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Systematics and character evolution of capitate hydrozoans

December 2023
Cladistics 40(2):1-28

December 2023
40(2):1-28

DOI:10.1111/cla.12567

License
CC BY 4.0

Authors:

Davide Maggioni

Università degli Studi di Milano-Bicocca

Peter Schuchert

Natural History Museum of Geneva

Andrew N. Ostrovsky

Saint Petersburg State University & University of Vienna

Andrea Schiavo

Politecnico di Milano

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Capitate hydrozoans are a morphologically and ecologically diverse hydrozoan suborder, currently including about 200 species. Being grouped in two clades, Corynida and Zancleida, these hydrozoans still show a number of taxonomic uncertainties at the species, genus and family levels. Many Capitata species established symbiotic relationships with other benthic organisms, including bryozoans, other cnidarians, molluscs and poriferans, as well as with planktonic dinoflagellates for mixotrophic relationships and with bacteria for thiotrophic ectosymbioses. Our study aimed at providing an updated and comprehensive phylogeny reconstruction of the suborder, at modelling the evolution of selected morphological and ecological characters, and at testing evolutionary relationships between the sym-biotic lifestyle and the other characters, by integrating taxonomic, ecological and evolutionary data. The phylogenetic hypotheses here presented shed light on the evolutionary relationships within Capitata, with most families and genera being recovered as monophyletic. The genus Zanclea and family Zancleidae, however, were divided into four divergent clades, requiring the establishment of the new genus Apatizanclea and the new combinations for species in Zanclea and Halocoryne genera. The ancestral state reconstructions revealed that symbiosis arose multiple times in the evolutionary history of the Capitata, and that homoplasy is a common phenomenon in the group. Correlations were found between the evolution of symbiosis and morphological characters, such as the perisarc. Overall, our results highlighted that the use of genetic data and a complete knowledge of the life cycles are strongly needed to disentangle taxo-nomic and systematic issues in capitate hydrozoans. Finally, the colonization of tropical habitat appears to have influenced the evolution of a symbiotic lifestyle, playing important roles in the evolution of the group.

Morphological diversity of the Zancleida species. (a) Reduced and polymorphic polyps of Halocoryne epizoica associated with Schizobrachiella sanguinea (Tricase, Italy, Mediterranean Sea), and (b) Zanclea pirainoid associated with Robertsonidra sp. (Magoodhoo, Maldives, Indian Ocean). (c) Polyps of Zanclea giancarloi associated with Adeonella sp. (Corfu, Greece, Mediterranean Sea). (d) Millepora dichotoma (Yanbu, Saudi Arabia, Red Sea). (e) Solanderia gracilis (Bocas del Toro, Panama, Caribbean Sea). (f) Porpita porpita (Magoodhoo, Maldives, Indian Ocean). (g) Sphaerocoryne bedoti associated with a sponge (Magoodhoo, Maldives, Indian Ocean), and (h) Pennaria disticha (Daranboodhoo, Maldives, Indian Ocean). Medusa stages of (i) Zanclea sp. (Villefranche-sur-Mer, France, Mediterranean Sea), (j) Pseudozanclea timida (Daranboodhoo, Maldives, Indian Ocean) and (k) Apatizanclea sp. 1 (Magoodhoo, Maldives). Medusoids of (l) Halocoryne epizoica (Tricase, Italy, Mediterranean Sea), and (m) Millepora dichotoma (Thuwal, Saudi Arabia, Red Sea). Medusoids (arrowheads) attached to parental polyps of (n) Cladocoryne haddoni (Bileddhoo, Maldives, Indian Ocean), (o) Solanderia secunda (Thuwal, Saudi Arabia, Red Sea), and (p) Pennaria disticha (Bocas del Toro, Panama, Caribbean Sea).

…

Bayesian phylogenetic hypothesis of the Capitata, obtained from the concatenated dataset and including one specimen per species. Numbers at nodes represent BPP, ML BS, and MP BS, respectively. Asterisks denote maximal support for all analyses, whereas dashes (–) indicate nodes that were not supported in the corresponding analyses. The scale bar indicates the mean number of nucleotide substitutions per site

…

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Systematics and character evolution of capitate hydrozoans

Davide Maggioni*

a,b,c

, Peter Schuchert

, Andrew N. Ostrovsky

e,f

Andrea Schiavo

, Bert W. Hoeksema

h,i

, Daniela Pica

, Stefano Piraino

k,l,m

Roberto Arrigoni

, Davide Seveso

b,c,m

, Enrico Montalbetti

b,c

, Paolo Galli

b,c,m

and Simone Montano

b,c,m

Department of Biotechnology and Biosciences (BtBs), University of Milano-Bicocca, Milan 20126, Italy;

Department of Earth and Environmental

Science (DISAT), University of Milano-Bicocca, Milan 20126, Italy;

Marine Research and Higher Education (MaRHE) Center, University of

Milano-Bicocca, Faafu Magoodhoo Island 12030, Maldives;

Mus

eum d’Histoire Naturelle, Geneva 1208, Switzerland;

Department of Invertebrate

Zoology, Faculty of Biology, Saint Petersburg State University, Saint Petersburg 199034, Russia;

Department of Palaeontology, Faculty of Earth

Sciences, Geography and Astronomy, University of Vienna, Vienna 1090, Austria;

Department of Electronics, Information and Bioengineering,

Polytechnic University of Milan, Milan 20133, Italy;

Marine Evolution and Ecology Group, Naturalis Biodiversity Center, Leiden 2333 CR, The

Netherlands;

Groningen Institute for Evolutionary Life Sciences, University of Groningen, Groningen 9747 AG, The Netherlands;

Department of

Integrative Marine Ecology, Stazione Zoologica Anton Dohrn, Calabria Marine Centre, Amendolara 87071, Italy;

Department of Biological and

Environmental Sciences and Technologies (DiSTeBA), University of Salento, Lecce 73100, Italy;

National Interuniversity Consortium for Marine

Science (CoNISMa), Rome 00196, Italy;

National Biodiversity Future Center (NBFC), Palermo 90133, Italy;

Department of Biology and

Evolution of Marine Organisms (BEOM), Genoa Marine Centre (GMC), Stazione Zoologica Anton Dohrn –National Institute of Marine Biology,

Ecology and Biotechnology, Genoa 16126, Italy

Received 31 May 2023; Revised 6 October 2023; Accepted 19 November 2023

Abstract

Capitate hydrozoans are a morphologically and ecologically diverse hydrozoan suborder, currently including about 200 species.

Being grouped in two clades, Corynida and Zancleida, these hydrozoans still show a number of taxonomic uncertainties at the species,

genus and family levels. Many Capitata species established symbiotic relationships with other benthic organisms, including bryozoans,

other cnidarians, molluscs and poriferans, as well as with planktonic dinoﬂagellates for mixotrophic relationshipsand with bacteria for

thiotrophic ectosymbioses. Our study aimed at providing an updated and comprehensive phylogeny reconstruction of the suborder, at

modelling the evolution of selected morphological and ecological characters, and at testing evolutionary relationships between the sym-

biotic lifestyle and the other characters, by integrating taxonomic, ecological and evolutionary data. The phylogenetic hypotheses here

presented shed light on the evolutionary relationships within Capitata, with most families and genera being recovered as monophyletic.

The genus Zanclea and family Zancleidae, however, were divided into four divergent clades, requiring the establishment of the new

genus Apatizanclea and the new combinations for species in Zanclea and Halocoryne genera. The ancestral state reconstructions

revealed that symbiosis arose multiple times in the evolutionary history of the Capitata, and that homoplasy is a common phenomenon

in the group. Correlations were found between the evolution of symbiosis and morphological characters, such as the perisarc. Overall,

our results highlighted that the use of genetic data and a complete knowledge of the life cycles are strongly needed to disentangle taxo-

nomic and systematic issues in capitate hydrozoans. Finally, the colonization of tropical habitat appears to have inﬂuenced the evolu-

tion of a symbiotic lifestyle, playing important roles in the evolution of the group.

Introduction

The Hydrozoa is one of the six currently accepted

classes of the phylum Cnidaria (Kayal et al., 2018;

WoRMS Editorial Board, 2023), showing an incredible

diversity of taxa, morphologies, life cycles and ecological

preferences (Bouillon et al., 2006). This class has been

estimated to be the second most species-rich within Cni-

daria, after Anthozoa (Appeltans et al., 2012), and new

taxa are continually being added (e.g. Toshino

*Corresponding author:

Email: davide.maggioni@unimib.it

Cladistics

Cladistics (2023) 1–28

doi: 10.1111/cla.12567

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.

et al., 2019; Galea and Maggioni, 2020; Maggioni et al.,

2021;T

€

odter et al., 2023). Yet, part of its diversity is still

unknown (Appeltans et al., 2012), also as a consequence

of the pervasive occurrence of morphologically cryptic

species (Postaire et al., 2016,2017; Maggioni et al.,

2020a,2022a). However, recent broad-scale molecular

studies have started to shed light on the evolutionary

afﬁnities of genera, families, and taxa at a higher level,

clarifying phylogenetic relationships that were unclear

or established solely based on morphology (e.g. Mar-

onna et al., 2016; Munro et al., 2018; Bentlage and

Collins, 2021).

The evolutionary history of some hydrozoan taxa

remains elusive, owing to low sampling effort com-

pared to extant diversity or to ineffective molecular

markers that are not able to disentangle deeper rela-

tionships. An example is the suborder Capitata, cur-

rently containing about 200 accepted extant species

(Schuchert, 2023), and showing several uncertainties at

the species, genus and family levels. This suborder was

also previously thought to include the now separated

group Aplanulata, which was demonstrated to diverge

from Capitata s.s., constituting a different suborder

also including the model organisms Hydra spp. (Col-

lins et al., 2005). Within Capitata, two major clades

were identiﬁed by Nawrocki et al. (2010), namely the

Zancleida and Corynida. On the one hand, the previ-

ous phylogenetic assessments of Capitata mostly

focused on Corynida (Collins et al., 2005; Nawrocki

et al., 2010), which contains two families, Corynidae

Johnston, 1836 and Cladonematidae Gegenbaur, 1857.

Despite several taxonomic and systematic issues being

clariﬁed by Nawrocki et al. (2010), some relationships

are still uncertain and many genera and species are

currently lacking DNA data. Zancleida, on the other

hand, shows a greater family and genus diversity, and

some recent works focused on the diversity and evolu-

tion of certain families or clades (Miglietta et al., 2019;

Maggioni et al., 2021,2022b). A striking example of

taxonomic and systematic uncertainties in Zancleida is

represented by the family Zancleidae Russell, 1953, the

most diverse of the clade, which has been demon-

strated to be polyphyletic and to contain several cryp-

tic species (Maggioni et al., 2018,2020a,2022a). The

taxonomy of this family is challenging for several rea-

sons, including the conserved general morphology, the

presence of poor-quality descriptions, and the limited

knowledge of the life cycles for several species (Mag-

gioni et al., 2018).

Capitata shows a large variation in morphologies of

polyps and medusae, habitats in which they live,

and ecological relationships that they establish. For

instance, regarding the morphology, different types of

polyps and colonies evolved in the group, including sto-

lonal, erect, or even completely pelagic colonies, and

some species also show polymorphic polyps

(Petersen, 1990; Boero et al., 2000; Bouillon

et al., 2006). Most species have a chitinous structure,

the perisarc, covering at least the hydrorhiza, whereas

some Zancleida species completely lost this structure

(Bouillon et al., 2006). The cnidome also is variable

among Capitata groups, and for some taxa it is consid-

ered an important diagnostic character (Gravili

et al., 1996). The reproductive stages show variable

levels of reduction, ranging from completely developed,

freely swimming and feeding medusae, to reduced

medusoids that can be released or not from the parental

polyps, and to sporosacs (Petersen, 1990; Schu-

chert, 2001; Bouillon et al., 2006). The geographical dis-

tribution of Capitata as a whole is circumglobal, from

polar to tropical regions and from shallow to deep

waters (Pe~

na Cantero et al., 2013; Ronowicz

et al., 2013; Montano et al., 2015a; Mastrototaro

et al., 2016), with some species showing wide distribu-

tional ranges, and others only known from restricted

localities (e.g. Maggioni et al., 2017,2021,2022b;

Miglietta et al., 2019).

Another source of variation in the Capitata is repre-

sented by the substrate on which polyps live. Indeed,

many species establish symbiotic relationships with

other benthic organisms, living on or partially embed-

ded in their hosts, which can be algae, crustaceans, bryo-

zoans, molluscs, polychaetes, poriferans, or other

cnidarians such as scleractinian corals and octocorals

(Puce et al., 2008). The host speciﬁcity of these symbi-

otic species is variable, with both generalist and special-

ist taxa (Maggioni et al., 2022a,b), and in some cases

the relationships appear to be very intimate, with the

hosts showing speciﬁc structural modiﬁcations (Puce

et al., 2005; Manca et al., 2019; Maggioni et al., 2020b).

Both mutualistic and parasitic associations are known,

with some species in the genus Zanclea Gegenbaur,

1856, for example, protecting their hosts from predators

(Osman and Haugsness, 1981; Montano et al., 2017)

and at least one Halocoryne species feeding on the loph-

ophores of the host bryozoans (Piraino et al., 1992). In

most cases, the nature of these associations is still

unclear. Additional types of mutualistic symbiotic rela-

tionships are known in capitate hydrozoans: for exam-

ple, Millepora spp. and Velella velella (Linnaeus, 1758)

hosting dinoﬂagellate symbionts in their endodermal

cells (Banaszak et al., 1993; Di Camillo et al., 2017), or

the thiotrophic ectosymbiosis of two sulfur-oxidizing

bacteria attached to the tentacles of Cladonema sp.

hydroids (Abouna et al., 2015).

The establishment of symbiotic relationships has

been hypothesized to have inﬂuenced the evolution of

the involved organisms, in terms of morphological and

behavioural adaptations. Piraino et al. (1992) put for-

ward the hypothesis that, at least in the symbiotic rela-

tionships between hydroids and bryozoans, mutualism

originated from simple epibiosis, and parasitism from

2D. Maggioni et al. / Cladistics 0 (2023) 1–28

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mutualism. Accordingly, Boero et al. (2000) proposed

an evolutionary trend in Zancleidae species, from non-

symbiotic, monomorphic species with a typical Zanclea

morphology in both polyp and medusa, to obligate

symbiotic species with modiﬁed polymorphic polyps, a

medusa that is more or less reduced, and behavioural

integrations with the host. Later, Puce et al. (2002)

pinpointed another interesting trend in zancleid symbi-

otic species related to modiﬁcations of the hydrorhiza.

Speciﬁcally, they hypothesized a shift from generalist

species growing on a variety of substrates, and with a

hydrorhiza protected by the chitinous perisarc, to spe-

cialist species (e.g. associated with bryozoans) with the

hydrorhiza growing inside/being surrounded by the

host skeleton and without protective perisarc as a con-

sequence of the defensive action exerted by the host

(also discussed in Bogdanov et al., 2022). These possi-

ble adaptations to the symbiotic lifestyle also may

have appeared convergently in other hydrozoan groups

(Puce et al., 2008). For instance, polymorphic colonies,

reduced polyps and absence of perisarc covering the

hydrorhiza are known in symbiotic ﬁliferan species

associated with molluscs, crustaceans, polychaetes and

ﬁsh (Hand and Hendrickson, 1950; Boero et al., 1991;

Puce et al., 2004; Miglietta and Cunningham, 2012).

Likewise, all species speciﬁcally associated with the

seagrass Posidonia oceanica (Linnaeus) Delile, 1813

lack the ability to produce free-swimming medusae

(Boero, 1987). Interestingly, Lecl

ere et al. (2009)

showed that the evolution of the life cycles in leptothe-

cate hydrozoans does not follow a phyletic gradualism

model, with, for instance, medusa loss preceded by

medusa reduction in the form of a medusoid. Instead,

the evolution of life cycles is characterized by indepen-

dent events of simpliﬁcation and re-acquisition of the

medusa stage (Boero and Sar

a, 1987; Lecl

ere

et al., 2009), as exempliﬁed by the re-invention of the

medusa in Obelia (P

eron & Lesueur, 1810), with its

peculiar apomorphies including the absence of a

velum, the chordal tentacles and the special orientation

of striated muscle cells (Boero and Sar

a, 1987). This

evolutionary scenario also may apply to the evolution

of other hydrozoan characters.

In order to clarify the occurrence and origin of life-

history traits within Capitata, the present study has

two main aims, namely (i) the clariﬁcation of its phy-

logenetic relationships, and (ii) the assessment of the

evolution of different ecological and morphological

features in the group. In this case, ecological traits

mainly concern symbiotic relationships with other

organisms, as also shown by, for example, corallivor-

ous parasitic snails (Gittenberger and Hoeksema, 2013;

Potkamp et al., 2017), symbiotic palaemonid shrimps

(Hork

a et al., 2016), coral-dwelling barnacles (Dreyer

et al., 2022) and anthozoan zoantharians (Kise

et al., 2023). New genetic data were collected and

analysed together with available DNA sequences to

produce the most comprehensive phylogenetic hypoth-

esis for the suborder. The obtained phylogeny recon-

struction was then used to model the evolution of

selected characters, including the morphological fea-

tures, the symbiotic lifestyle and the habitat type.

Finally, evolutionary relationships between the symbi-

otic lifestyle and the other characters were tested.

Material and methods

Sampling and morphological assessment

Sampling was carried out by snorkelling and SCUBA diving (0–

30 m deep) between October 2014 and December 2019 in various

localities across the Indo-Paciﬁc, Atlantic and Mediterranean Sea

(Table S1), and mostly targeted polyp and medusa stages of the Zan-

cleida (Fig. 1). When hydrozoan polyps were observed, fragments of

the substrate and associated hydroids were collected using hammer

and chisel or a diving knife. Before manipulation, hydroids were

anaesthetized using menthol crystals, photographed, and detached

from the substrate using forceps, syringe needles and micropipettes,

and ﬁxed in 99% ethanol for genetic analyses and in 10% formalin

(=water solution of 4% formaldehyde) for morphological assess-

ments. When possible, polyps were reared in oxygenated bowls to

allow for medusae release, but in a few cases, medusae were directly

collected from the environment.

Specimens were observed and photographed under a EZ4 D ste-

reo microscope (Leica, Wetzlar, Germany) to perform species iden-

tiﬁcation and to describe their general morphology, and under a

Axioskop 40 compound microscope (Zeiss, Oberkochen, Germany)

to characterize ﬁne-scale structures of polyps and medusae and

cnidocysts. Both microscopes were equipped with a PowerShot G7

X Mark II camera (Canon, Tokyo, Japan). Measurements were

performed using the ImageJ 1.52p software (see Schneider

et al., 2012).

When symbiotic species occurred, hosts were identiﬁed to the low-

est taxonomic level possible. In particular, to identify host bryozoan

species, tissues were removed by immersion in a 10% sodium hypo-

chlorite solution for up to 12 h, skeletons were rinsed, air-dried,

sputter-coated with gold, and observed under a Gemini SEM 500

scanning electron microscope (Zeiss, Oberkochen, Germany) (see

Grischenko et al., 2022).

The present work is registered in ZooBank under: http://zoobank.

org/urn:lsid:zoobank.org:pub:3D9D9A6D-183C-4397-8D60-

810E2B6166D2.

DNA extraction, sequencing and dataset assembly

Genomic DNA was extracted from single ethanol-ﬁxed polyps or

medusae following the protocol described in Maggioni et al. (2022a)

and six gene regions were ampliﬁed. Speciﬁcally, portions of the

mitochondrial large ribosomal RNA (16S rRNA), cytochrome coxi-

dase subunit I (COX1), cytochrome coxidase 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) were ampliﬁed using the

primers and protocols described in Maggioni et al. (2020a). Ampliﬁ-

cation success was assessed through 1.5% agarose electrophoretic

runs, PCR products were puriﬁed with Illustra ExoStar (GE Health-

care, Amersham, UK) and ﬁnally sequenced with an ABI 3730xl

DNA Analyser (Applied Biosystems, Carlsbad, CA, USA) in both

D. Maggioni et al. / Cladistics 0 (2023) 1–28 3

Fig. 1. Morphological diversity of the Zancleida species. (a) Reduced and polymorphic polyps of Halocoryne epizoica associated with Schizobra-

chiella sanguinea (Tricase, Italy, Mediterranean Sea), and (b) Zanclea pirainoid associated with Robertsonidra sp. (Magoodhoo, Maldives, Indian

Ocean). (c) Polyps of Zanclea giancarloi associated with Adeonella sp. (Corfu, Greece, Mediterranean Sea). (d) Millepora dichotoma (Yanbu,

Saudi Arabia, Red Sea). (e) Solanderia gracilis (Bocas del Toro, Panama, Caribbean Sea). (f) Porpita porpita (Magoodhoo, Maldives, Indian

Ocean). (g) Sphaerocoryne bedoti associated with a sponge (Magoodhoo, Maldives, Indian Ocean), and (h) Pennaria disticha (Daranboodhoo,

Maldives, Indian Ocean). Medusa stages of (i) Zanclea sp. (Villefranche-sur-Mer, France, Mediterranean Sea), (j) Pseudozanclea timida (Daran-

boodhoo, Maldives, Indian Ocean) and (k) Apatizanclea sp. 1 (Magoodhoo, Maldives). Medusoids of (l) Halocoryne epizoica (Tricase, Italy,

Mediterranean Sea), and (m) Millepora dichotoma (Thuwal, Saudi Arabia, Red Sea). Medusoids (arrowheads) attached to parental polyps of (n)

Cladocoryne haddoni (Bileddhoo, Maldives, Indian Ocean), (o) Solanderia secunda (Thuwal, Saudi Arabia, Red Sea), and (p) Pennaria disticha

(Bocas del Toro, Panama, Caribbean Sea).

4D. Maggioni et al. / Cladistics 0 (2023) 1–28

directions. Geneious 7.1.9 (Biomatters, Auckland, New Zealand) was

used to check, correct and assemble the chromatograms, and to

translate the protein-coding genes (COX1 and COX3) to control for

the presence of stop codons. The obtained consensus sequences were

deposited in GenBank (Table S1).

Additional sequences were downloaded from GenBank, including

all available sequenced specimens for the family Zancleidae and one

specimen for each other Capitata species (Table S1). Even though

many species were represented by only one or a few sequences

obtained from GenBank, they were included in the analyses in order

to obtain the most comprehensive dataset possible in terms of taxo-

nomic diversity at the species, genus and family levels.

Sequences of each DNA region were aligned using MAFFT 7.110

(Katoh and Standley, 2013) with the E-INS-i option, after adding

Eudendrium racemosum (Cavolini, 1785) as outgroup. The 16S, 18S, 28S

and ITS alignments were run through Gblocks (Castresana, 2000; Tala-

vera and Castresana, 2007) using the ”less stringent” settings to remove

ambiguously aligned regions (Table S2). Finally, alignments were

concatenated using Mesquite 3.2 (Maddison and Maddison, 2006).

Initially, all sequences were included in the downstream phyloge-

netic analyses to assess the phylogenetic position of the Zancleidae

species within Capitata, owing to the known polyphyly of the family

(Maggioni et al., 2018). Subsequently, a single specimen per species

was maintained to obtain the presented phylogenetic hypotheses for

the Capitata. For each Zancleidae clade, phylogenetic analyses were

also run separately including all available sequenced specimens and

using Porpita porpita (Linnaeus, 1758), Millepora dichotoma For-

ssk



al, 1775 and Pseudozanclea timida (Puce, Di Camillo & Baves-

trello, 2008) as outgroups.

Phylogenetic analyses

Substitution models and partitions were determined with Partition-

Finder 1.1.1 (Lanfear et al., 2012) using the Akaike information crite-

rion (AIC), resulting in partitioning by gene and codon (for protein-

coding genes) and in the selection of the GTR +I+G for all partitions.

Phylogeny reconstructions were performed using maximum parsimony

(MP), maximum likelihood (ML) and Bayesian inference (BI). MP ana-

lyses were carried out using tree searches performed in TNT 1.6 (Golob-

off, 1999; Nixon, 1999; Goloboff et al., 2008) with 10000 random

addition sequences, each employing 100 cycles of sectorial searches,

ratcheting, drifting and tree fusing. Gaps were treated as missing data.

Resampling was performed with 10000 bootstrap replicates. ML ana-

lyses were run using RAxML 8.2.12 (Stamatakis, 2014) with 1000 non-

parametric bootstrap replicates. BI analyses were conducted using

BEAST 1.8.2 (Drummond et al., 2012), setting a Yule process tree prior

and an uncorrelated lognormal relaxed clock. Three replicate analyses

were run for 10

million generations each, sampling every 10000th tree,

and were combined using LogCombiner 1.8.2 (Drummond et al., 2012)

with a burn-in of 25%, after checking the stationarity for effective sam-

pling size and unimodal posterior distribution using Tracer v.1.6 (Ram-

baut et al., 2014). Maximum clade credibility trees were obtained using

TreeAnnotator 1.8.2 (Drummond et al., 2012). ML and BI analyses

were run on the CIPRES server (Miller et al., 2010). The nodal supports

of all phylogenetic reconstructions were mapped onto the BI tree. We

considered the node support as maximal when bootstrap values for MP

(MP BS) and ML (ML BS) analyses were =100 and Bayesian posterior

probabilities (BPP) were =1, high when BS ≥75 and BPP ≥0.9, and low

when BS <75 and BPP <0.9. Nodes with low support for all the ana-

lyses were collapsed.

Genetic distances within and between Zancleidae species were cal-

culated with MEGA X (Kumar et al., 2018) as % uncorrected p-

distances with 1000 nonparametric bootstrap replicates and were

presented as density plots of intra- and interspeciﬁc genetic distances

for each Zancleidae clade using the ggplot2 package (Wickham, 2009)

in the R environment (R Core Team, 2020).

Phylogenetic comparative methods

The evolution of selected discrete morphological and ecological

traits was assessed performing ancestral state reconstructions on the

Capitata phylogeny using stochastic mapping (Huelsenbeck

et al., 2003). The studied characters (Table S3) were: (i) symbiotic life-

style (presence, absence), (ii) host (generalist, bryozoan, scleractinian

coral, octocoral, sponge, mollusc), (iii) perisarc (presence, absence),

(iv) polymorphism, intended as colonies with gastrozooids and dacty-

lozooids (presence, absence), (v) heteronemes, intended as cnidocysts

of the eurytele and mastigophore type (presence, absence), (vi) colony

type (stolonal, erect, solitary, pelagic), (vii) reproductive stage

(medusa, medusoid, sporosac) and (viii) habitat (tropical, nontropical,

both). Hydrozoan species were considered as ”symbiotic” when living

as epibionts or partial epibionts of other organisms, and ”generalist”

when living associated to more than one of the host groups listed

above or living on abiotic substrates. Probable realizations of the evo-

lution of the characters were mapped onto the Capitata phylogeny

using the ”make.simmap” function in the phytools package

(Revell, 2012) in R (R Core Team, 2020). Because the character states

were unknown for some of the tips, the input character state vector

was converted into a matrix of prior probabilities for tip states, and

equal probabilities for each state were assigned for these unknown tip

states (e.g. it is unknown whether the polyp stage of Zanclea mayeri

Schuchert & Collins, 2021 lives in symbiosis with other organisms and

therefore the tip states ”presence” and ”absence” were assigned equal

probabilities of 0.5 for the character ”symbiotic lifestyle”). Recon-

structions were run under the ”equal rates” (ER) and ”all rates differ-

ent” (ARD) models, comparing the ﬁt of the models with a likelihood

ratio test using the function ”pchisq” and resulting in the selection of

the ARD model for all characters except habitat (ER model). For each

reconstruction, 10000 stochastic mapping replicates were run and the

results were summarized with pie charts representing the posterior

probability of each internal node being in each state.

The presence of evolutionary correlation between some of the

characters was also tested using the Pagel’s method (Pagel, 1994),

which, by ﬁtting different models to the data (e.g. two traits evolve

independently vs. the evolution of two characters is correlated) and

performing a likelihood ratio test, assesses whether the difference

between models is signiﬁcant. The tested relationships were between

the character ”symbiotic lifestyle” and all other characters but

”host”, to assess whether the evolution of symbiosis inﬂuenced the

evolution of morphological traits and to test whether living in tropi-

cal environments favoured the shift to a symbiotic lifestyle. Tests

were carried out using the ”ﬁtPagel” function in the phytools pack-

age (Revell, 2012) after re-coding character states to binary

(Table S2) and after pruning the tree from species with unknown

states. For each test, four models of coevolution between characters

xand ywere ﬁtted to data (independent evolution; the evolution of

xdepends on the evolution of y; the evolution of ydepends on the

evolution of x;xand yevolve interdependently) and results were

compared using AIC weights (AICw) and performing a likelihood

ratio test between each model assuming coevolution and the model

assuming independent evolution.

Alignments, raw phylogenetic trees, and R scripts and ﬁles used

for the phylogenetic comparative methods are available at the fol-

lowing link: https://ﬁgshare.com/s/ff00a8fe9e61b1bde4e3.

Results

Molecular phylogenetics

Sampling activities resulted in the collection of 60

Capitata species, with most species belonging to the

D. Maggioni et al. / Cladistics 0 (2023) 1–28 5

superfamily Zancleida (Fig. 1; Table S1). All speci-

mens were identiﬁed to the lowest taxonomic level pos-

sible, but in some cases a species identiﬁcation was not

possible, owing to the lack of suitable morphological

diagnostic characters and incomplete knowledge of the

life cycle (i.e. only the medusa or polyp stage was

available). These species are dealt with in the ”Taxo-

nomic account” section.

The assembled genetic dataset included 223 speci-

mens, 115 species, 35 genera and 14 families of Capitata

hydrozoans, representing the most complete dataset

employed so far for phylogenetic analyses of the subor-

der. The MP, BI and ML reconstructions were broadly

concordant and the nodal supports of each analysis

were mapped onto the BI tree (Fig. 2). The Capitata

were fully supported and divided into the two clades

Zancleida and Corynida, both showing high support in

almost all analyses, with the only exception being the

Zancleida, which was not supported by the MP analysis.

A clade composed of the families Pennariidae McCrady,

1859, Hydrocorynidae Rees, 1957, Halimedusidae Arai

& Brinckmann-Voss, 1980, and Moerisiidae Poche,

1914 was a sister to all other Zancleida taxa. Within this

clade, Pennariidae and Hydrocorynidae were sister

groups, and so were Halimedusidae and Moerisiidae.

Notably, Tiaricodon orientalis Yamamoto & Toshino,

2021 had a higher afﬁnity with the Moerisiidae than

with the Halimedusidae, to which it is currently

ascribed, but further sampling is needed to clarify this

issue. Zancleopsidae Bouillon, 1978 and Sphaerocoryni-

dae Pr

evot, 1959 were highly supported and, together,

placed as a sister group to the remaining Zancleida, fol-

lowed by Cladocorynidae Allman, 1872 and Porpitidae

Goldfuss, 1818. The families Milleporidae Fleming,

1828 and Solanderiidae Marshall, 1892 formed two fully

supported, reciprocally monophyletic clades, even if the

support of this sister-group relationship was low.

Finally, Zancleidae resulted to be polyphyletic in all

analyses and, overall, cryptic species were common in

different Zancleida groups, namely in Apatizanclea

divergens (Boero, Bouillon & Gravili, 2000), coral-

associated Zanclea spp., Solanderia secunda (Inaba,

1892), Pteroclava krempﬁ (Billard, 1919) and Pennaria

disticha Goldfuss, 1820.

Regarding the Zancleidae, several Zanclea species

formed a highly supported group corresponding to the

”true” family Zancleidae and the genus Zanclea (Figs 2

and 3a), including the type species Zanclea costata

Gegenbaur, 1857. The other included species were

Z. tipis Puce, Cerrano, Boyer, Ferretti & Bavestrello,

2002, Z. giancarloi Boero, Bouillon & Gravili, 2000,

Z. sessilis (Gosse, 1853), Z. implexa (Alder, 1856),

coral-associated Zanclea species, including Z. sango

Hirose & Hirose, 2011 and Z. gallii Montano,

Maggioni & Puce, 2015, Zanclea cf. migottoi, ﬁve uni-

dentiﬁed species from the Mediterranean, Florida,

China, Caribbean and Maldives, and Zanclea pirainoid

(Boero, Bouillon & Gravili, 2000) comb.n., previously

ascribed to the genus Halocoryne Hadzi, 1917.

Sequences of Z. migottoi Galea, 2008 from GenBank,

and three Zanclea specimens from the Caribbean and

the Maldives formed a fully supported group, with a

moderate intragroup genetic distance of 1.5%

(Table S4) and were named Z. cf. migottoi, pending fur-

ther sampling to clarify if they belong to the same spe-

cies or are a species complex (see ”Taxonomic account”

section). Sequences from China were downloaded from

GenBank but no associated data were available. The

other undetermined species could not be identiﬁed to

the species level as a consequence of a lack of informa-

tion about the polyp or medusa stage and are described

in the ”Taxonomic account” section. The intra- and

interspeciﬁc genetic distances of Zanclea species did not

overlap, showing nevertheless a very narrow gap

(Fig. 3d; Table S4). The other species ascribed to the

genus Zanclea formed three divergent groups. A ﬁrst

was composed of Zanclea prolifera Uchida and

Sugiura, 1976 alone, showing a sister-group relationship

with the Asyncorynidae Kramp, 1949 (Fig. 2), even if

with low support. For a second clade, including the spe-

cies Zanclea divergens,Zanclea mayeri,Zanclea exposita

Puce, Cerrano, Boyer, Ferretti & Bavestrello, 2002,

Zanclea sp. 1 and Zanclea sp. 2 (sensu Maggioni

et al., 2018), the new genus Apatizanclea gen.n. is

erected, resulting in the new combinations Apatizanclea

divergens,Apatizanclea mayeri,Apatizanclea exposita,

Apatizanclea sp. 1 and Apatizanclea sp. 2 (Figs 2and

3b). This clade showed uncertain intragroup phyloge-

netic relationships, but all species were well-deﬁned, also

according to genetic distance analysis (Fig. 3d;

Table S4), with a clear separation between intra- and

interspeciﬁc distances. A third, fully supported group

was composed of Halocoryne protecta (Hastings, 1932)

comb.n.,Halocoryne eilatensis (Pica, Bastari & Puce,

2017) comb.n. and Halocoryne epizoica Hadzi, 1917

(Fig. 3c), with the ﬁrst two, previously ascribed to the

genus Zanclea, being sister species (Fig. 3c) and sharing

almost identical morphologies (Pica et al., 2017; Mag-

gioni et al., 2020b). Intra- and interspeciﬁc genetic dis-

tances showed some overlapping values in this group,

due to the high intraspeciﬁc divergence of H. eilatensis

(Fig. 3d; Table S4).

Fig. 2. Bayesian phylogenetic hypothesis of the Capitata, obtained from the concatenated dataset and including one specimen per species. Num-

bers at nodes represent BPP, ML BS, and MP BS, respectively. Asterisks denote maximal support for all analyses, whereas dashes (–) indicate

nodes that were not supported in the corresponding analyses. The scale bar indicates the mean number of nucleotide substitutions per site.

6D. Maggioni et al. / Cladistics 0 (2023) 1–28

D. Maggioni et al. / Cladistics 0 (2023) 1–28 7

Fig. 3. Phylogenetic hypotheses of (a) Zanclea, (b) Apatizanclea and (c) Halocoryne. Numbers at nodes represent BPP, ML BS, and MP BS,

respectively. Asterisks denote maximal support for all analyses, whereas dashes (–) indicate nodes that were not supported in the corresponding

analyses. Scale bars indicate the mean number of nucleotide substitutions per site. (d) Density plots of the genetic distances (uncorrected p-

distances in %) of the three clades shown in the phylogenetic trees, with intraspeciﬁc distances in yellow and interspeciﬁc distances in light blue.

8D. Maggioni et al. / Cladistics 0 (2023) 1–28

The superfamily Corynida encompasses the families

Corynidae and Cladonematidae, which both were

highly supported in the phylogenetic hypotheses pre-

sented here (Fig. 2). In the family Corynidae, the group

composed of Sarsia Lesson, 1843 and Slabberia Forbes,

1846 formed a well-supported monophyletic group, but

the internal relationships were not completely resolved.

Despite most Sarsia species forming a cohesive clade,

the position of Sarsia bella Brinckmann-Voss, 2000

was unclear, as well as the validity of the genus Slab-

beria. A more confusing situation occurred within Cla-

donematidae, where, despite the genus Cladonema

Dujardin, 1843 being monophyletic and well-supported,

the relationships among Staurocladia Hartlaub, 1917

and Eleutheria Quatrefages, 1842 were unclear (Fig. 2)

and insufﬁciently resolved to understand the validity of

the two genera.

Taxonomic account

In this section the descriptions of undetermined or

dubious Zanclea species are provided, together with

diagnoses of the genera Zanclea,Halocoryne and Apa-

tizanclea. The family Zancleidae was not split in differ-

ent families because phylogenetic relationships were

not fully resolved, especially in relation to the families

Asyncorynidae, Millerporidae and Solanderiidae.

Owing to the need for genetic data to solve the phylo-

genetic position of all zancleid species, generic diagno-

ses are based solely on the species included in the

molecular analyses, and afﬁnities with species not

included are discussed in the ”Remarks” sections.

Hydrozoa Owen, 1843

Capitata Kuhn, 1913

Zancleidae Russel, 1953

Zanclea Gegenbaur, 1856

Diagnosis: Polyps colonial, stolonal with creeping

hydrorhiza, with or without perisarc, frequently associ-

ated with other organisms including bryozoans, scler-

actinian corals, molluscs and algae. Polyps

monomorphic or polymorphic, including gastrozooids

and dactylozooids. Gastrozooids cylindrical or clavi-

form with oral and aboral capitate tentacles or without

tentacles. Dactylozooids elongated and contractile,

with or without capitate tentacles. Cnidome composed

of stenoteles generally of two size classes, with or with-

out macrobasic euryteles or mastigophores. Medusae

with bell-shaped umbrella, four radial canals, and four

exumbrellar perradial cnidocyst patches or lines with

stenoteles. Manubrium bearing gonads inter-radially,

ending with a mouth simple and circular. Four bulbs

with no ocelli, forming two or four tentacles equipped

with cnidophores containing bean-shaped macrobasic

euryteles.

Species currently included in the genus:Z. costata

(type species), Z. migottoi,Z. giancarloi,Z. sessilis,

Z. implexa,Z. pirainoid comb.n.,Z. tipis,Zgallii,

Z. sango, other undetermined species associated with

scleractinian corals and bryozoans or known from the

medusa stage only.

Remarks: Zancleidae is a species-rich family com-

posed of taxa in many cases hardly distinguishable or

with partially unknown life cycles, making it difﬁcult

to assess their systematic afﬁnities without genetic

data. The many species described based on the polyp

or medusa alone and without DNA data contributed

to fuelling the taxonomic confusion of this family. For

this reason, it is challenging to assess whether the

many Zanclea species not included in the phylogenetic

analyses belong to this genus. The family has been pre-

viously subdivided into the three genera Zanclea,

Halocoryne and Zanclella Boero & Hewitt, 1992, even

if the validity of the latter two has been questioned,

owing to the overlap of the morphological characters

used in their diagnoses (Schuchert, 1996,2010). On the

one hand, the type species of Zanclea,Z. costata,

belongs to a well-supported clade that we deﬁned as

the ”true” Zancleidae and Zanclea, together with

many other species. On the other hand, the type spe-

cies of Halocoryne,H. epizoica, is divergent from this

group and the genus should therefore not be consid-

ered as part of the Zancleidae anymore. Further sam-

pling is needed to address this issue. Nevertheless, a

species previously ascribed to Halocoryne has been

demonstrated to belong to the genus Zanclea, and,

likewise, species previously ascribed to Zanclea were

moved to the genus Halocoryne, hampering clear mor-

phological diagnoses for both genera. The situation

remains even more uncertain for Zanclella, with Zan-

clella diabolica Boero, Bouillon & Gravili, 2000 shar-

ing strong similarities with Apatizanclea species.

However, no molecular data are available for the type

species Zanclella bryozoophila Boero & Hewitt, 1992.

For this reason, for the moment being, we consider

Zancleidae as a polyphyletic family.

Zanclea cf. migottoi.Figure 4

Material examined. BT001: Panama, Bocas del Toro

(9.3509°N, 82.2548°W), August 2015, attached to a

ﬂoating Sargassum sp.—MA0117152 (DNA code:

MA334): Maldives, Faafu Atoll, Adangau Island

(3.1429°N, 73.0121°E), 18.II.2017, 10 m deep, on

Parasmittina egyptiaca (Waters, 1909).—MA0117155

(DNA code: MA335): Maldives, Faafu Atoll, Adan-

gau Island (3.1429°N, 73.0121°E), 18.II.2017, 10 m

deep, on a coralline alga.

Description. Colony stolonal, growing on different

substrates including Sargassum algae (Fig. 4a), bryo-

zoans (Fig. 4b) and coralline algae (Fig. 4c). Hydro-

rhiza with a thick perisarc, reticular, crawling on the

substrate, and often with epiphytes (Fig. 4d–f). Colo-

nies monomorphic, gastrozooids tubular to clavate, up

D. Maggioni et al. / Cladistics 0 (2023) 1–28 9

to 2 mm long, with a pedicel covered by perisarc

(Fig. 4a,d–f), slightly corrugated in sample BT001

(Fig. 4d); a distal and circular mouth surrounded by

4–6 oral capitate tentacles, and with up to 30 aboral

capitate tentacles scattered over ⅔or ¾of the polyp

column (Fig. 4a,e,f). Capitula up to 45 lm in diame-

ter. Living polyps transparent with a whitish mouth

(Fig. 4b,c). Medusa buds observed in sample BT001

(Fig. 4a) and MA0117155, at the base of gastrozooids.

Medusae not observed. Cnidome composed of steno-

teles of two size classes (Fig. 4g,i,k) abundant in capit-

ula, and macrobasic apotrichous euryteles (Fig. 4h,j,k)

in the hypostome, distributed in the hydranth and in

some cases concentrated at the base of tentacles, and

in the hydrorhiza. In the Caribbean sample euryteles

are rare and found only in the hydrorhiza. Large

Fig. 4. Zanclea cf. migottoi from (a, d, g, h) Panama associated with Sargassum sp. (sample BT001), (b, e, i, j) Maldives associated with a bryo-

zoan (sample MA0117152) and (c, f, k) Maldives associated with a red alga (sample MA0117155). (a–c) Polyps of living colonies on various sub-

strates. (d) Detail of a corrugated pedicel. (e, f) Polyps showing moderately long pedicels. (g) Stenoteles and (h) eurytele of BT001. (i) Stenoteles

and (j) eurytele of MA0117152. (k) Stenoteles and eurytele of MA0117155. Scale bars: (a–c) 0.2 mm, (d–f) 50 lm, (g–k) 5 lm.

10 D. Maggioni et al. / Cladistics 0 (2023) 1–28

stenoteles are 10–13 99–10 lm in sample

MA0117152, 8–11 99–10 lm in sample MA0117155

and 9–10 97–9lm in sample BT001. Small stenoteles

are 6–794–5lm in sample MA0117152, 5–693–

5lm in sample MA0117155 and 5–694–5lmin

sample BT001. Euryteles are 21–22 98–9lm in sam-

ple MA0117152, 21–22 98–9lm in sample

MA0117152 and 16 99lm in sample BT001.

Distribution. Samples analysed here came from Carib-

bean Panama and from Faafu Atoll, Central Maldives.

Records of Z. migottoi are from Guadeloupe (Galea,

2008), Yucat

an, Mexico (Mendoza-Becerril et al., 2018),

and Sergipe, Brasil (Castro Mendonc

ßa et al., 2022).

Remarks.Zanclea migottoi was described by

Galea (2008) to accommodate Zanclea polyps previ-

ously attributed to Z. cf. alba and Z. costata (Migotto,

1996; Vervoort, 2006) but differing from these two

species by the cnidome composition. Our Caribbean

specimen comes very close to Z. alba (Meyen, 1834) in

general morphology and in its association with the

alga Sargassum sp. However, the presence of euryteles

in the hydrorhiza, even if rare, points against its

assignment to Z. alba and makes it more similar to

Z. migottoi. Indeed, euryteles were never found in

Z. alba (Calder, 1988; Galea, 2008)andZ. migottoi

shows euryteles of a size comparable to our Caribbean

material, even if in the present material euryteles were

not found at the base of tentacles, as reported in the

Z. migottoi description (Galea, 2008). Maldivian speci-

mens showed a similar morphology, which also is simi-

lar to other monomorphic Zanclea species with

euryteles, and were associated with other substrates,

namely a red alga, similar to the colony described in

Migotto (1996), and a bryozoan. Genetically, these

specimens were closely related to our Caribbean mate-

rial and to a sequence obtained from GenBank (Acces-

sion number: MF538731) formerly identiﬁed as

Z. migottoi (Mendoza-Becerril et al., 2018). Recent re-

analysis of this latter specimen revealed the absence of

euryteles in the polyp (Mendoza-Becerril, pers.

comm.), ﬁtting therefore the scope of Z. alba. Given

the presence of euryteles in our specimens, we provi-

sionally identiﬁed them as Z. cf. migottoi, but it might

be possible that Z. migottoi and Z. alba are the same

species, being the presence of euryteles variable and

not diagnostic for this species. A focused morpho-

molecular assessment is needed to disentangle this

issue. Whatever the species identity of this lineage, it

shows a wide distribution, spanning the Atlantic and

Indian oceans, and is generalist in terms of substrate

on which the polyps grow.

Zanclea sp. (Maldives). Figure 5

Material examined. MA0318071 (DNA code:

MA464): Maldives, Faafu Atoll, Adangau Island

(3.1429°N, 73.0121°E), 19.III.2018, 25 m deep.

Description. Colony stolonal, growing in association

with the cheilostome bryozoan Plesiocleidochasma

laterale (Harmer, 1957) (Fig. 5a–c). Hydrorhiza with a

thin perisarc, reticular, crawling on (and possibly

under) the host surface. Colonies monomorphic, gas-

trozooids tubular, up to 1 mm long, with a distal and

circular mouth, surrounded by 5 oral capitate tenta-

cles, and with up to 25 aboral capitate tentacles scat-

tered over all the length of the polyp column (Fig. 5a–

e), with 2–4 short tentacles often found at the base of

the polyp (Fig. 5f). Capitula larger in oral tentacles

(up to 50 lm in diameter) and smaller in aboral tenta-

cles (up to 40 lm in diameter) (Fig. 5e). Living polyps

transparent with a whitish mouth (Fig. 5a–d). Medusa

buds originating in the proximal portion of the gastro-

zooid. Medusa not observed. Cnidome composed of

stenoteles of two size classes (Fig. 5g) and macrobasic

apotrichous euryteles (Fig. 5h,i). Large (9–12 98–

10 lm) and small stenoteles (5–694–5lm) abundant

in capitula and hydrorhiza, euryteles (21–24 98–

12 lm) present around the hypostome and scattered in

the hydranth.

Distribution. Known only from Faafu Atoll, Central

Maldives.

Remarks. The only analysed colony can hardly be

distinguished from other monomorphic Zanclea species

with macrobasic apotrichous euryteles. Among these

species, some show different host preferences, such as

Z. fanella Boero, Bouillon & Gravili, 2000 growing on

mollusc shells in the Western Paciﬁc and coral-

associated Zanclea. Other similar species are

Z. giancarloi,Z. implexa and Z. migottoi, which never-

theless clearly differ from a genetic point of view and

are generalists in the substrate on which they settle.

Several Zanclea species were described from the Indo-

Paciﬁc based on their medusa stage only (Maggioni

et al., 2018) and, given also the unknown medusa stage

for the Maldivian specimen, we were unable to assign

it to any known or new species.

Zanclea sp. (Curac

ßao). Figure 6

Material examined. CU024: Curac

ßao, St. Michiel’s

Bay (12.1481°N, 69.0003°W), 10.VI.2017, 22 m

deep.—CU041: Curac

ßao, Playa Marie Pampoen

(12.0901°N, 68.9052°W), 12.VI.2017, 27 m deep.—

CU043: Curac

ßao, Substation (12.0844°N,

68.8983°W), 12.VI.2017, 22 m deep.—CU055: Cur-

ßao, Caracas Bay, Tugboat Beach, (12.0681°N,

68.8622°W), 13.VI.2017, 15 m deep.—CU061: Cur-

ßao, Playa Kalki (12.3750°N, 69.1578°W),

14.VI.2017, 15 m deep.—CU082: Curac

ßao: Coral

Estate (12.1958°N, 69.0795°W), 17.VI.2017,

30 m deep.

Description. Colony stolonal, exclusively growing in

association with the cheilostome bryozoan Trematooe-

cia aviculifera (Canu & Bassler, 1923) (Fig. 6a–f).

D. Maggioni et al. / Cladistics 0 (2023) 1–28 11

Hydrorhiza devoid of perisarc, reticular, crawling

under the host skeleton or among the grooves of the

autozooids and projecting out for some of its length.

Colonies polymorphic, composed of gastrozooids

(Fig. 6a–d) and dactylozooids (Fig. 6e,f).

Gastrozooids tubular, ≤1 mm long, with a distal and

circular mouth, surrounded by 5 or 6 oral capitate ten-

tacles, and with up to 40 aboral capitate tentacles scat-

tered over all the length of the polyp column (Fig. 6g).

Capitula larger in oral tentacles (up to 60 lm in diam-

eter) and smaller in aboral tentacles (up to 40 lmin

diameter) (Fig. 6g). Dactylozooid thin and very exten-

sible, up to 2.5 mm long, without a mouth, and with 4

distal capitate tentacles (capitula up to 90 lm in diam-

eter) (Fig. 6h,i). Living gastrozooids transparent with

a whitish mouth, dactylozooids transparent (Fig. 6a–

d). Medusa buds originating directly from the hydro-

rhiza. Medusa not observed. Cnidome composed of

Fig. 5. Zanclea sp. from Maldives associated with Plesiocleidochasma laterale.(a–d) Polyps of living colonies on the host bryozoan. (e) Gastro-

zooid and (f) detail of two tentacles at the base of the polyp. (g) Capitulum with small and large size stenotele. (h) Undischarged and (i) dis-

charged macrobasic apotrichous eurytele capsules. Scale bars: (a–d) 0.2 mm, (e, f) 50 lm, (g–i) 5 lm.

12 D. Maggioni et al. / Cladistics 0 (2023) 1–28

stenoteles of two size classes (Fig. 6j) and macrobasic

apotrichous euryteles (Fig. 6k). Large (10–14 98–

11 lm) and small stenoteles (5–794–5lm) abundant

in capitula, euryteles (15–20 97–9lm) in the hydro-

rhiza, at the base of gastrozooids, and in the distal

portion of the dactylozooids.

Distribution. Known only from Curac

ßao,

Caribbean Sea.

Remarks. A few Zanclea species are reported to have

tentaculated dactylozooids, namely Z. sessilis from the

Mediterranean and Atlantic, Z. polymorpha Schu-

chert, 1996 from New Zealand, Z. bomala Boero,

Bouillon & Gravili, 2000 from California and

Z. hirohitoi Boero, Bouillon & Gravili, 2000 from

Papua New Guinea (Schuchert, 1996; Boero et al.,

2000; Altuna, 2016). The specimens analysed herein

differ genetically from Z. sessilis and also show some

differences compared to the other species. Zanclea

bomala lacks euryteles and Z. hirohitoi has tentaculo-

zooids that are absent in the present specimens.

Fig. 6. Zanclea sp. from Curac

ßao associated with Trematooecia aviculifera.(a–d) Gastrozooids and (e, f) dactylozooids showing over the host

bryozoan colony. (g) Gastrozooid and (h) dactylozooid, with (i) detail of the four dactylozooid tentacles. (j) Capitulum with small and large size

stenoteles and (k) euryteles. Scale bars: (a–f) 0.2 mm, (g, h) 50 lm, (i, j) 5 lm.

D. Maggioni et al. / Cladistics 0 (2023) 1–28 13

Zanclea polymorpha is the most similar species, never-

theless showing a lower number of aboral tentacles

and larger euyteles, and being associated with a differ-

ent bryozoan species, namely Rhynchozoon larreyi

(Audouin, 1826). A few Zanclea-like medusae, whose

polyp stage is still unknown, have been reported from

the Atlantic Ocean, such as Apatizanclea mayeri and

Zanclea sp. from Florida (Schuchert and Collins, 2021)

—but these two species clearly differ genetically from

this species (Figs 2and 3), Z. sagittaria (Haeckel,

1879) from Cuba (Haeckel, 1879), and

Z. medusopolypata Boero, Bouillon & Gravili, 2000

from Venezuela and Brazil (Boero et al., 2000). How-

ever, no genetic data are available for the latter two

species. Given the more or less distinct morphological

differences, the genetic differences, and the disjunct

geographical distributions, we refer to our specimens

as Zanclea sp. pending further sampling and analyses.

Zanclea sp. (Mediterranean). Figure 7

Material examined. Vfr14-01 (DNA code:

DNA0977), male: France, Bay of Villefranche-sur-Mer

(43.6856°N, 7.3178°E), 28.IV.2014, 0–70 m deep.—-

Vfr14-02 (DNA code: DNA0978), female: France, Bay

of Villefranche-sur-Mer (43.6856°N, 7.3178°E),

28.IV.2014, 0–70 m deep.—Vfr16-11 (DNA code:

DNA1152), male: France, Bay of Villefranche-sur-Mer

(43.6856°N, 7.3178°E), 28.IV.2016, 0–20 m deep.—-

Vfr16-15 (DNA code: DNA1154), male: France, Bay

of Villefranche-sur-Mer (43.6856°N, 7.3178°E),

29.IV.2016, 0–75 m deep. All specimens were collected

with plankton net by PS.

Fig. 7. Zanclea sp. medusa with four tentacles from western Mediterranean. (a) Female (left) and male (right) medusae and higher magniﬁcation

of the same (b) male and (c) female specimens. (d) Another male individual. Scale bars: 0.5 mm.

14 D. Maggioni et al. / Cladistics 0 (2023) 1–28

Description. Mature medusa ~5 mm high and 4 mm

wide, umbrella bell-shaped, apex slightly pointed.

Exumbrella with 4 perradial nematocyst bands,

extending from circular canal to apex. Manubrium

large, extending for ¾of subumbrellar cavity, with a

larger upper part and a thinner, cylindrical mouth

region. Upper part of manubrium cruciform in cross-

section, with gonads separated perradially. Four radial

canals ending in 4 tentaculated bulbs. Four thick ten-

tacles, armed with spherical cnidophores. No ocelli

present. Living medusae transparent, with orange

manubrium and bulbs and radial canals showing green

ﬂuorescence. Cnidome composed of stenoteles and

bean-shaped euryteles.

Distribution. Only known from the western

Mediterranean Sea.

Remarks. Contrary to most zancleid species, all of

the specimens belonging to this undetermined species

have four tentacles. Zanclea costata is known to pro-

duce two-tentacled newly liberated medusae that can

develop two more tentacles at maturity (Schu-

chert, 2010), and its four-tentacled medusa is hardly

distinguishable from our specimens. Zanclea costata is

currently thought to be a Mediterranean endemic and

was described based on the four-tentacled medusa

(Gegenbaur, 1857). In this species, all medusae

observed after release from polyps and then cultivated

showed two tentacles only (Cerrano et al., 1997; Schu-

chert, 2010), including the specimen described by

Schuchert (2010) that we have analysed too. The latter

showed a high genetic divergence from Zanclea sp.,

with a 16S genetic distance of 7%. Two- and four-

tentacled medusae were also found together in plank-

ton samples, and it was hypothesized that the latter

was a more developed stage of the same species

(Browne, 1906). However, because no direct transition

from two- to four-tentacle stages have been observed

so far, the issue remains unsolved. Zanclea bomala is

another species with four tentacles in the adult medusa

stage after being reared in laboratory (Boero et al.,

2000). It shows nevertheless differences in the general

morphology, especially in the perradial oval nemato-

cyst pouches, that were absent in the present species.

Finally, Altuna (2016) observed Z. sessilis medusae

with a variable number of tentacles, spanning zero to

three at liberation, and two to ﬁve at maturity. Adult

Z. sessilis medusae also show slight differences with

the present species, such as the median swellings of the

radial canals, and the two species are genetically differ-

ent, with a 16S genetic distance of 3.6%. Because the

polyp stage of Zanclea sp. is unknown, we cannot

assign it to any known or new Zanclea species.

Halocoryne Hadzi, 1917. Diagnosis: Polyps colonial,

stolonal with creeping hydrorhiza, without perisarc,

exclusively associated with bryozoans. Polyps

monomorphic or polymorphic, including gastrozooids

and gono-dactylozooids. Gastrozooids cylindrical or

claviform with oral and aboral capitate tentacles,

or reduced, without tentacles. Gono-dactylozooids

elongated, without tentacles. Cnidome composed of

stenoteles of two size classes. Reproduction by

medusae or medusoids. Medusae with bell-shaped

umbrella, 4 radial canals, and 4 exumbrellar perradial

cnidocyst patches with stenoteles. Manubrium bearing

gonads interradially, ending with a mouth simple and

circular. Four bulbs with no ocelli, originating 2

tentacles equipped with cnidophores containing bean-

shaped macrobasic euryteles. Medusoids with bell-

shaped umbrella, manubrium without mouth and with

gonads in a single mass, 4 radial canals, 4 perradial

bulbs without ocelli and without tentacles.

Species currently included in the genus:H. epizoica

(type species), H. eilatensis comb.n.,H. protecta

comb.n.

Remarks: The family Halocorynidae Picard, 1957

was erected by Picard (1957) to accommodate the spe-

cies H. epizoica, later transferred to the family Zanclei-

dae. The family Halocorynidae may need to be

resurrected as a result of the clear divergence of

H. epizoica from the Zancleidae, but owing to the phy-

logenetic uncertainties in our phylogenetic hypothesis

we prefer to conservatively avoid this to prevent fur-

ther taxonomic confusion. Three other species were

described as belonging to the genus Halocoryne,

namely H. pirainoid,H. frasca Boero, Bouillon &

Gravili 2000 and H. orientalis (Browne, 1916). The

ﬁrst species was demonstrated in the present work to

belong to the genus Zanclea, despite having a polyp

morphology similar to H. epizoica and H. frasca.

Regarding the two other species, it is currently not

possible to ascertain their belonging to the genus Halo-

coryne owing to lack of DNA data. On the one hand,

the polyp stage of H. frasca has an overall morphol-

ogy similar to H. epizoica but possesses macrobasic

holotrichous euryteles, a feature absent in the type spe-

cies and other Zanclea-like species now ascribed to the

genus. The presence of these euryteles makes H. frasca

similar to species in the newly erected genus Apatizan-

clea, even if none of these species is polymorphic. Also

Z. costata is known to possess this type of cnidocyst.

On the other hand, H. orientalis is known only from

the medusa stage and its systematic afﬁnities remain

even more doubtful. Finally, in our phylogeny recon-

structions (Figs 2and 3c), we found that the former

Z. eilatensis and Z. protecta were closely related to

Halocoryne epizoica, and the two species are thus here

transferred to the genus Halocoryne.

Apatizanclea Maggioni gen.n..http://zoobank.

org/urn:lsid:zoobank.org:act:F80CFD68-B3DC-

4A89-80F5-F53C254AEA26

D. Maggioni et al. / Cladistics 0 (2023) 1–28 15

Diagnosis: Polyps colonial, stolonal with creeping

hydrorhiza, without perisarc, exclusively associated

with bryozoans. Polyps monomorphic. Gastrozooids

cylindrical or claviform with oral and aboral capitate

tentacles. Cnidome composed of stenoteles of two size

classes and macrobasic holotrichous euryteles. Medu-

sae with umbrella bell-shaped, 0 or 4 radial canals,

and 0 or 4 exumbrellar perradial cnidocyst patches

with stenoteles. Manubrium bearing gonads interra-

dially, ending with a mouth simple and circular with

or without oral arms. Two tentaculated bulbs with no

ocelli, originating 2 tentacles equipped with cnido-

phores containing bean-shaped macrobasic euryteles.

No atentaculate bulbs present.

Etymology:Apatizanclea derives from the combina-

tion of ”ἀpasάx”, meaning ”to deceive, mislead” in

ancient Greek, and -zanclea, referring to the fact that

species in this genus can be easily confused with Zan-

clea species.

Species currently included in the genus:A. divergens

comb.n. (type species, here designated), A. mayeri

comb.n.,A. exposita comb.n., and the two undeter-

mined species Apatizanclea sp. 1 and

Apatizanclea sp. 2.

Remarks: Our phylogeny reconstructions (Figs 2and

3b) demonstrated that the lineage here described as

Apatizanclea diverged from the genus Zanclea, forming

a cohesive group and requiring the establishment of

the new genus and new combinations. This genus is

mostly based on genetic data. but similarities among

the species are present in both the polyp and medusa

stages, when known. Speciﬁcally, all known polyps

possess macrobasic holotrichous euryteles, are devoid

of perisarc, and are exclusively associated with bryo-

zoans mostly belonging to Celleporaria species. The

adult medusa is known only for A. mayeri, whereas

newly released medusae are known for A. divergens,

Apatizanclea sp. 1, and Apatizanclea sp. 2, but not for

A. exposita. However, all of them share the presence

of only two tentaculate bulbs and the absence of aten-

taculate bulbs. Adult medusae of A. mayeri and newly

released medusae of A. divergens are typical Zanclea-

like medusae. Newly released medusae of Apatizanclea

sp. 1 and Apatizanclea sp. 2 show instead striking simi-

larities with Zanclella diabolica, the latter also having

polyps very similar to Apatizanclea sp. 1. As pointed

out by Maggioni et al. (2018), Apatizanclea sp. 1 may

actually correspond to Z. diabolica. Also Zanclella

glomboides Boero, Bouillon & Gravili, 2000 possesses

similar newly released medusae, and both young and

adult medusae have only two bulbs, equipped with

tentacles. The polyp stage of the latter species is

reduced and polymorphic, contrarily to the known

polyps of Apatizanclea. At the same time, it is associ-

ated with bryozoans, has no perisarc and shows

macrobasic euryteles (even if it is still unknown

whether they are holotrichous), thus possibly matching

the diagnosis for the Apatizanclea genus. Zanclella is a

problematic genus, and its validity has been questioned

previously (Schuchert, 1996,2010). The type species is

Z. bryozoophila, showing reduced, polymorphic

polyps, no perisarc, macrobasic holotrichous euryteles

and reproduction via medusoids. Unfortunately, no

genetic data are available for this species, and there-

fore the validity of the genus Zanclella remains uncer-

tain. Four other species have known medusae with

two bulbs only, namely Halocoryne frasca, which also

possesses polyps with macrobasic holotrichous eury-

teles, Zanclea hirohitoi,Z. medusopolypata and

Z. tipis.

Range expansions and morphological modiﬁcations

associated with the symbiotic lifestyle

In this work we report the expansion of geographical

and host ranges for some species. In particular, the dis-

tribution of Zanclea pirainoid was extended westward,

being reported for the ﬁrst time in the Maldives, after its

description from Papua New Guinea (Boero

et al., 2000). Apatizanclea exposita was reported from

Eastern Australia, after its description in North Sula-

wesi, Indonesia (Puce et al., 2002) and the distribution

of Halocoryne protecta was extended to the Central Red

Sea, being previously only known from the Indo-Paciﬁc

(Boero et al., 2000; Maggioni et al., 2020b). Finally,

specimens identiﬁed as Zanclea cf. migottoi were

reported from the Indian Ocean, whereas this species

was previously known only from the Western Atlantic

Ocean (Galea, 2008; Mendoza-Becerril et al., 2018;

Mendonc

ßa et al., 2022). Regarding the extension of host

ranges, Z. pirainoid is here reported associated with the

bryozoan Robertsonidra sp., while the host bryozoan

was not identiﬁed in the original description. Two Zan-

clea species appeared to be more generalist than previ-

ously thought, namely Z. sessilis, which was found

growing on bare rock, red algae and bryozoans, and the

Zanclea cf. migottoi group, found on Sargassum sp. in

the Caribbean and on a red alga and the bryozoan Para-

smittina egyptiaca in the Maldives. Halocoryne eilatensis

was reported growing in association with the bryozoan

Adeonella sp. in the Central Red Sea, whereas it was

previously only known in association with Parasmittina

species in the Northern Red Sea. Finally, H. protecta

was found for the ﬁrst time on the bryozoans Parasmit-

tina raigii (Audouin, 1826), P. egyptiaca,Calyptotheca

sp. and Steginoporella sp.

Morphological modiﬁcations of the skeleton of the

hosts were observed in several symbiotic species,

namely coral-associated Zanclea species (with the

exception of the Acropora-associated ones), as already

shown in Manca et al. (2019), and in Maggioni

et al. (2020b,2022a) and in bryozoan-associated

16 D. Maggioni et al. / Cladistics 0 (2023) 1–28

species. Speciﬁcally, modiﬁcations of the bryozoan

skeleton were observed in colonies hosting H. epizoica,

H. protecta,Z. pirainoid,Z. tipis,Zanclea sp. (Cur-

ßao), the A. divergens species complex, Apatizanclea

sp. 1 and Apatizanclea sp. 2 (Fig. 8). Skeletal modiﬁca-

tions were mostly observed as a secondary

calciﬁcation that covered or partially covered the

hydrorhiza and base of the symbiotic hydroids

(Fig. 8a,d–h). However, in Z. tipis only holes from

where hydrozoan polyps protruded were observed

(Fig. 8c), and in Z. pirainoid modiﬁcations were repre-

sented by the formation of a smooth and open canal

between the bryozoan zooids, where the hydrorhiza

was accommodated (Fig. 8b).

In other symbiotic associations involving host

sponges, octocorals, bivalves and algae, no obvious

modiﬁcations either of the hydrozoans or the hosts

were observed.

Fig. 8. Skeletal modiﬁcations in bryozoans hosting symbiotic hydrozoans. (a) Schizobrachiella sanguinea hosting Halocoryne epizoica. (b) Robert-

sonidra sp. hosting Zanclea pirainoid. (c) Triphyllozoon inornatum hosting Zanclea tipis. (d) Trematooecia aviculifera hosting Zanclea sp. (Cur-

ßao). (e) Celleporaria pigmentaria hosting Apatizanclea sp. 1. (f) Celleporaria sp. hosting Apatizanclea sp. 2. (g) Parasmittina cf. spondylicola

hosting Zanclea protecta. (h) Celleporaria cf. pigmentaria hosting Apatizanclea divergens (lineage IIb). Arrowheads indicate examples of skeletal

modiﬁcation in each image. Scale bars: 100 lm.

D. Maggioni et al. / Cladistics 0 (2023) 1–28 17

Fig. 9. Ancestral state reconstructions of the characters (a) symbiotic lifestyle, (b) host, (c) perisarc and (d) polymorphism. Pie charts represent

the posterior probability of each node being in each state. Grey circles at tips represent unknown states.

18 D. Maggioni et al. / Cladistics 0 (2023) 1–28

Fig. 10. Ancestral state reconstructions of the characters (a) heteronemes, (b) colony type, (c) reproduction and (d) habitat. Pie charts represent

the posterior probability of each node being in each state. Grey circles at tips represent unknown states.

D. Maggioni et al. / Cladistics 0 (2023) 1–28 19

Phylogenetic comparative methods

The ancestral state reconstructions and the tests for

evolutionary correlation between characters shed light

on the evolutionary patterns of the analysed ecological

and morphological characters (Figs 9and 10; Tables 1

and S5), although some of the reconstructed ancestral

states were supported by low Bayesian posterior proba-

bility (BPP) values and must therefore be considered

with caution. Additionally, it must be taken into

account that the inclusion of other taxa, genetic data

and information in this type of analysis may reshape the

observed patterns. The symbiotic lifestyle is likely to

have been absent in the most recent common ancestor

(MRCA) of the Capitata (node number referring

to Fig. S1,nn=1, BPP =0.69) and Corynida (nn =76,

0.62) (Fig. 9a) and in the latter group it appeared at

least three times. Contrarily, in Zancleida it may already

have been present in the MRCA of all taxa but Pennar-

iidae, Hydrocorynidae, Halimedusidae and Moerisiidae

(nn =3, BPP =0.73), and was then lost several times,

for instance in the MRCA of Cladocoryne Rotch, 1871

(nn =60, BPP =0.8), Porpitidae (nn =53,

BPP =0.94), Milleporidae (nn =10, BPP =1), Solan-

deriidae (nn =19, BPP =1), Asyncoryne ryniensis War-

ren, 1908, and some Zanclea species. Regarding the

host (Fig. 9b), the only reported associations in the

Corynida are with sponges and molluscs, and the asso-

ciation with Porifera also convergently evolved in the

Sphaerocorynidae +Zancleopsidae clade (nn =61,

BPP =0.65). Also the association with molluscs

appeared independently twice, in Zanclea costata and in

Coryne epizoica Stechow, 1921. Both the association

with octocorals and scleractinian corals appeared only

once, in the MRCA of Pteroclava +Pseudozanclea

(nn =55, BPP =0.83) and in the MRCA of coral-

associated Zanclea (nn =39, BPP =1), respectively.

The evolution of the association with bryozoans is made

uncertain by the low resolution of some phylogenetic

relationships among bryozoan-associated species. This

association is common in Zancleidae but it is not clear

whether it was present in the MRCA of the family and

the associations with other organisms represented sec-

ondary host shifts, or whether it evolved convergently

in different taxa. The association with Bryozoa was

already present in the MRCA of Halocoryne (nn =30,

BPP =0.97) and in the MRCA of Apatizanclea

(nn =23, BPP =0.99).

Regarding the evolution of morphological characters,

the perisarc was present in the MRCA of the Capitata

(nn =1, BPP =1) and was subsequently lost multiple

times in several Zancleida symbiotic lineages (Fig. 9c),

especially in species associated with bryozoans and

other cnidarians. The appearance of polymorphic polyps

Table 1

Results of the Pagel’s tests for correlated evolution of selected characters

Correlation tested Metrics Independent Dependent xDependent yDependent x&y

Symbiosis (x)—Perisarc (y) log-L 63.2738 58.2949 55.8664 54.8848

AICw 0.0031 0.0606 0.6878 0.2484

L-ratio 9.9577 14.8147 16.7779

p-value 0.0069 0.0006 0.0021

Symbiosis (x)—Heteronemes (y) log-L 75.2465 74.0568 74.0559 72.5561

AICw 0.4630 0.2059 0.2061 0.1250

L-ratio 2.3796 2.3812 5.3808

p-value 0.3043 0.3040 0.2504

Symbiosis (x)—Polymorphism (y) log-L 62.0238 61.1327 61.5771 60.2644

AICw 0.6069 0.2002 0.1284 0.0646

L-ratio 1.7821 0.8934 3.5188

p-value 0.4102 0.6397 0.4750

Symbiosis (x)—Reproduction (y) log-L 57.7026 54.8692 54.5473 53.9137

AICw 0.1373 0.3158 0.4358 0.1111

L-ratio 5.6667 6.3106 7.5777

p-value 0.0588 0.0426 0.1083

Symbiosis (x)—Colony (y) log-L 53.2162 45.6010 42.9474 42.9035

AICw 0.0002 0.0581 0.8250 0.1167

L-ratio 15.2304 20.5375 20.6253

p-value 0.0005 3.5e

5

0.0004

Symbiosis (x)—Habitat (y) log-L 80.1354 76.3706 79.4014 76.1916

AICw 0.1240 0.7239 0.0350 0.1171

L-ratio 7.5296 1.4681 7.8876

p-value 0.0232 0.4750 0.0958

The highest AICw for each analysis is in bold, as well as all statistically signiﬁcant models. Independent, the two characters evolve indepen-

dently; Dependent x, evolution of xdepends on evolution of y; Dependent y, evolution of ydepends on evolution of x; Dependent x&y,xand y

evolve interdependently; log-L, log-likelihood; AICw, Akaike information criterion weights; L-ratio, likelihood ratio.

20 D. Maggioni et al. / Cladistics 0 (2023) 1–28

only occurred among Zancleida (Fig. 9d), possibly in

the MRCA of Milleporidae +Solanderiidae +

Zancleidae +Asyncorynidae +Porpitidae (nn =5,

BPP =0.71), but the evolutionary patterns are obscured

by phylogenetic uncertainties and by the fact that the

character state is still unknown for several species. The

heteronemes, here including eurytele and mastigophore

capsules only, were present in the MRCA of the Zan-

cleida (nn =2, BPP =1) and were then lost a few times

in both symbiotic and nonsymbiotic species (Fig. 10a).

Contrarily, heteronemes were more probably absent in

the MRCA of Corynida (nn =76, BPP =0.69) and then

evolved again a few times. The type of colony seems to

be already stolonal in the MRCA of Capitata (nn =1,

BPP =0.75) and the erect state convergently evolved in

both Corynida and Zancleida, namely in some Coryne

species, in Stauridiosarsia cliffordi (Brinckmann-Voss,

1989), and in Pennariidae (nn =75, BPP =0.9), Mille-

poridae (nn =10, BPP =1) and Solanderiidae (nn =19,

BPP =1) (Fig. 10b). Two peculiar colony types, which

are solitary and pelagic, evolved singly in the MRCA of

Moerisia +Odessia (nn =71, BPP =0.84) and in the

MRCA of Porpitidae (nn =53, BPP =0.95), respec-

tively. The reproductive stage, here coded as free-

swimming medusa, medusoid and sporosac, was

a completely developed medusa in the MRCA of the

Capitata (nn =1, BPP =0.94), Zancleida (nn =2,

BPP =0.94) and Corynida (nn =76, BPP =1)

(Fig. 10c). A reduction was observed convergently in the

MRCA of a monophyletic Coryne clade (as sporosac)

(nn =82, BPP =1) and in different Zancleida lineages

(as medusoid), namely in the MRCA of Pennariidae

(nn =75, BPP =0.9), Cladocoryne (nn =60, BPP =

0.83), Solanderiidae (nn =19, BPP =1) and Millepori-

dae (nn =10, BPP =1), and also in the species Hetero-

coryne caribbensis Wedler & Larson, 1986.

Finally, we reconstructed the evolution of coloniza-

tion of tropical habitats and the MRCA of Corynida

appeared to live in nontropical (mostly temperate)

environments (nn =76, BPP =0.98) and only a few

Corynida species currently live in tropical waters

(Fig. 10d). However, the MRCA of all symbiotic Zan-

cleida species is likely to have been tropical (nn =3,

BPP =0.94), and most of the extant species included

in the analyses currently live in tropical or subtropical

environments.

The tests for correlation between the evolution of a

symbiotic lifestyle and the other investigated characters

revealed signiﬁcant relationships (Table 1). Speciﬁcally,

the correlations between the evolution of symbiosis and

perisarc were all statistically signiﬁcant with the best-

supported model being that the evolution of perisarc

depended on the evolution of symbiosis (AICw =

0.6878, p=0.0006). In a similar manner, also the evo-

lution of the reproductive stage and the colony type

seems to have been inﬂuenced by the evolution of

symbiosis. In the ﬁrst case, the only supported model,

even if with quite high p-value, was that the evolution

of the reproductive stage depended on the evolution of

symbiosis (AICw =0.4358, p=0.0426), whereas in the

second case all correlations were statistically signiﬁcant,

with the best-supported model being that the evolution

of colony type depended on the evolution of symbiosis

(AICw =0.8250, p=3.5e

5

). The evolution of symbio-

sis depended on the habitat type (AICw =0.7239,

p=0.0232), whereas the models of independent evolu-

tion of symbiosis in regards to heteronemes and poly-

morphism were preferred.

Discussion

Phylogenetic relationships within Capitata

The dataset assembled for this study is the most

complete employed so far for phylogenetic analyses of

the Capitata. Nevertheless, many taxa still remain

unsampled from a genetic point of view, leaving their

phylogenetic position and validity unresolved, includ-

ing 110 species, 13 genera and four families (Schu-

chert, 2023). The Capitata was recovered as a

monophyletic group composed of the two clades Zan-

cleida and Corynida, in agreement with previous work

(Nawrocki et al., 2010). Internal relationships within

Corynida were also in agreement with Nawrocki

et al. (2010), with the two monophyletic families Cory-

nidae and Cladonematidae. However, our sampling

mostly focused on Zancleida species, in order to disen-

tangle the remaining uncertainties in the phylogenetic

relationships within this group. By including previ-

ously unsampled species and genera, and by employing

a wider set of DNA regions compared to previous

studies, we were able to shed light on the phylogeny of

Zancleida and on the relationships among different

families.

Most of the analysed families resulted to be mono-

phyletic. Exceptions are represented by the

Halimedusidae +Moerisiidae clade and by the Zan-

cleidae. In the ﬁrst group, Tiaricodon orientalis, cur-

rently included in Halimedusidae, showed higher

afﬁnities with Moerisiidae. It must be taken into

account, however, that the two other Halimedusidae

species included (i.e. Urashimea globosa Kishinouye,

1910 and Octorhopalona saltatrix Toshino, Yamamoto

& Saito, 2022) were sequenced only in their 16S rRNA

gene and that the other species and genera (e.g. Hali-

medusa typus Bigelow, 1916, the type species of

Halimedusidae) should be sequenced to obtain a com-

prehensive overview of these two families.

The family Zancleidae is already known to be a

problematic group (Nawrocki et al., 2010; Maggioni

et al., 2018). The polyphyletic nature of this family

D. Maggioni et al. / Cladistics 0 (2023) 1–28 21

was further supported by our new data, with the

recovery of four distinct clades. Indeed, the general

morphology of the genus Zanclea,asseeninZ. costata,

is likely to represent an ancestral plesiomorphic condi-

tion that was retained in many species, but from which

much more derived groups such as Milleporidae and

Solanderiidae also evolved. The ”true” Zanclea genus

and, by consequence, the ”true” Zancleidae family

formed a cohesive group with high nodal support in all

analyses. This clade contained both well-established

species and undeterminable or cryptic species, such as

the coral-associated Zanclea complex (Maggioni et al.,

2022a), that could not be properly described owing to

the lack of suitable morphological characters or

to incomplete knowledge of the life cycles. Many Zan-

clea species were previously described rather superﬁ-

cially and incompletely, and without DNA data. These

issues exacerbated an already problematic taxon, com-

plicating the description of new species. It is now clear

that new species descriptions must always include

genetic data, to assess the divergence with already

sequenced taxa and to make possible future compari-

sons and identiﬁcations (Tautz et al., 2003). Concomi-

tantly, ﬂexible nomenclature systems have also been

proposed when species descriptions are not possible, for

instance when dealing with cryptic species, making pos-

sible the transfer of DNA-based taxonomic and biodi-

versity knowledge across disciplines (Morard

et al., 2016). Given these reasons, we stress the impor-

tance of avoiding new species descriptions without com-

plete morphological information and/or DNA data.

The position of the remaining zancleid species clus-

tered with members of the families Asyncorynidae,

Milleporidae and Solanderiidae in a clade whose inter-

nal phylogenetic relationships were not fully resolved.

Zanclea prolifera showed a sister-group relationship

with Asyncoryne ryniensis, although with low statistical

support. Nawrocki et al. (2010) suggested that

Z. prolifera may indeed need to be transferred to

Asyncorynidae, also because this species is known only

from its medusa stage (Uchida and Sugiura, 1976) and

Zanclea and Asyncoryne species have almost identical

medusae (Migotto, 1996). This issue currently could

not be resolved, and only the discovery of the

Z. prolifera polyps together with a wider sampling

could clarify the systematic afﬁnities of this species.

Two other zancleid clades were recovered from our

analyses, but their phylogenetic relationships could not

be resolved. For this reason, and because of the inter-

grading morphologies shared by all zancleid species, it

was impossible to split the Zancleidae into different

families, and taxonomic actions were undertaken only

at the genus level. Speciﬁcally, a ﬁrst group included

Halocoryne epizoica and two species previously

assigned to Zanclea (Hastings, 1932; Pica et al., 2017)

and now transferred to Halocoryne. These three species

showed some common traits that also are found in

some other zancleid species; however, they lack peri-

sarc and euryteles and are exclusively associated with

bryozoans, whose skeleton is modiﬁed by the presence

of the symbionts.

The last zancleid group comprised species previously

ascribed to the genus Zanclea (Puce et al., 2002; Mag-

gioni et al., 2018; Schuchert and Collins, 2021)and

showing some peculiarities. Given their genetic afﬁnities

and divergence from other species, they were included

in the newly erected genus Apatizanclea. Interestingly,

all known polyps have macrobasic holotrichous eury-

teles, no perisarc, and are associated with bryozoans,

whereas all known medusae have two bulbs and two

tentacles. These features are shared with a few other

species (Boero et al., 2000), but never together, and

constitute diagnostic characters for the genus.

Finally, Milleporidae and Solanderiidae were recov-

ered as sister groups, even if with moderate nodal sup-

port. These two families have modiﬁed erect colonies,

either with production of a calcium carbonate skeleton

or with the perisarc forming an internal chitinous skel-

eton, respectively. Other taxa not sampled here may be

related to Milleporidae and Solanderiidae species,

showing speciﬁc structures in their colonies, such as

internal (Pseudosolanderia Bouillon & Gravier-Bonnet,

1988) or external (Rosalinda Totton, 1949, Teissiera

Bouillon, 1974) chitinous skeletons. In a similar way

to the Solanderiidae, the genus Pseudosolanderia has

erect ﬂabellate colonies with an internal chitinous skel-

eton, which nevertheless shows differences compared

to Solanderia skeletons. Also, their cnidomes are dif-

ferent (Bouillon and Gravier-Bonnet, 1987; Bouillon

et al., 1992). Teissiera and Rosalinda are characterized

by stolonal colonies, often associated with other

organisms, both genera have chitinous basal plates,

and, in a similar way to Pseudosolanderia, have hetero-

nemes in their cnidome (Bouillon, 1974; Bouillon and

Gravier-Bonnet, 1987; Petersen, 1990; Gil et al., 2021).

As noted by Petersen (1990), it is not always clear

whether these heteronemes are euryteles or mastigo-

phores, which also has been observed in the genus

Millepora (Arrigoni et al., 2018). Teissiera is further

differentiated by having polymorphic polyps and

medusae with ocelli (Bouillon, 1974,1978). The inclu-

sion of these three genera in future molecular analyses

will hopefully help clarifying their phylogenetic posi-

tion, and the relationships among the Asyncorynidae,

Milleporidae, Solanderiidae and Zancleidae, and will

shed light on the evolution of colony modiﬁcations.

Character evolution in Capitata

Cnidarians, and especially hydrozoans, are known

for their large morphological diversity and variety of

life cycles (Bouillon et al., 2006; Cartwright and

22 D. Maggioni et al. / Cladistics 0 (2023) 1–28

Nawrocki, 2010). All three life stages (i.e. larva, polyp

and medusa) are extremely diversiﬁed in the Hydro-

zoa, and morphological simpliﬁcation or even reduc-

tion is a widespread phenomenon in the class

(Bouillon et al., 2006). This large variability, together

with the common intergrading morphologies between

species, genera and even families, hamper the under-

standing of both taxonomy and evolution of several

hydrozoan taxa. However, the use of molecular data

to test the evolution of morphological and ecological

traits has proven very useful in different taxa,

highlighting the frequent presence of convergent and

parallel evolutionary patterns (Lecl

ere et al., 2007,

2009; Cartwright and Nawrocki, 2010; Maggioni

et al., 2022a,b). Indeed, despite the presence of synap-

omorphies characterizing many taxa, homoplasy is

quite a common phenomenon in hydrozoans, as dem-

onstrated by, among others, the reduction of the

medusa stage, the presence of polymorphic polyps and

the colony type (Cartwright and Nawrocki, 2010).

Our results are in line with these previous ﬁndings,

because all of the examined characters showed some

degree of convergent or parallel evolution. Indeed,

regarding morphological characters, our ancestral state

reconstructions showed that the perisarc, polymorphic

polyps, heteronemes, stolonal colonies and reduced

medusa were frequently and independently lost among

the Capitata. Likewise, the establishment of a symbi-

otic lifestyle was acquired multiple times and then lost

again in certain taxa. The MRCA of Capitata seem-

ingly had nonsymbiotic stolonal polyps with perisarc

and heteronemes, and a free-swimming medusa,

whereas the presence of polymorphic polyps remained

unclear. Symbiosis, when present, is in most cases obli-

gate for the capitate hydrozoans (exceptions are, for

instance, Z. giancarloi,Z. sessilis,Zanclea cf. migot-

toi). Interestingly, coral-associated Zanclea species rep-

resent the only known case of hydrozoans associated

with scleractinian corals, whereas associations with

octocorals, sponges, molluscs, bryozoans and other

organisms are found not only in other noncapitate

hydrozoans, but also in other cnidarian taxa (e.g. Puce

et al., 2008; Kise et al., 2023).

Our analyses proved that the establishment of a

symbiotic lifestyle may have inﬂuenced the evolution

of other morphological characters, as already hypothe-

sized by previous authors (Boero et al., 2000; Puce

et al., 2002). Speciﬁcally, on the one hand, the best-

supported models of correlated evolution of the char-

acters were that the evolution of perisarc, reproductive

stage and colony type depended on the evolution of

the symbiotic lifestyle. On the other hand, the indepen-

dent evolution of heteronemes and polymorphism

related to the evolution of symbiosis were most sup-

ported. In our view, the most obvious result relates to

the evolution of perisarc, with the loss of this structure

being associated with the symbiotic lifestyle. The loss

of perisarc convergently occurred in some zancleid spe-

cies associated with bryozoans and scleractinian corals,

and in a cladocorynid species associated with octocor-

als. All of these associations are likely to be obligate

for the hydrozoan, and the loss of a protective perisarc

may have been promoted by a higher integration with

the hosts, which often cover the hydrorhiza with skele-

ton or tissue, and may be indicative of a high speciﬁc-

ity of the association (Boero et al., 2000; Puce et al.,

2002; Montano et al., 2015a,b; Maggioni et al.,

2020c). The loss of the perisarc, for instance, allows

the species Zanclea margaritae Panthos & Bythell,

2010 to ﬁrmly, but dynamically, attach to the host

coral skeleton using desmocytes, in a similar fashion

to how the coral tissue attaches to its skeleton (Pantos

and Hoegh-Guldberg, 2011). Previous works, focusing

on other hydrozoan species, also found a correlated

evolution of certain characters in relation to the sym-

biotic lifestyle. For instance, in hydractiniid species,

Miglietta and Cunningham (2012) detected a relation-

ship between the medusa loss and the specialization of

polyps to a single type of host and a relationship

between the colony type (e.g. encrusting or reticulate)

and the ability to live on speciﬁc hosts, thus highlight-

ing the importance of symbiosis in the evolution of

phenotypic and ecological traits and vice versa. How-

ever, the same authors did not ﬁnd a clear relationship

between the evolution of the medusa stage and the col-

ony type, opposite to what was found by Lecl

ere

et al. (2009) for leptothecate hydrozoans. Indeed, the

medusa stage has been repeatedly lost during hydro-

zoan evolution, and a class-wide analysis is needed to

possibly clarify the drivers of medusa loss or reduc-

tion. In this sense, important steps forward have

recently been made, with the discovery that the loss of

the homeobox gene Tlx is correlated with medusa sup-

pression in multiple clades (Travert et al., 2023).

Finally, we found a correlation between the evolu-

tion of the symbiotic lifestyle and the colonization of

tropical habitats, with most symbiotic species inhabit-

ing the latter environments. Tropical coral reefs are

biodiversity hotspots, showing an incredible diversity

of species and exploitable habitats (Fisher et al., 2015;

Hoeksema, 2017), and several symbiotic associations

are known to involve coral reef benthic organisms as

hosts (Montano, 2020), one of which is unique by

involving one Capitata species as the host and a spe-

cies of the hydrozoan suborder Filifera as an epibiont

(Montano et al., 2020). The richness of symbiotic rela-

tionships in these environments may be promoted by

the vast biodiversity, the three-dimensional habitats

provided by corals and other organisms, and the com-

petition for space and resources (Gates and Ains-

worth, 2011). Therefore, the colonization of tropical

habitats may have spurred the establishment of

D. Maggioni et al. / Cladistics 0 (2023) 1–28 23

symbiotic relationships between capitate hydrozoans

and other benthic organisms, and this, in turn, also

may have resulted in adaptive radiations in symbiotic

species, as already known for different coral-associated

organisms (e.g. Gittenberger and Gittenberger, 2005,

2011; Tsang et al., 2014; van der Meij et al., 2015;

Kunihiro et al., 2019; Fritts-Penniman et al., 2020;

Mehrotra et al., 2020), driven for instance by host

shifts. However, a more comprehensive sampling and

a time-calibrated phylogeny of the Capitata, at the

moment still not possible because of the lack of reli-

able fossil records, are needed to test this hypothesis.

Conclusions

The molecular phylogenetic hypothesis obtained here

represents the most comprehensive scenario produced so

far for the Capitata, shedding light on the systematic

afﬁnities and evolution of the group. Most of the families

were recovered as monophyletic, with a striking excep-

tion represented by the polyphyletic family Zancleidae,

making necessary the transfer of species in different gen-

era and the establishment of a new genus. The taxonomic

and systematic uncertainties in this family are exacer-

bated by the paucity of diagnostic morphological charac-

ters and the partially unknown life cycles, and future

integrative research targeting both already sampled and

unsampled taxa is strongly needed to unravel the com-

plex diversity and evolution of this group.

The analyses of the evolution of morphological and

ecological characters revealed high levels of convergent

or parallel evolution in divergent taxa, with indepen-

dent losses of several traits. In particular, symbiosis

appeared multiple times in the evolutionary history of

the Capitata, especially in the Zancleida, which is

likely to have been inﬂuenced by the colonization of

tropical habitats, and likely to have inﬂuenced the evo-

lution of other morphological characters, highlighting

the importance of symbiosis in animal evolution.

Acknowledgements

The authors wish to thank all the people involved in

collecting material or organizing sampling campaigns,

including Luca Fallati (University of Milano-Bicocca),

Luca Saponari (Nature Seychelles), Stephen Keable (Aus-

tralian Museum), Penny Berents (Australian Museum),

Anne Hoggett (Australian Museum), Lyle Vail (Austra-

lian Museum), Francesca Strano (Victoria University of

Wellington), Valerio Micaroni (Victoria University

of Wellington), and the staff of CARMABI Marine

Research Center at Curac

ßao. Permissions relevant to

undertaking the research have been obtained from the

applicable governmental agencies. The work was partially

funded by PADI Foundation grant nos 28634 and 14384

to DM and SM. SM is grateful to Naturalis Biodiversity

Center for providing Martin Fellowships, which sup-

ported ﬁeldwork in Curac

ßao. Samples from Eilat (Israel)

were collected during the HyDRa Project funded by the

EU FP7 Research Infrastructure Initiative ‘ASSEMBLE’

(grant no. 227799) to DP. Financial support to DP for

collecting samples at Lizard Island (Australia) was pro-

vided by the 2018 John and Laurine Proud Fellowship

and the Australian Museum’s Lizard Island Research

Station. Finally, we would like to thank Maria Mendoza

Becerril for re-analysing material in the collection: Cni-

darians of the Gulf of Mexico and Mexican Caribbean

”Lourdes Segura” (Faculty of Science, Multidisciplinary

Teaching and Research Unit, Sisal, Yucat

an).

Conﬂict of interest

None declared.

Data availability statement

The genetic data underlying this article are available

in the GenBank Nucleotide Database and can be

accessed with accession numbers OQ955308–OQ9

55478, OQ956203–OQ956256, OQ984068–OQ984146,

OQ992945–OQ993031. Alignments, raw phylogenetic

trees, and R scripts and ﬁles used for the

phylogenetic comparative methods are available at:

https://ﬁgshare.com/s/ff00a8fe9e61b1bde4e3.

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Supporting Information

Additional supporting information may be found

online in the Supporting Information section at the

end of the article.

Table S1. List of the samples included in the analy-

sis with associated data on the sampled life stage, sub-

strate, sampling locality and coordinates, GenBank

accession numbers, and whether they were included in

D. Maggioni et al. / Cladistics 0 (2023) 1–28 27

the Capitata analyses. Newly deposited sequences are

in bold.

Table S2. Length of each single-locus and

concatenated dataset used for the Capitata phyloge-

netic reconstructions, before and after the Gblocks

treatment.

Table S3. Morphological and ecological character

state for all species. States in brackets refer to the

recoding to make character states binary for correla-

tion analyses. 0, absence; 1, presence; g, generalist; b,

bryozoan; c, scleractinian coral; o, octocoral; s,

sponge; m, mollusc; ma, medusa; md, medusoid; sp,

sporosac; st, stolonal; er, erect; so, solitary (vs. colo-

nial); pe, pelagic; tr, tropical; nt, nontropical; wd, both

tropical and nontropical; ol?, unknown.

Table S4. Pairwise average genetic distances within

and between species belonging to Zanclea,Apatizan-

clea and Halocoryne calculated as % uncorrected p-

distances. Interspeciﬁc distances are reported in the

lower left portions, whereas standard deviations in the

upper right portions; intraspeciﬁc distances and rela-

tive standard deviations are reported along the

diagonal.

Table S5. Posterior probabilities for ancestral char-

acter states reconstructed at each node, for all exam-

ined characters. Node numbers refer to Fig. S1.

Figure S1. Bayesian phylogenetic hypothesis of the

Capitata showing the node numbers to which Table S1

refers.

28 D. Maggioni et al. / Cladistics 0 (2023) 1–28

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May 2022

Twenty-four Recent species of the boreal-Arctic and Pacific cheilostome bryozoan genus Rhamphostomella are described. The species R. tatarica and R. pacifica are transferred to Rhamphostomella from Posterula and Porella, respectively. Eight species are new: R. aleutica n. sp., R. aspera n. sp., R. commandorica n. sp., R. echinata n. sp., R. microavicularia n. sp., R. morozovi n. sp., R. multirostrata n. sp. and R. obliqua n. sp. Neotypes are selected for six species, and lectotypes for eight species. Mixtoscutella n. gen. is established for several Rhamphostomella-like species, including M. androsovae [formerly Smittina androsovae Gontar], M. cancellata [formerly Escharella porifera forma cancellata Smitt], M. harmsworthi [formerly Schizoporella harmsworthi Waters], M. ovata [formerly Cellepora ovata (Smitt)], and M. ussowi [formerly Schizoporella ussowi (Kluge)]. In addition to taxonomic revision, the morphology (frontal shields, ovicells and multiporous septula), ecology and zoogeography of these cheilostomes are discussed, and identification keys are presented. Most species of Rhamphostomella have broad bathymetric distributions. Some have long protuberances on their basal walls that allow them to grow elevated above allelopathically active substrates such as sponges. The diversity of Rhamphostomella peaks in the northwestern Pacific.

Integrative systematics illuminates the relationships in two sponge-associated hydrozoan families (Capitata: Sphaerocorynidae and Zancleopsidae)

Article

Full-text available

Nov 2021
CONTRIB ZOOL

An integrated approach using morphological and genetic data is needed to disentangle taxonomic uncertainties affecting the hydrozoan families Sphaerocorynidae and Zancleopsidae. Here we used this approach to accurately characterise species in these families, identify the previously unknown polyp stages of the genera Euphysilla and Zancleopsis, which were originally described exclusively based on the medusa stages, describe a new sphaerocorynid genus and species, and assess the phylogenetic position of the two families within the Capitata. The monotypic genus Astrocoryne was found to be a synonym of Zancleopsis. Astrocoryne cabela was therefore transferred to the genus Zancleopsis as Zancleopsis cabela comb. nov. The new polyp-based genus and species Kudacoryne diaphana gen. nov. sp. nov. was erected within the Sphaerocorynidae. Both taxa are primarily based on genetic data, but the introduction of this new genus was made necessary by the fact that it clustered with the genera Heterocoryne and Euphysilla, despite showing Sphaerocoryne-like polyps. Interestingly, the species analysed in this work showed contrasting biogeographical patterns. Based on our data and literature records, some species appear to have a wide circumtropical range, whereas others are limited to few localities. Overall, these results lay the ground for future investigations aimed at resolving the taxonomy and systematics of these two enigmatic families.

Evolutionary patterns of host switching, lifestyle mode, and the diversification history in symbiotic zoantharians

Article

Feb 2023
MOL PHYLOGENET EVOL

Symbioses play important roles in forming the structural and distributional patterns of marine diversity. Understanding how interspecies interactions through symbioses contribute to biodiversity is an essential topic. Host switching has been considered as one of the main drivers of diversification in symbiotic systems. However, its process and patterns remain poorly investigated in the marine realms. Hexacoral species of the order Zoantharia (=zoantharians) are often epizoic on other marine invertebrates and generally use specific taxa as hosts. The present study investigates the patterns of host switching and the diversification history of zoantharians based on the most comprehensive molecular phylogenetic analyses to date, using sequences from three mitochondrial and three nuclear markers from representatives of 27 of 29 genera. Our results indicate that symbiotic zoantharians, in particular those within suborder Macrocnemina, diversified through repeated host switching. In addition, colonization of new host taxa appears to have driven morphological and ecological specialization in zoantharians. These findings have important implications for understanding the role of symbioses in the morphological and ecological evolution of marine invertebrates.

The Parsimony Ratchet, a New Method for Rapid Parsimony Analysis

Article

Dec 1999
CLADISTICS

Kevin C. Nixon

The Parsimony Ratchet1 is presented as a new method for analysis of large data sets. The method can be easily implemented with existing phylogenetic software by generating batch command files. Such an approach has been implemented in the programs DADA (Nixon, 1998) and Winclada (Nixon, 1999). The Parsimony Ratchet has also been implemented in the most recent versions of NONA (Goloboff, 1998). These implementations of the ratchet use the following steps: (1) Generate a starting tree (e.g., a "Wagner" tree followed by some level of branch swapping or not). (2) Randomly select a subset of characters, each of which is given additional weight (e.g., add 1 to the weight of each selected character). (3) Perform branch swapping (e.g., "branch-breaking" or TBR) on the current tree using the reweighted matrix, keeping only one (or few) tree. (4) Set all weights for the characters to the "original" weights (typically, equal weights). (5) Perform branch swapping (e.g., branch-breaking or TBR) on the current tree (from step 3) holding one (or few) tree. (6) Return to step 2. Steps 2-6 are considered to be one iteration, and typically, 50-200 or more iterations are performed. The number of characters to be sampled for reweighting in step 2 is determined by the user; I have found that between 5 and 25% of the characters provide good results in most cases. The performance of the ratchet for large data sets is outstanding, and the results of analyses of the 500 taxon seed plant rbcL data set (Chase et al., 1993) are presented here. A separate analysis of a three-gene data set for 567 taxa will be presented elsewhere (Soltis et al., in preparation) demonstrating the same extraordinary power. With the 500-taxon data set, shortest trees are typically found within 22 h (four runs of 200 iterations) on a 200-MHz Pentium Pro. These analyses indicate efficiency increases of 20×-80× over "traditional methods" such as varying taxon order randomly and holding few trees, followed by more complete analyses of the best trees found, and thousands of times faster than nonstrategic searches with PAUP. Because the ratchet samples many tree islands with fewer trees from each island, it provides much more accurate estimates of the "true" consensus than collecting many trees from few islands. With the ratchet, Goloboff's NONA, and existing computer hardware, data sets that were previously intractable or required months or years of analysis with PAUP* can now be adequately analyzed in a few hours or days.

Analyzing Large Data Sets in Reasonable Times: Solutions for Composite Optima

Article

Dec 1999
CLADISTICS

Pablo A. Goloboff

New methods for parsimony analysis of large data sets are presented. The new methods are sectorial searches, tree-drifting, and tree-fusing. For Chase et al.'s 500-taxon data set these methods (on a 266-MHz Pentium II) find a shortest tree in less than 10 min (i.e., over 15,000 times faster than PAUP and 1000 times faster than PAUP*). Making a complete parsimony analysis requires hitting minimum length several times independently, but not necessarily all "islands" for Chase et al.'s data set, this can be done in 4 to 6 h. The new methods also perform well in other cases analyzed (which range from 170 to 854 taxa).

Systematics and character evolution of capitate hydrozoans

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