3
18S*
28S
ITS
H3*
5
5
4
3
3
3
3
4
4
4
3
3
3
3
5
4
4
2
3
2
3
3
1
1
ABGD
ASAP
mtPTP
stGMYC
bGMYC
(a)
5
4
5
1
4
5
5
3
3
3
3
Fig. 3. Single-locus species delimitation summary of Pteroclava krempfi. (a) Number of species hypotheses obtained analysing different DNA
regions and using different methods. (b) Five possible scenarios, ranging from one to five species within P. krempfi, as obtained with species
delimitation analyses. *Clade IIb not sequenced.
D. Maggioni et al. / Cladistics 0 (2021) 1–25 9
Morphometry of the Pteroclava polyp cnidome
The cnidome of P. krempfi polyps was composed of
stenoteles of two size classes (small and large) located
in the tentacles and macrobasic apotrichous euryteles
below the hypostome. A total of 5816 capsules were
measured (Table S7), and the raw data reflected simi-
larities in cnidocyst size among the three investigated
clades (Ia, IIa, and III), as shown in Table S8. The
eurytele length of clade III was slightly larger than Ia
and IIa, even if the ranges of the three clades were
partially overlapping. Furthermore, eurytele width was
slightly larger in clade III. All other measurements
were not noticeably different among the investigated
clades.
The distributions of cnidocyst length for each cnido-
cyst type and clade are reported in the density plots in
Fig. 5a–c, showing the general overlapping of the
length distributions and the higher values for eurytele
length in clade III (Fig. 6a). Fig. 5d–f show the strati-
fied box plots of the length measurements for each
cnidocyst type, clade, host, and locality investigated.
The larger size of euryteles of clade III can be
observed in Fig. 6d, whereas clade Ia from the Red
Sea showed the smallest size. The size of both large
and small stenoteles appeared to be very similar
Odessia maeotica
Pteroclava krempfi
Cladocoryne floccosa
Zanclea gallii
Asyncoryne ryniensis
Pennaria disticha
Sphaerocoryne sp.
Cladocoryne haddoni
Zanclea divergens
Pseudozanclea timida
Solanderia secunda
Heterocoryne caribbensis
Millepora alcicornis
Absent
Euryteles
Stenoteles
Exumbrellar nematocyst pouches (medusa)
Odessia maeotica
Pteroclava krempfi
Cladocoryne floccosa
Zanclea gallii
Asyncoryne ryniensis
Pennaria disticha
Sphaerocoryne sp.
Cladocoryne haddoni
Zanclea divergens
Pseudozanclea timida
Solanderia secunda
Heterocoryne caribbensis
Millepora alcicornis
Generalist
Symbiotic
Substrate (polyp)
Odessia maeotica
Pteroclava krempfi
Cladocoryne floccosa
Zanclea gallii
Asyncoryne ryniensis
Pennaria disticha
Sphaerocoryne sp.
Cladocoryne haddoni
Zanclea divergens
Pseudozanclea timida
Solanderia secunda
Heterocoryne caribbensis
Millepora alcicornis
Not organised
Clusters or rings
Euryteles organisation (polyp)
Odessia maeotica
Pteroclava krempfi
Cladocoryne floccosa
Zanclea gallii
Asyncoryne ryniensis
Pennaria disticha
Sphaerocoryne sp.
Cladocoryne haddoni
Zanclea divergens
Pseudozanclea timida
Solanderia secunda
Heterocoryne caribbensis
Millepora alcicornis
None
Octocorals
Host (polyp)
Sponges
Bryozoans
Scleractinians
(a) (b)
(c) (d)
Fig. 4. Stochastic character maps showing the ancestral state reconstruction of the characters (a) eurytele organization in polyps, (b) exumbrellar
nematocyst pouches in medusae, (c) substrate on which polyp colonies grow, (d) host of polyp colonies. Pie charts represents the posterior prob-
ability of each node being in each state.
10 D. Maggioni et al. / Cladistics 0 (2021) 1–25
among the three clades from different hosts and locali-
ties (Fig. 5e, f), with the largest values observed in
specimens belonging to clade IIa associated with
Melithaea in the Red Sea.
The best model produced by model selection was:
Cnidocyst sizes ~Clade +(1|Polyp) +(1|Colony) +e.
For all cnidocyst types, both ‘Polyp’ and ‘Colony’
were significant, whereas ‘Locality’ and ‘Host’ were
not (Table S9). For euryteles and large stenoteles, a
LMM was fitted according to the normality of the
residuals (P=0.4805 and P=0.2207, respectively;
a=0.05) (Fig. S2) from the best models. For small
stenoteles, the residuals of the best LMM did not fit
the normal distribution (P=0.001; a=0.05), there-
fore a GLMM was fitted. The best models for eurytele
and stenotele large (LMMs), and stenotele small
(GLMM) were significant versus the null models
(Table 5). Estimates and confidence intervals (CIs) of
the models for each clade are shown in Table 6, and
the standard deviation of the random effects ‘Polyp’
and ‘Colony’ for each cnidocyst type are shown in
Table S10.
Euryteles of the clade III showed an estimated value
for cnidocyst length larger than Ia and IIa. Moreover,
the CIs for clade III did not overlap with the CIs of
clade Ia, and only partially overlapped with the CIs of
clade IIb. On the other hand, estimates for clade Ia
and Ib were very similar, with almost totally
overlapping CIs (Table 6). Although both ‘Polyp’ and
‘Colony’ as random effects were significant, their stan-
dard deviations were small, around 1 µm and 2 µm,
respectively (Table S10), suggesting no considerable
variation within polyps and colonies. The model esti-
mates for both small and large stenoteles were almost
identical for the three clades, with completely overlap-
ping CIs and very small standard deviations of ‘Polyp’
and ‘Colony’ (Table 6; Table S10). Thus, no differ-
ences were detected in the length of stenoteles among
clades.
Taxonomic account
Acronyms for the museums hosting cladocorynid
material are as follows: Museo Civico di Storia Natu-
rale, Cnidaria Collection, Milano (MSNMCOE), Nat-
ural History Museum, London (NHMUK), Mus
eum
National d’Histoire Naturelle, Paris (MNHN), Aus-
tralian Museum (AU), Museo di Storia Naturale Gia-
como Doria, Genova (MSNG).
Hydrozoa Owen, 1843
Capitata K€
uhn, 1913
Cladocorynidae Allman, 1872
Amended diagnosis: Stem simple or slightly
branched, arising from a creeping hydrorhiza. Oral
tentacles capitate or moniliform, aboral tentacles
10
1
Ib
IIb IIb
Ib
Clade I - Host Clade I - Locality
Clade II - Host Clade II - Locality
(a) (b)
(c) (d)
Ia Ia
IIa IIa
Sinularia
Rhytisma
Sarcophyton
Lobophytum
Paralemnalia
HOST:
Ctenocella
Melithaea
Dendronephthya
Paraplexaura
Astrogorgia
Red Sea
Indian Ocean
Pacific Ocean
LOCALITY:
Acanthogorgia
ALCYONIIDAE
NEPHTHEIDAE
ELLISELLIDAE
PLEXAURIDAE
MELITHAEIDAE
ACANTHOGORGIIDAE
NEPHTHEIDAE
Clade I
Clade II
Fig. 5. 16S rRNA most parsimonious median-joining haplotype networks of Pteroclava krempfi (a, b) clade I and (c, d) clade II. Haplotypes are
coloured by (a, c) host and (b, d) sampling locality for both clades, as shown in the legend, and sub-clades are delimited by dashed lines. Each
cross-bar represents a single nucleotide change, and small black circles represent missing haplotypes.
D. Maggioni et al. / Cladistics 0 (2021) 1–25 11
moniliform, capitate, or branched capitate. Euryteles
organized in patches or in a ring. Fixed cryptomedu-
soids or free-living medusae with exumbrellar pouches
containing euryteles.
Cladocoryne Rotch, 1871
Type species: Cladocoryne floccosa Rotch, 1871
Diagnosis: Stem long arising from a creeping hydro-
rhiza covered by a perisarc. Polyps with oral whorl of
short capitate tentacles, and one to four aboral whorls
of branched-capitate tentacles. Euryteles organized in
patches on hydranth body. Fixed cryptomedusoids.
Cladocoryne floccosa Rotch, 1871
See Schuchert (2006) for a complete synonymy.
Type locality: Herm, Channel Islands, United King-
dom
Type material: Holotype could not be located.
Newly deposited material: Colony collected in
Corfu, Greece, 2 August 2018, fixed in formalin 10%
(MSNMCOE348) and ethanol 99% (MSNMCOE349),
DNA name GR003.
Description: Colonies growing on rock (Fig. 7a).
Unbranched stems, sparingly annulated at some points
45
0.0
0.2
0.4
0.6
678
Stenotele small length (μm)
Density Density
Eurytele length (μm)
8910
11 12 13
0.0
0.2
0.4
0.6
Stenotele large length (μm)
Density
25 30 35 40 45
0.00
0.05
0.25
0.20
0.15
0.10
Clade Ia Clade IIa Clade III
(a)
(b)
(c)
Eurytele length (μm)
MA PA SA SG EUX
Clade Ia
MA PA SA SG EUX
Clade IIa
MA PA SA SG EUX
Clade III
25
30
35
40
(d)
Locality
Stenotele large length (μm)
(e)
Locality
Stenotele small length (μm)
(f)
MA PA SA SG EUX MA PA SA SG EUX MA PA SA SG EUX
8
9
10
12
11
Locality
EUX
4
5
6
7
MA PA SA SG MA PA SA SG EUX MA PA SA SG EUX
Sinularia
Rhytisma
Sarcophyton
Lobophytum
Ctenocella
Melithaea
Astrogorgia
Paraplexaura
Antillogorgia
MA:
PA:
SA:
SG:
EUX:
Maldives
Panama
Saudi Arabia
Singapore
Sint Eustatius
Clade Ia Clade IIa Clade III
Clade Ia Clade IIa Clade III
Fig. 6. Kernel density plots of the (a) euryteles, (b) large stenoteles, and (c) small stenoteles length for each Pteroclava krempfi clade (Ia, IIa,
III). Stratified box plots for the length of (d) euryteles, (e) large stenoteles, and (f) small stenoteles, for each P. krempfi investigated clade, local-
ity, and host.
12 D. Maggioni et al. / Cladistics 0 (2021) 1–25
(Fig. 7b), up to 0.5 cm long and 140–200 µm wide,
arising from creeping hydrorhiza. Perisarc stopping at
the base of polyp. Polyps (Fig. 6c, d) up to 1.5 mm
long with an oral whorl of 4–5 adnate capitate tenta-
cles, up to 250 µm long, and three whorls of 4–6 rami-
fied tentacles, up to 1.1 mm long, with up to 21
capitula disposed spirally and usually with three termi-
nal capitula (Fig. 6e, f). Oral capitula larger than abo-
ral capitula (~90 µm and ~55 µm wide, respectively),
both with stenoteles of two size classes, with large
stenoteles rare in aboral capitula. Up to five patches
of macrobasic apotrichous euryteles among oral tenta-
cles, up to three among aboral tentacles in the median
whorl (not always present), and up to four among
aboral tentacles in the proximal whorl. Polyps bearing
up to four immature cryptomedusoids among the dis-
tal and median whorls of aboral tentacles (Fig. 7c), up
to 0.35 mm long and 0.29 mm wide. Young cryptome-
dusoids without canals and bulbs, but with two exum-
brellar pouches with euryteles (Fig. 7g). Living polyps
transparent, sometimes with a reddish gastroderm and
a white hypostome (Fig. 7a), living cryptomedusoids
transparent with reddish manubrium.
Cnidome: Small stenoteles in capitula (5–794–
6lm) (Fig. 7h). Large stenoteles common in oral
capitula and rare in aboral capitula (13–17 911–
14 lm) (Fig. 7i). Macrobasic apotrichous euryteles in
patches among oral and aboral tentacles and in
pouches on the exumbrella (28–35 913–19 lm; dis-
charged shaft: 190–200 lm) (Fig. 7j). All cnidocyst
types found also in the stem and hydrorhiza and rarely
in the hydranth.
Cladocoryne haddoni Kirkpatrick, 1890
See Schuchert (2003) for a complete synonymy.
Type locality: Murray Island, Torres Strait, Aus-
tralia
Type material: Holotype –1890.11.22.15-20
(NHMUK).
Newly deposited material: Colony collected in Cen-
tral Maldives, 10 October 2015, fixed in formalin 10%
(MSNMCOE346) and ethanol 99% (MSNMCOE347),
DNA name MA200.
Description: Colonies growing on a variety of sub-
strates, including rock, coralline algae, ascidians, and
sponges (Fig. 8a). Unbranched stems up to 1 cm long
and 185–195 lm wide, arising from creeping hydro-
rhiza. Perisarc stopping at the base of polyp. Polyps
(Fig. 7b, c) up to 1.8 mm long with an oral whorl of
6–7 adnate capitate tentacles, up to 350 µm long, and
two whorls of 5–8 ramified tentacles up to 1.2 mm
long (Fig. 8d), with up to 30 capitula disposed spirally
and usually with three terminal capitula (Fig. 8e). Oral
capitula larger than aboral capitula (~90 µmand
~50 µm wide, respectively), both with stenoteles of two
size classes, with large stenoteles rare in aboral capit-
ula. Up to five patches of large macrobasic apotric-
hous euryteles in the area between oral and aboral
tentacles (Fig. 8c). Smaller macrobasic apotrichous
euryteles at the base or along some of the aboral ten-
tacles. Polyps bearing up to two cryptomedusoids
(only males observed), below eurytele patches and
above aboral tentacles, up to 0.9 mm long and
0.6 mm wide (Fig. 8f). Cryptomedusoids with four
rudimentary perradial canals ending in reduced tentac-
ular bulbs with no tentacles, a rudimentary circular
canal and pouches with large euryteles between bulbs
(Fig. 8g). Manubrium large, bearing a white mass of
Table 5
ANOVA for the best models versus null models for each Pteroclava krempfi cnidocyst type
Model npar AIC logLik deviance Chisq Df Pr(>Chisq)
Eurytele length–Species
♦
4 6232.1 3112.1 6224.1
Eurytele length–Species +(1|Polyp) +(1|Colony)
▲
6 4916.9 2452.4 4904.9 1319.2 2 <2.2E-016***
Stenotele large length–Species
♦
4 4919.7 2455.9 4911.7
Stenotele large length–Species +(1|Polyp) +(1|Colony)
▲
6 4206.6 2097.3 4194.6 717.16 2 <2.2E-016***
Stenotele small length–Species
◊
4 3995.7 1993.9 3987.7
Stenotele small length–Species +(1|Polyp) +(1|Colony)
►
6 3258.7 1623.3 3246.7 741.04 2 <2.2E-016***
AIC, Akaike Information Criterion for the model evaluated as - *(logLik -npar); logLik, log-likelihood for the model; npar, number of
parameters; ♦null LMM; ▲best LMM; ◊null GLMM; ▶best GLMM.
***Significance level: 0.001.
Table 6
Pteroclava krempfi cnidocyst length (µm) estimates and confidence
intervals (CIs) from the models for each cnidocyst type and clade
Cnidocyst type/Clade Estimate Lower-95 Upper-95
Eurytele (n=1335)
Ia 32.45985 30.510483 34.409216
IIa 34.107214 29.399497 38.814931
III 40.025497 35.307838 44.743154
Stenotele large (n=2248)
Ia 9.8692909 9.5357731 10.20280875
IIa 10.069944 9.2647662 10.87512181
III 9.4704293 8.6652515 10.27560714
Stenotele small (n=2233)
Ia 5.7324277 5.4382122 6.0602992
IIa 5.5764185 4.9518472 6.3812832
III 5.6185172 4.983667 6.4387212
D. Maggioni et al. / Cladistics 0 (2021) 1–25 13
(a) (b)
(d) (e)
(f)
(j)(g) (h)
(i)
(c)
14 D. Maggioni et al. / Cladistics 0 (2021) 1–25
sperm. Stenoteles of two size classes and small euryte-
les sparingly scattered on the exumbrella. Living
polyps transparent, with a reddish gastroderm and a
white hypostome (Fig. 8a, b, f), cryptomedusoids
transparent with reddish manubrium (Fig. 8f).
Cnidome: Small stenoteles in capitula and exum-
brella (5–693–5lm) (Fig. 8h). Large stenoteles com-
mon in oral capitula and rare in aboral capitula, and
on exumbrella (10–14 99–11 lm) (Fig. 8i). Large
macrobasic apotrichous euryteles in patches below oral
tentacles and in pouches on the exumbrella (61–
72 922–27 lm; discharged shaft: ~1.4 mm) (Fig. 8j).
Small macrobasic apotrichous euryteles in tentacles
and exumbrella (24–29 916–20 lm; discharged shaft:
~150 lm) (Fig. 8k). All cnidocyst types found also in
the stem and hydrorhiza and rarely in the hydranth.
Pteroclava Weill, 1931
Type species: Pteroclava krempfi (Billard, 1919)
Diagnosis: Colonies growing on octocorals, with
perisarc-covered hydrorhiza embedded in host tissues.
Polyps with oral and aboral moniliform tentacles.
Euryteles organized in patches on hydranth body.
Medusae with pouches containing euryteles above
atentaculate bulbs.
Pteroclava krempfi (Billard, 1919)
See Boero et al., (1995) and Puce et al., (2008) for a
complete synonymy.
Type locality: Nha-Trang Bay, Vietnam.
Type material: Holotype –Type H.L. 020 (MNHN).
Newly deposited material: Colony collected in Cen-
tral Red Sea, associated with Sinularia sp., 1 May
2017, fixed in formalin 10% (MSNMCOE350) and
ethanol 99% (MSNMCOE351), DNA name FB047
(clade Ia); Colony collected in the Great Barrier Reef,
Australia, associated with Sarcophyton sp., 13 Novem-
ber 2018, fixed in ethanol 99% (G18585; AM), DNA
name AU008 (clade Ib); Colony collected in Central
Maldives, associated with Paraplexaura sp., 13 April
2016, fixed in formalin 10% (MSNMCOE352) and
ethanol 99% (MSNMCOE353), DNA name MA234
(clade IIa); Colony collected in the Great Barrier Reef,
Australia, associated with Acanthogorgia sp., 14
November 2018, fixed in ethanol 99% (G18678; AM),
DNA name AU006 (clade IIb); Colony collected in
Caribbean Panama, associated with Antillogorgia
americana, 18 August 2015, fixed in formalin 10%
(MSNMCOE354) and ethanol 99% (MSNMCOE355),
DNA name BT006 (clade III).
Description: Colonies living as partial endosym-
bionts of a variety of octocorals (Table 2, Fig. S3).
Unbranched stems (up to 1.5 mm long) arising from
creeping hydrorhiza embedded in host tissues
(Fig. 9a). Perisarc stopping at the base of polyps
(Fig. 9b). Polyps claviform up to 2 mm high, with 4–5
oral (up to 0.5 mm long) and 6–24 aboral (up to
1 mm long) moniliform tentacles scattered on the
hydranth (Fig. 8a, b) with stenoteles of two size
classes. Up to four rounded patches of macrobasic
apotrichous euryteles below the oral tentacles, some-
times absent. Up to five medusa buds at the base or
half of the polyp body, polyps often showing repro-
ductive exhaustion (Fig. 9c). Newly liberated medusa
(Fig. 9d) with a bell-shaped umbrella (up to 800 lm
high and 850 lm wide), manubrium extending for 1/3
or 1/2 of the umbrella, and microbasic euryteles scat-
tered on the exumbrella. Four perradial canals ending
in two tentaculate bulbs and two smaller atentaculate
bulbs. Atentaculate bulbs with an exumbrellar cnido-
cyst pouch containing up to four euryteles (Fig. 9e),
the latter often found also in tentaculate bulbs. Ten-
tacular bulbs bearing one tentacle each (Fig. 9f), about
1 mm long, with up to 40 ciliated rounded cnido-
phores approximately 25 lm wide, armed with 4–5
bean-shaped macrobasic apotrichous euryteles. Living
polyps transparent, with a whitish hypostome and
sometimes a reddish gastroderm (Fig. 9a), medusae
transparent, with whitish or reddish manubrium.
Cnidome: Small stenoteles (4–893–7lm) and large
stenoteles (8–13 97–11 lm) in capitula (Fig. 8g, h).
Macrobasic apotrichous euryteles in patches below
oral tentacles, in exumbrellar pouches, and tentaculate
bulbs (24–44 911–21 lm; discharged shaft: ~500)
(Fig. 8e, i). Bean-shaped macrobasic apotrichous eury-
teles in cnidophores (6–10 94–6lm; discharged shaft:
91–98 lm) (Fig. 9j). Microbasic euryteles on exum-
brella (6–995–6lm; discharged shaft: 15–16 lm)
(Fig. 9k).
Pseudozanclea gen. nov. Maggioni & Montano
http://zoobank.org/ urn:lsid:zoobank.org:
act:12328071-CED1-4765-9745-D114F3F8D33E
Type species: Pseudozanclea timida (Puce, Di
Camillo and Bavestrello, 2008)
Diagnosis: Colonies growing on octocorals, with
perisarc-free hydrorhiza embedded in host tissues.
Polyps with oral and aboral capitate tentacles. Euryte-
les organized in a ring on hydranth body. Medusa
Fig. 7. Cladocoryne floccosa. (a) Living colony. (b) Proximal portion of the stem showing annulations (arrowhead). (c) Polyp (formalin-fixed)
with four immature cryptomedusoids. (d) Same polyp under a compound microscope. (e) Aboral branching tentacle with ramifications arranged
spirally. (f) Terminal ramifications of an aboral tentacle. (g) Close-up of two immature cryptomedusoids showing clusters of euryteles (arrow-
head). (h) Small stenoteles. (i) Large stenoteles. (j) Macrobasic apotrichous euryteles. Scale bars: (a) 1 mm; (b) 150 lm; (c, d) 350 lm; (e–g)
50 lm; (h–j) 5 lm.
D. Maggioni et al. / Cladistics 0 (2021) 1–25 15
(a) (b) (c)
(d) (e)
(g)
(i)
(h)
(k)
(j)
(f)
16 D. Maggioni et al. / Cladistics 0 (2021) 1–25
buds in tissue pockets until release. Medusae with
pouches containing euryteles above atentaculate bulbs.
Etymology: The generic name refers to the similarity
of the polyp stage to Zanclea polyps, and to the fact
that the type species was previously ascribed to Zan-
clea genus.
Remarks: The establishment of the new genus is
supported by both morphological and genetic data.
Indeed, the polyp stage of Pseudozanclea differs from
Pteroclava and Cladocoryne by showing a Zanclea-like
morphology (i.e., hydranths with oral and aboral capi-
tate tentacles) and the presence of peculiar tissue pock-
ets containing medusa buds. Similarities with
Pteroclava are observed in the newly released medusa,
with exumbrellar pouches containing euryteles, and in
the association with octocorals. These latter similarities
are also reflected in the proposed phylogeny of the
Cladocorynidae, with a sister group relationship
between Pteroclava and Pseudozanclea.
Pseudozanclea timida comb. nov. (Puce, Di Camillo
and Bavestrello, 2008)
Synonymy: Zanclea timida Puce et al., 2008: 1649,
figs. 5c-e, 7; Maggioni et al., 2020a: 1, figs. 1d-f
Type locality: Siladen, North Sulawesi, Indonesia
Type material: Holotype –MSNG54191.
Newly deposited material: Colony collected in Cen-
tral Maldives, associated with Bebryce cf. grandicalyx,
4 April 2016, fixed in formalin 10% (MSNMCOE344)
and ethanol 99% (MSNMCOE345), and medusae
released from the same colony fixed in formalin 10%
(MSNMCOE343), DNA name MA243.
Description: Maldivian colonies associated with the
octocoral Bebryce cf. grandicalyx and with a sponge in
the family Suberitidae (Fig. 10a). Indonesian colonies
associated with the octocoral Paratelesto sp. Perisarc-
free hydrorhiza with clusters of macrobasic apotric-
hous euryteles (Fig. 10b), extending on the octocoral
surface and completely covered by the sponge, when
present. Polyps piriform, up to 0.9 mm long, highly
contractile (Fig. 9b, c), and partially covered by the
sponge (Fig. 10d). Five or six oral capitate tentacles
up to 200 lm long (capitula ~60 lm wide) and 9–12
aboral tentacles up to 180 lm long and with smaller
capitula (capitula ~45 lm wide). Capitula containing
stenoteles of two size classes. Polyps with a proximal
ring of macrobasic apotrichous euryteles (Fig. 10e)
corresponding to the point where the sponge stops sur-
rounding the polyps, approximately at one-third of the
polyp body. Up to four medusa buds borne basally,
kept inside a tissue pocket protected externally by the
ring of euryteles (Fig. 10e) and covered by the sponge.
Medusa buds exposed only at maturation, before
release. Newly liberated medusa (Fig. 9f, g) with a
bell-shaped umbrella (~750 lm high and ~800 lm
wide), manubrium extending for one-third or half of
the umbrella (Fig. 10g), and microbasic euryteles scat-
tered on the exumbrella. Four perradial canals ending
in two triangular tentaculate bulbs and two smaller
atentaculate bulbs (Fig. 9g, h). Atentaculate bulbs with
an exumbrellar cnidocyst pouch containing up to five
euryteles, and in some cases small-sized stenoteles
(Fig. 10i). Euryteles found also in tentaculate bulbs
(Fig. 10h). Tentacular bubs bearing one tentacle each,
about 700 lm long, with 50–100 rounded ciliated cni-
dophores approximately 30 lm wide, armed with 3–4
bean-shaped macrobasic apotrichous euryteles. Seven
days old medusa with similar size and longer tentacles
(up to 2 mm) equipped with more cnidophores. Living
polyps transparent (Fig. 9c, d), sometimes with a red-
dish gastroderm and living medusae transparent, with
reddish bulbs and a whitish manubrium (Fig. 10f).
Cnidome: Small stenoteles (5–794–5lm) and large
stenoteles (10–13 98–11 lm) in capitula (Fig. 10j).
Macrobasic apotrichous euryteles in a ring at the base
of polyps, in clusters in hydrorhiza, in exumbrellar
pouches, and in bulbs (23–29 915–21 lm; discharged
shaft: 460–500 lm) (Fig. 9i, k, l). Bean-shaped mac-
robasic apotrichous euryteles in cnidophores (6–
894–6lm; discharged shaft: 48–54 lm) (Fig. 10m).
Microbasic euryteles on exumbrella (8–11 96–9lm;
discharged shaft: 19–21 lm) (Fig. 10n).
Discussion
An updated phylogeny of the Cladocorynidae
In this work, a phylogenetic hypothesis of the family
Cladocorynidae was proposed and, according to multi-
ple lines of evidence, the new genus Pseudozanclea was
established to accommodate Zanclea timida. Various
phylogenetic analyses consistently placed Pseudozan-
clea timida as the sister group of Pteroclava and as
phylogenetically distant from other Zanclea species.
The description, for the first time, of the newly
released medusa of P. timida demonstrated that there
are strong similarities between the Pseudozanclea and
Pteroclava medusae, both in the general morphology
Fig. 8. Cladocoryne haddoni. (a) Living colony. (b, c) Polyps showing large euryteles patches below oral tentacles (arrowhead). (d) Polyp showing
the two whorls of aboral tentacles. (e) Branching aboral tentacles showing ramifications spirally arranged with three terminal branches. (f) Polyp
with a mature male cryptomedusoid. (g) Mature male cryptomedusoid detached from parental colony, with euryteles clusters on exumbrella (ar-
rowhead). (h) Small stenoteles. (i) Large stenoteles. (j) Large macrobasic apotrichous eurytele. (k) Small macrobasic apotrichous eurytele. Scale
bars: (a) 1.5 mm; (b–g) 200 lm; (h–k) 5 lm.
D. Maggioni et al. / Cladistics 0 (2021) 1–25 17
(a) (b) (c)
(d) (e)
(g) (i)
(h) (k)
(j)
(f)
18 D. Maggioni et al. / Cladistics 0 (2021) 1–25
and in the unique presence of exumbrellar cnidocyst
pouches containing euryteles. The polyp stage also
shares a similarity with those of other cladocorynid
taxa, which is the presence of macrobasic apotrichous
euryteles organized in a specific pattern. Finally, simi-
larly to Pteroclava,Pseudozanclea is an obligate sym-
biont of octocorals.
The family Cladocorynidae was initially erected to
accommodate the genus Cladocoryne (Allman, 1872),
whereas only later Petersen (1990) and Boero et al.,
(1995) proposed to include the genus Pteroclava. This
conclusion was justified by the synapomorphy repre-
sented by the presence of patches of large macrobasic
euryteles in the polyp stages of both genera. However,
similar patches are found at least in another non-
cladocorynid species, Eudendrium glomeratum Picard,
1952, in which patches can form an irregular band
(Schuchert, 2008). The presence of eurytele patches in
Cladocorynidae and E. glomeratum clearly represents
morphological convergence, with the trait emerging
twice independently in two phylogenetically distant
suborders (Maronna et al., 2016). As demonstrated in
this work, Pseudozanclea timida shows aggregations of
macrobasic euryteles in the polyp stage, even if orga-
nized in a ring rather than patches, and this organiza-
tion may derive from the coalescence of the patches
observed in Pteroclava and Cladocoryne. Interestingly,
specimens from Indonesia and associated with Parate-
lesto possess macrobasic apotrichous mastigophores
instead of euryteles, even if the heteronemes of the two
populations show similarity in shape and overlap in
size. Maldivian heteronemes have an enlargement of
the distal part of the shaft, typical of euryteles, and
similar to that occurring in Pteroclava and Cladoco-
ryne, i.e., with a zig-zag pattern and covered with
spines (see for instance Maggioni et al., 2016: 488,
Fig. 7 and Bouillon et al., 1987: 283: Fig. 7). On the
other hand, the shaft figured by Puce et al. (2008b:
1652: Fig. 7g) appears to be the same diameter for all
its length, a typical feature of mastigophores. This
could be the case, but it is noteworthy that the figured
distal portion of the shaft seems damaged, for instance
with spines not covering the end of the shaft, and this
may have caused an incorrect classification of the
cnidocyst occurring in Indonesian specimens.
Whatever the cnidocyst type found in the ring, the
function may relate to the polyp defence, since P. tim-
ida hydroids are able to retract within their basal cup,
exposing only the cnidocyst ring and the tentacular
capitula (Puce et al., 2008b). Moreover, the immature
medusa buds are kept inside a tissue pocket below the
cnidocyst ring, and the latter may provide protection
to developing medusae. Pseudozanclea timida has sto-
lonal cnidocyst clusters, which have been hypothesized
to protect the perisarc-derived stolons from predators
(Puce et al., 2008b). However, the stolons of Maldi-
vian specimens were always covered by a sponge, con-
trary to those described from Indonesia. Therefore, the
defensive function of stolonal cnidocyst clusters does
not seem to apply for the Maldivian specimens of P.
timida. The associated sponge also covers the proximal
portion of P. timida hydranths, including the medusa
buds, and this may result in a further protection of the
immature medusae before release (Maggioni et al.,
2020a). The sponge was never observed to cover P.
timida polyps above the cnidocyst ring, and the aggre-
gation of euryteles may prevent an excessive over-
growth of the hydroids by the sponge.
Another synapomorphy of the family is the presence
of exumbrellar pouches with eurytele capsules, a pecu-
liarity never found in other hydrozoan species. The
medusae of both Pseudozanclea timida and Pteroclava
krempfi clearly possess this feature on atentaculate
bulbs. Cladocoryne species also show exumbrellar eury-
tele aggregations on their cryptomedusoids. The pres-
ence of these exumbrellar cnidocyst clusters was
confirmed herein for both C. floccosa and C. haddoni,
and are apparently present also in C. minuta (Watson,
2005), even if described as scattered rather than
grouped. Regarding the other two Cladocoryne species,
the reproductive structures of C. travancorensis are
unknown, whereas the cnidome of the C. littoralis
cryptomedusoids was not reported in the original
description (Mammen, 1963). It is therefore likely that
the exumbrellar cnidocyst pouches containing euryteles
are found in all cladocorynid species, but future re-
analysis of the poorly described species will be neces-
sary to address this issue.
The clade Pseudozanclea +Pteroclava is composed
of obligate octocoral symbionts, and this association is
likely to have arisen in the MRCA of the two genera.
The species Pteroclava crassa was described growing
on the hydroid Macrorhynchia philippina Kirchen-
pauer, 1872 (Pictet, 1893), but the information on this
species is very scant and the only difference with P.
krempfi appears to be the host. Therefore, it remains
unclear whether the two Pteroclava species are con-
specific or not. Within the superfamily Zancleida,
Fig. 9. Pteroclava krempfi. (a, b) Polyps showing moniliform tentacles and a perisarc-covered pedicel (arrowheads). (c) Polyp with a mature
medusa bud, showing reproductive exhaustion. (d) Newly released medusa. (e) Exumbrellar cnidocyst pouch with an eurytele. (f) Tentaculate
bulb. (g) Small stenoteles. (h) Large stenotele. (i) Macrobasic apotrichous eurytele. (j) Cnidophores with bean-shaped macrobasic apotrichous
euryteles. (k) Microbasic eurytele. Scale bars: (a–d) 0.2 mm; (e, g–k) 5 lm; (f) 50 lm.
D. Maggioni et al. / Cladistics 0 (2021) 1–25 19
(a) (b) (c)
(d)
(e) (g)
(i)
(h)
(k)(j)
(l) (n)(m)
(f)
20 D. Maggioni et al. / Cladistics 0 (2021) 1–25
another species was described growing on octocorals,
Zanclea cubensis Varela, 2012, described from the Car-
ibbean Sea (Varela, 2012). Zanclea cubensis is very
similar to other zancleid species, having a claviform
hydranth with oral and aboral capitate tentacles, and
stenoteles and euryteles as cnidocysts. The medusa was
not described, and the species was named mostly based
on the combination of the type of euryteles (macroba-
sic holotrichous) and the association with octocorals.
Considering the lack of genetic data and the partial
description of the species, along with the polyphyletic
nature of the family Zancleidae and the genus Zanclea
(Maggioni et al., 2018), the phylogenetic position of Z.
cubensis remains unknown. Therefore, it is currently
not possible to establish how many times the associa-
tion with octocorals evolved in the Zancleida.
Taxonomic uncertainties in the Cladocorynidae
Previous studies suggested the presence of three
cryptic species within Pteroclava krempfi, based on the
analysis of four DNA regions of Maldivian and Carib-
bean samples (Maggioni et al., 2016; Montano et al.,
2017). In the present work, several colonies associated
with multiple hosts and from different localities were
analyzed to better comprehend the genetic structure of
the P. krempfi species complex. Despite the conserved
general morphology across samples, our phylogenetic,
genetic distance, and single- and multi-locus species
delimitation analyses confirmed the presence of highly
divergent genetic lineages. The number of possible spe-
cies hypotheses varied according to the method used
and DNA region analyzed, with mitochondrial and
multi-locus analyses resulting in the highest numbers
of species hypotheses. The higher number of species
found based on mitochondrial regions is concordant
with the higher rates of nucleotide substitution found
in mitochondrial DNA of hydrozoans compared to
other phylogenetically informative nuclear regions
(e.g., Maggioni et al., 2020b, c). It therefore remains
unclear how many possible species exist under the
name P. krempfi, also because clades Ib and IIb are
herein represented by a limited number of samples.
Unfortunately, it is currently not possible to formally
describe the clades due to the lack of P. krempfi mate-
rial suitable for genetic analyses from the type locality
(Vietnam) and associated with the type’s host (Cla-
diella krempfi). Additionally, Cladocoryne floccosa may
be another species complex, given the high intra-
specific genetic distances found in this study for all
mitochondrial and some nuclear regions. Cladocoryne
floccosa has a wide distribution, from tropical to tem-
perate areas (Schuchert, 2006; Gravili et al., 2015),
and only a thorough sampling across the whole geo-
graphic range and an accurate morpho-molecular
assessment would clarify the possible presence of cryp-
tic lineages or species.
The analysis of cnidocyst size data of P. krempfi
revealed partial differences among the genetic clades,
since a clear size variation was found only for euryte-
les, showing a larger size in organisms from the Carib-
bean (clade III). However, no differences were
detected in eurytele size between clade Ia and IIa and
stenotele size among all clades. The statistical treat-
ment of cnidocyst size data was previously used to
search for fine-scale variations in other hydrozoan spe-
cies, revealing in some cases no differences (Wollschla-
ger et al., 2013) or significant differences among
genetic clades (Arrigoni et al., 2018; Manca et al.,
2019; Maggioni et al., 2020c). Similar studies were per-
formed also on sea anemones, in which only some
cnidocysts, coming from particular structures, varied
among morphs (Gonz
alez-Mu~
noz et al., 2017), or did
not vary at all (Gonz
alez-Mu~
noz et al., 2018), showing
a high intraspecific variability (Garese et al., 2016).
The results obtained in this work are therefore in line
with previous works showing limited, but in some
cases still useful, power of discrimination of cnidocyst
size among closely related species or populations. Nev-
ertheless, to present, there is no evidence that can
explain the intraspecific variation on cnidocysts.
The obtained P. krempfi clades showed different
host preferences. Clades Ia and IIb showed overlap-
ping host octocoral genera, and all clade I colonies but
one were found living on genera belonging to the fam-
ily Alcyoniidae. The only exception was a colony
found on Paralemnalia (family Nephtheidae). No host
overlapping was found between clade I and II at the
genus level, with a single exception at the family level
being a clade IIa colony associated with Dendroneph-
thya, another genus of the family Nephtheidae. All
other clade II colonies were associated with several
octocoral genera and families, and no host overlapping
between clades IIa and IIb was observed at both the
genus and family level, even if clade IIb is currently
composed of a single colony. Finally, clade III was
Fig. 10. Pseudozanclea timida. (a) Living colony growing on Bebryce cf. grandicalyx. (b) Polyp with a portion of the hydrorhiza showing an
euryteles cluster (arrowhead). (c, d) Living polyps partially overgrown by a sponge in the family Suberitidae (arrowhead). (e) Medusa buds (ar-
rowhead) surrounded by polyp tissue equipped with euryteles capsules arranged in a ring. (f, g) Newly released medusa. (h) Tentacular bulb with
an eurytele (arrowhead). (i) Exumbrellar cnidocyst pouch with an eurytele and stenoteles. (j) Small stenoteles (white arrowhead) and large
stenoteles (black arrowhead). (k) Macrobasic apotrichous eurytele. (l) Terminal portion of the shaft of the macrobasic apotrichous eurytele. (m)
Cnidophore with bean-shaped macrobasic apotrichous euryteles. (n) Microbasic eurytele. Scale bars: (a) 2 mm; (b, e, h) 100 lm; (c, d, f–g)
0.2 lm; (i–n) 5 lm.
D. Maggioni et al. / Cladistics 0 (2021) 1–25 21
consistently found associated with Antillogorgia (Mon-
tano et al., 2017; Miglietta et al., 2018). Another
P. krempfi record from the Caribbean Sea reports a
colony associated with Plexaurella grisea Kunze, 1916
(Varela and Cabrales Caballero, 2010), suggesting a
wider host range for clade III. Overall, a certain
degree of host specificity can be detected, mostly at the
family level, even if with some exceptions. Host speci-
ficity has recently been demonstrated for several other
coral-reef invertebrates associated with a variety of
benthic organisms, such as parasitic gastropods (Git-
tenberger and Gittenberger, 2011; Gittenberger and
Hoeksema, 2013; Potkamp et al., 2017; Fritts-
Penniman et al., 2020), coral-dwelling barnacles
(Malay and Michonneau, 2014; Tsang et al., 2014),
commensal shrimps (Hork
a et al., 2016), copepods
(Korzhavina et al., 2019), benthic ctenophores (Ala-
maru et al., 2017), coral gall-crabs (Garc
ıa-Hern
andez
et al., 2020), acoel flatworms (Kunihiro et al., 2019),
and hydrozoans (Maggioni et al., 2020b, c). Host-
specificity of associated species can be very weak as
observed in various coral-dwelling copepods living on
mushroom corals (Ivanenko et al., 2018) and in one
serpulid worm living on a wide range of Caribbean
scleractinians (Hoeksema and Hove, 2017). Hence, the
causes (and consequences) of these, more or less speci-
fic, associations largely remain unclear and need fur-
ther research. Additionally, colonies belonging to the
same clade (clade I) displayed variable host preference,
with some Alcyoniidae genera showing higher preva-
lence of the symbiosis, and these prevalences also var-
ied according to the studied locality and reef type
(Montano et al., 2017; Seveso et al., 2020). Therefore,
despite a single clade being associated with multiple
octocoral genera, it is likely that, at least in some
localities, some of the associations are rarer than
others, or even occasional, and that the association is
host-reliant (Montano et al., 2017).
In conclusion, this work sheds new light on the evo-
lution and diversity of the poorly known family Clado-
corynidae, providing a new well-supported
phylogenetic reconstruction of the family, establishing
a new genus, and demonstrating the presence of sev-
eral DNA-based species hypotheses. Moreover, the
new data on the host specificity and geographic distri-
bution of P. krempfi provide valuable insights for
future research on how the current diversity patterns
have evolved and on the study of the nature of the
hydrozoan-octocoral symbioses.
Acknowledgements
The authors thank all the people involved in collect-
ing/providing material or organising sampling cam-
paigns: Peter Schuchert (MHNG, Switzerland), Tullia
Isotta Terraneo (KAUST, Saudi Arabia), Malek Amr
Gusti (KAUST, Saudi Arabia), Timothy Ravasi
(OIST, Japan), the captain and crew of the MV
Dream-Master (Saudi Arabia), the KAUST Coastal
and Marine Resources Core Lab, Inga Dehnert
(UNIMIB, Italy), Nicholas WL Yap (NUS, Singa-
pore), Sudhanshi S Jain (NUS, Singapore), Stephen
Keable (Australian Museum), Penny Berents (Aus-
tralian Museum), Anne Hoggett (Australian Museum),
Lyle Vail (Australian Museum). Additionally, we wish
to thank Leen P. van Ofwegen (Naturalis, The Nether-
lands) for his valuable help in identifying the octocoral
Paralemnalia sp., Peter Schuchert for his comments on
an earlier version of the manuscript, and two anony-
mous referees for their thorough revision of this work.
Permissions relevant to undertake the research have
been obtained from the applicable governmental agen-
cies. Fieldwork at St. Eustatius was funded through a
Martin Fellowship from Naturalis Biodiversity Center
to SM, while logistic support was supplied by St. Eus-
tatius Marine Parks (STENAPA), the Caribbean
Netherlands Science Institute (CNSI) and Scubaqua
Dive Centre. Samples from Eilat (Israel) were collected
during the HyDRa Project funded by the EU FP7
Research Infrastructure Initiative ‘ASSEMBLE’
(Grant #227799) to DP. Financial support to DP for
collecting samples at Lizard Island (Australia) was
provided by the 2018 John and Laurine Proud Fellow-
ship and the Australian Museum’s Lizard Island
Research Station. Fieldwork in Mozambique was con-
ducted during the Green Bubbles financed by EU’s
H2020 research and innovation programme to DP,
under the Marie Sklodowska-Curie grant agreement
no 643712 (Permit n°09/2018 ANAC). Fieldwork in
Singapore was partially funded by the National
Research Foundation, Prime Minister’s Office, Singa-
pore under its Marine Science R&D Programme
(MSRDP-P03) to DH.
Conflict of interest
None declared.
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Supporting Information
Additional supporting information may be found
online in the Supporting Information section at the
end of the article.
Fig. S1. Phylogentic reconstructions based on the
multi-locus dataset according to (a) Maximum parsi-
mony, (b) Maximum likelihood, (c) Bayesian infer-
ence.
Fig. S2. Graphical evaluation of normality of resid-
ual to the fit of a linear model. QQ-plots (left) and dis-
persion diagram plots (right) for each cnidocyst type.
Fig. S3. Overview of the association between Ptero-
clava krempfi and alcyonancean octocorals.
Table S1. Table S1. Information on the specimens
included in the analyses. Newly obtained sequences are
in bold.
Table S2. Partitions and substitution models based
on AIC for the multi-locus dataset.
Table S3. Support values for the clades recovered by
single- and multi-locus analyses, based on maximum
parsimony (MP), maximum likelihood (ML), Bayesian
inference (BI). Highly supported nodes are in green
(MP and ML bootstrap values ≥75, BI posterior
probabilities ≥0.9).
Table S4. Genetic distances shown as % uncorrected
p-distance (standard deviation) among and within
cladocorynid species/clades.
Table S5. Outputs of the A11 BPP analyses based
on different sets of priors and algorithms. Posterior
probabilities (PP) are shown when >0.
Table S6. Molecular diagnostic characters for each
Pteroclava krempfi clade in the 3- and 5- species
hypotheses for (a) 16S rRNA, (b) COX1, (c) COX3,
(d) 18S rRNA, (e) 28S rRNA, (f) ITS, and (g) H3
markers. n.s. not sequenced.
Table S7. Measurements of cnidocyst length and
width in Pteroclava krempfi.
Table S8. Descriptive statistical parameters of each
Pteroclava krempfi cnidocyst type for clades Ia, IIa, III
reported in µm as range (mean SD).
Table S9. Evaluation of the significance of the ran-
dom effects by deleting one by one from the complete
model.
Table S10.Pteroclava krempfi cnidocyst standard
deviation (SD) of random effects from the best mod-
els.
File S1. R scripts used for the LMM and GLMM.
D. Maggioni et al. / Cladistics 0 (2021) 1–25 25
