Zoological Studies 62:49 (2023)
doi:10.6620/ZS.2023.62-49

Open AccessIntegrative Systematics and Biogeography ofthe Hydrozoans (Leptothecata: Eirenidae) Eirene
menoni Kramp, 1953 and Eirene lacteoides
Kubota and Horita, 1992 from Japan and China
with Comments on Pacific Ocean Distributions
Gerald L. Crow1,* , Brenden S. Holland2 , Gaku Yamamoto3, Shuhei Ikeda4, Aya Adachi3, andKelley Niide5
1

Ocean Research Explorations, P. O. Box 235926, Honolulu, Hawaii 96823. *Correspondence: E-mail: gcrow@hawaii.edu (Crow)
Department of Natural Sciences, College of Natural and Computer Sciences, Hawaii Pacific University, 45-045 Kamehameha Highway, Kaneohe,
Hawaii 96744. E-mail: bholland@hpu.edu (Holland)
3
Enoshima Aquarium, 2-19-1 Katase Kaigan, Fujisawa, Kanagawa Prefecture 251-0035, Japan. E-mail: g-yamamoto@enosui.com (Yamamoto);
adati@enosui.com (Adachi)
4
Tsuruoka City Kamo Aquarium, 656 Okubo Imaizumi, Tsuruoka, Yamagata Prefecture 997-1206, Japan. E-mail: s.ikeda@kamo-kurage.jp (Ikeda)
5
Waikiki Aquarium, University of Hawaii at Manoa, 2777 Kalakaua Avenue, Honolulu, Hawaii 96815. E-mail: kelleyl@hawaii.edu (Niide)
2

Received 4 November 2022 / Accepted 4 August 2023 / Published 5 October 2023
Communicated by James D. Reimer

The hydrozoan family Eirenidae is known scientifically for its morphological plasticity and challenges in
species identification. We used an integrative taxonomic approach based on morphological, molecularand life history evidence to systematically assess field-collected medusae of Eirene menoni Kramp
1953 and captive raised polyps of both E. menoni and E. lacteoides Kubota and Horita 1992. Following
morphological review, we updated the genus description to include the presence of rudimentary bulbs(warts) on the ring canal in at least eight of the 24 valid Eirene species. We propose the potential for themature E. menoni hydrotheca to develop into a gonotheca. However, this proposal will require additional
study for verification. We provide validated distribution records from the Indo-Pacific Ocean for E. menoni,
and updated collection records for E. lacteoides from the Yellow and East China Seas, and public
aquaria-cultured specimens from Japan and Hawaii, using cytochrome c oxidase I (COI) sequences that
we generated and compared with those from GenBank. The COI gene reliably separated four species,
each forming a monophyletic clade with strong bootstrap support and low mean intraspecific moleculardivergences (≤ 1%) within clades. However, some of the deeper nodes of the tree remained poorly
resolved, and our analysis failed to demonstrate monophyly among eirenid genera Eirene and Tima. Our
integrative taxonomic approach is essential in confirming species identity within the family Eirenidae andgenus Eirene, and we have also identified a likely range expansion of E. lacteoides to Hawaii.
Key words: Hydrozoa, Leptomedusae, Plasticity, Campanulinida hydroid, COI mtDNA(Bouillon et al. 2006; Moura et al. 2008; He et al.
2015; Maronna et al. 2016). Genetic barcoding has
provided a useful tool for gelatinous zooplankton
identification, enhancing the ability to rapidly assess

BACKGROUND
Hydrozoans are well known for their phenotypic
plasticity and challenging taxonomic identification

Citation: Crow GL, Holland BS, Yamamoto G, Ikeda S, Adachi A, Niide K. 2023. Integrative systematics and biogeography of the hydrozoans(Leptothecata: Eirenidae) Eirene menoni Kramp, 1953 and Eirene lacteoides Kubota and Horita, 1992 from Japan and China with comments on
Pacific Ocean distributions. Zool Stud 62:49. doi:10.6620/ZS.2023.62-49.
© 2023 Academia Sinica, Taiwan

1Zoological Studies 62:49 (2023)

regional biodiversity, reveal the presence of invasive
taxa, and delineate species distributions (Holland et al.
2004; Zhou et al. 2013; Scorrano et al. 2017). However,misidentifications in the GenBank database (www.ncbi.
nlm.nih.gov/genbank/) have led to sequences that do notaccurately represent the associated binomials (Moura etal. 2008; Lindsay et al. 2017). An additional challenge
lies in the fact that at present, DNA sequences are rarely
published from species type localities, and we argue that
this should be standard taxonomic practice.
DNA barcoding typically relies on 16S rRNA
or COI mtDNA sequences for species identification,although both markers have shown polyphyletic andparaphyletic patterns in the hydrozoan genera Eirene
and Clytia (Zheng et al. 2014; He et al. 2015; Zhang
et al. 2015). Due to the challenges posed by jellyfish
species taxonomy and frequent-misidentifications,accurate species recognition should include
morphological descriptions of polyps, juvenile andadult medusae, documentation of complete life history,
and molecular systematics (Straehler-Pohl et al. 2011;
Lindsay et al. 2017; Toshino et al. 2019; Lawley et al.
2021). The 16S gene has been recommended for basal
clade and species identification in this group (Zheng
et al. 2014; Maronna et al. 2016). However, the COI
gene also provides a reliable species-level identification(Laakmann and Holst 2014; He et al. 2015; Zhang et al.
2015; Bucklin et al. 2021; Calder et al. 2021). Folmer etal. (1994) and Bucklin et al. (2021) reported that COI:
1) is broadly applicable due to availability of universal
primers, effective in amplifying this fragment froma wide range of phyla; 2) tends to show informative
phylogenetic signals over a range of taxonomic levels; 3)
evolves rapidly enough to allow for the discriminationof very closely related species; and 4) tends to reveal
phylogeographically significant intraspecific variation
associated with fine scale distribution patterns.
The hydrozoan family Eirenidae has proven to
be a taxonomically difficult group to classify, due tomultiple factors, including a lack of comprehensive
species descriptions, a scarcity of detailed knowledge oflife cycle information for most species, and unusuallyhigh evolutionary distances between congenericspecies (e.g., Calder et al. 2021). This has resulted ininadequate family level assessment (Maronna et al.
2016), problematic generic relationships among Eirene,
Clytia (Schuchert 2017), and Tima (Calder et al. 2021),
and even misidentifications in species groupings (He etal. 2015; Maronna et al. 2016).
The Eirene genus comprises three documented
species from Japan: E. hexanemalis (Goette, 1886), E.
lacteoides (with the type locality being Toba Aquarium),and E. menoni. Additionally, there is an as-yetundescribed species (Kubota and Gravili 2007) within© 2023 Academia Sinica, Taiwan

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the genus. But since misidentifications continue toplague this group, our objective here is to use historical
records to revise morphological descriptions and use
molecular tools to improve species identifications.
We also recommend that other researchers focusing
on hydromedusan biology follow the same approach.
We worked with preserved specimens obtained froma regional museum collection, captive bred aquarium
samples, and field collected samples. We examinedpublished biogeographic and taxonomic descriptions,medusa and polyp morphology, and phylogenetic
analyses of E. menoni and E. lacteoides to verify
taxonomic status and distribution. Eirene menoni
was originally described from a single hydromedusa
collected off of the Great Barrier Reef and was
subsequently documented throughout the Indo-Pacific(Kramp 1953; Kramp 1968; Gershwin et al. 2010;
Zheng et al. 2014). In Japanese waters, E. menoni has
been documented from Kumamoto Prefecture, Kyushu(Sugiura 1979), Enoshima, Kanagawa Prefecture,
Honshu (Sakiyama and Adachi 2001), Hamanako (Lake
Hamana), Shizuoka Prefecture, Honshu (Okamoto etal. 2016), and northern Oita Prefecture, Kyushu (Iwai
2021). Eirene lacteoides was described from medusae
cultured in the Toba Aquarium, Mie Prefecture, Honshu,
Japan (Kubota and Horita 1992) and cultured polyps at
the Qingdao Marine Science Museum, Qingdao, China(Huang et al. 2009). We reconfirmed the identities of
COI sequences of E. lacteoides from GenBank which
had been previously misidentified as Tima formosa
Agassiz, L. 1862. These sequences had been collectedin the field from Chanjiang River Estuary, China[JQ71666-67] and Jiaozhou Bay, China [JQ71616870], and from cultured samples at the Waikiki
Aquarium, Oahu, Hawaii. Schuchert (2017) reported
16S [FJ418650] and 18S [FJ418671] sequences fromhydromedusan specimens collected in the “South
China Sea” (submitted to GenBank by Zheng, exact
geographic source unknown) as Eirene lacteoides. We
again utilized an integrative taxonomic approach (Dayrat2005) that included descriptions of the entire life cycle,
documentation of geographic sources, phylogenetic
reconstruction, and morphological descriptions.
Finally, in this study, we verify the phylogeographic
distributions of these two species in the Pacific Ocean.
MATERIALS AND METHODS
Collecting and culturing
Wild specimens of Eirene menoni were collected(12 Sep 2021) using a rectangular dipnet (32 cm ×19 cm with 0.5 mm mesh) or an open-top plastic 1.5 L

Zoological Studies 62:49 (2023)

container on a pole from shallow waters (0–1 m)
at Shonan Fishing Port, Sagami Bay, Kanagawa
Prefecture, Japan. Cultured specimens of E. lacteoides
that originated at the Toba Aquarium and produced
multiple generations at both the Enoshima Aquarium
and Tsuruoka City Kamo Aquarium (Kamo Aquarium)
were examined. This species also was discovered in
sea water tanks at the Waikiki Aquarium in July 2019.
Cultured medusae and polyp samples were placed insmall plastic containers (4 L) supplied with aeration
to ensure growth and were transferred to (3 L) kreisel
aquaria (Hamner 1990; Raskoff et al. 2003). Hydroids

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and hydromedusae were maintained on a diet of either
Vietnamese (Japan) or Great Salt Lake (Hawaii) strainsof Artemia nauplii, fed once per day. Mean watertemperatures were maintained at 20°C and salinity
range was kept between 30–35 psu. Field salinity
was measured with a ATAGO ® (MASTER-S/milla)
refractometer. Water quality was maintained via regularwater changes. Medusae and hydroids were preserved
in 3% formalin seawater solution for anatomicalevaluation and 90% ethanol for DNA extraction andsequencing.

Fig. 1. Anatomy diagram of a typical Eirenidae medusa and polyp with labels. Some sections cut away. Medusa not to scale. Drawing of medusa(1A) by Brenden Holland, image of ring canal with marginal tentacles and rudimentary bulbs (1B, C) by Gerald Crow, and image of polyps (1D) by
Shuhei Ikeda, Tsuruoka City Kamo Aquarium.
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Morphology and systematics
Basic terminology and morphology of medusa
and polyps are provided in (Fig. 1). The formalin
specimens were morphologically examined to verify
species identity as Eirene menoni and Eirene lacteoides
and measured with a Scienceware® Bel-Art Products
dial caliper to the nearest 0.1 mm. An Olympus
SZX16 Stereomicroscope with a Teledyne Lumenera
INFINITY3-1 ® , 1.4 megapixel CCD microscopy
camera, was used for morphological descriptions andimages. We utilized a 0.5X camera objective with
0.7X to 13.5X magnification for viewing and imaging.
Medusa bell size, morphology, and marginal tentacle
numbers were compared to the original descriptions foreach species. We also examined and imaged hydroids
from both the Enoshima and Kamo Aquaria, and used
Bouillon et al. (2006) for anatomical terminology todescribe the medusae and hydroids for both species.
Museum specimens of E. menoni at the South
Australian Museum, Adelaide (SAMA), South Australia
were inspected for geographic location, taxonomic
verification, and images were recorded. Voucher
specimens from Japan for both species were deposited
into the National Museum of Nature and Science Tokyo(NSMT), Tsukuba, Japan and samples of E. lacteoides
from Hawaii were deposited into the Bernice P. Bishop
Museum (BPBM) Honolulu, Hawaii.
DNA extraction, PCR amplification, and DNA
sequencing
The hydromedusae specimens from Japan were
supplied in an ethanol storage buffer. These specimens
were carefully extracted and rinsed with deionizedautoclaved water. Genomic DNAs were extracted
from each whole organism using a Macherey-Nagel
NucleoSpin® Mini kit for DNA from cells and tissue
according to the manufacturer’s protocol. Genomic
DNA was eluted in 200 µL of deionized autoclaved
water and stored at -20°C.
We used existing and additional GenBank COI
gene fragments to examine the species level andgenus level phylogenetic position of Eirenidae species(Table 1). Fragments of 613 basepairs (bp) of themitochondrial DNA (mtDNA) cytochrome c oxidase
I (COI) gene were amplified by polymerase chain
reaction (PCR) using the primers LCO1490/HCO2198(Folmer et al. 1994). Target fragments were amplified
using a MyCycler® Thermal Cycler (Bio-Rad, Hercules,

Table 1. Taxa included in COI phylogenetic analysis, with GenBank accession numbers, sampling locations, and GPS
coordinates. Note that geographic source of collection is not always known with confidence for specimens obtained
from public aquaria, and for two gene sequences from China. The GenBank entries that lacked geographic source
information are marked as unknown. In cases where more than one COI sequence was included from a location, thenumber of samples is indicated (n)Species

GenBank Accession # (n)Location

Geographic Coordinates

Eirene menoni
Eirene menoni
Eirene menoni
Eirene menoni
Eirene menoni
Eirene hexanemalis
Eirene lacteoides

FJ418662
JQ716132
JQ716133-34 (2)JQ716135-37 (3)OP175931-32 (2)JQ716149-51 (3)OP175929-30 (2)China
Zhujiang River Estuary, China
Beibu Gulf, China
Xaimen Bay, China
Shonan Fishing Port, Enoshima, Japan
Beibu Gulf, China
Enoshima Aquarium, Japan (captive culture)

Eirene lacteoides
Eirene lacteoides
Eirene lacteoides (as Tima formosa)Eirene lacteoides (as Tima formosa)Eirene viridula
Tima bairdii

OP175926-28 (3)FJ418661
JQ716166-67 (2)JQ716168-70 (3)KC44018-19 (2)MG935038
MF000509
MW490678
MW490677
MW490679
KM200330
LC198742

Waikiki Aquarium, Hawaii (captive culture)
China
Changjiang River Estuary, China
Jiaozhou Bay, China
German Bight, North Sea
Skagerrak, Sweden
Fanafjord, Norway
Sendai, Japan
Fukushima, Japan
Fukushima, Japan
Waikiki Beach, Oahu
Palau, Federated States of Micronesia

Unknown
22.51°N, 113.69°E
20.85°N, 109.26°E
24.39°N, 118.14°E
35.10°N, 139.50°E
20.95°N, 108.76°E
Originally from
Toba Aquarium
Unknown
Unknown
31.50°N, 122.20°E
36.10°N, 120.30°E
54.20°N, 7.90°E
58.35°N, 9.93°E
58.84°N, 6.30°E
38°N, 120.25°E
36.90°N, 140.90°E
36.90°N, 140.90°E
21.28°N, 157.83°W
7.515°N, 134.58°E

Tima nigroannulata

Alatina alata
Cassiopea sp.

© 2023 Academia Sinica, Taiwan

Zoological Studies 62:49 (2023)

CA, USA) with Conquest PCR Master Mix Optimizing
Pack reagents and buffers from Lamba Biotech (catalog#D911-Mix1234). Twenty µL PCR reactions were run
at Hawaii Pacific University’s Oceanic Institute, Oahu,
Hawaii, under the following conditions: 4 minutes at
94°C, followed by 33 cycles of 94°C for 40 seconds,
52°C for 30 seconds and 72°C for 90 seconds, with afinal 72°C extension for 5 minutes.
PCR fragments were purified with MachereyNagel NucleoSpin Gel and PCR clean-up columns, per
the manufacturer’s protocol, and visualized via agarose
minigel electrophoresis. Amplified mtDNA fragments
were sequenced using the forward primer. DNA
sequencing was performed at the Advanced Studies inGenomics, Proteomics and Bioinformatics (ASGPB) lab
at the University of Hawaii at Manoa, Oahu, Hawaii.
Phylogenetic analysis
Randomized Axelerated Maximum Likelihood[RAxML] (Stamatakis 2014) and Molecular
Evolutionary Analysis [MEGA X 11.0.13] (Tamura etal. 2021) were used for phylogenetic reconstruction(Table 1). The tree was generated with 1000 bootstrap
replicates using the maximum-likelihood optimality
criterion with the empirically determined best-fit
substitution model. The optimal model selected was
Tamura-Nei. Non-uniformity of evolutionary rates
among sites was modeled using a discrete gamma
distribution (+G) with 5 rate categories under theassumption that a specified fraction of sites were
evolutionarily invariable (+I), resulting in the model
TN93+G+I. This model had 66 parameters, BIC= 6461.115 (Bayesian Information Criterion), AICc
= 6461.12 (Akaike Information Criterion, corrected),
maximum likelihood score lnL = -2907.703, gamma
correction (G) = 1.40, and the proportion of invariantsites (I) = 0.49. Genetic distances among andwithin clades were determined using the Kimura
2-parameter substitution model. We selected species
for this phylogenetic analysis that were clearlymorphologically valid, and therefore reflect correct
binomial identifications. Intraspecific (within species)
and interspecific (between species) differentiation werecompared to aid our understanding of phylogenetic
relationships.
RESULTS
Taxonomic Account
Phylum Cnidaria Verrill, 1865
Subphylum Medusozoa Petersen, 1979
Class Hydrozoa Owen, 1843

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Subclass Hydroidolina Collins, 2000
Order Leptothecata Cornelius, 1992
Family Eirenidae Haeckel, 1879
Genus Eirene Eschscholtz, 1829
There are 24-valid Eirene species in the World
Registry of Marine Species [WoRMS database] (www.
marinespecies.org/aphia.php?p=taxdetails&id=117080
accessed 17 June 2022). Amended Genus description
based on (Kramp 1961 1968; Bouillon and Boero 2000;
Bouillon et al. 2006; Schuchert 2017; this study).
Typically small medusae ≤ 32 mm bell diameter,with a distinct gastric peduncle, no marginal or lateral
cirri, with or without excretory pores, numerous
statocysts present (may disappear in preserved
specimens), with or without rudimentary bulbs (warts),
these bulbs are present on the ring canal in at least eightspecies [E. conica Du, Xu, Huang and Guo, 2010, E.
elliceana (Agassiz and Mayer, 1902), E. hexanemalis,
E. kambara Agassiz and Mayer, 1899, E. lacteoides,
E. macrogonia Huang, Sun and Liu, 2019, E. palkensis
Browne, 1905, E. tenuis (Browne, 1905)], with 4–12
simple radial canals, gonads only on subumbrella
portions of radial canals that do not extend down thepeduncle.
Eirene menoni Kramp, 1953
Menon’s hydromedusa
Eirene-Kurage
Type locality. Great Barrier Reef, outside Trinity
Harbor, near Cairns, Queensland, Australia (~16°S,
146°E), water temperature 25.7°C, at 32 m depth. One
specimen captured 5 Dec 1928. Holotype deposited in
Natural History Museum, London, UK [British Museum
Natural History BMNH 1954.3.4.559] (Not seen).
Material examined
Medusae- SAMA H3614 (4 specimens) wildcollected Aldinga Reef, South Australia, 12 Feb
1961 [Fig. 2A] (see Kramp 1965). SAMA H3615(1 specimen, 10.6 mm bell width) wild collected
between Hopkins and Thistle Islands, South Australia,
01 Jan 1964 collected by Ronald Vernon Southcott (Fig.
2B). SAMA H1605 (1 specimen, current 13.38 mm
bell diameter) wild collected Pumicestone Passage,
Moreton Bay, Queensland, Australia (~27.6°S, 153°E),
Feb 2000- collected by Puk Petersen. NSMT-Co 1808
(9-specimens) wild collected Shonan Fishing Port,
Kanagawa Prefecture, Japan (35.17°N, 139.28°E),
collected near surface 12 Sept 2021, Gaku Yamamotocollector. NSMT-Co 1810 (1 specimen) medusa Kamo
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Zoological Studies 62:49 (2023)

Aquarium, Shuhei Ikeda collector. NSMT-Co 1811
(1 specimen) medusa Kamo Aquarium, Shuhei Ikeda
collector. NSMT-Co 1812 (1 specimen) medusa Kamo
Aquarium, S. Ikeda collector.
Polyps- NSMT-Co 1809 cultured colony Kamo
Aquarium preserved 1 Oct 2021, transferred from
Enoshima Aquarium 31 Jan 2007 [Shuhei Ikeda, perscomm].
GenBank sequences - for E. menoni (see Table 1).
Amended species description
After Kramp (1953) original description, modified
by Bouillon (1984) description of polyp, Bouillon(1995), and this study. Medusae-Bell more flat thanbell shaped; Bell diameter usually 12 mm may reach
20 mm, narrow tubular peduncle extending basicallysame size down to manubrium (Fig. 3A), manubriumand oral “lips” exceed bell height in living specimens,
oral “lips” strap-like in young specimens less than
3.2 mm bell diameter (Fig. 3B), four radial canals withgonads that begin to develop in mid-radial canal andextend to the bell ring canal (Fig. 3B–D), variabilityof gonad formation was described by Kramp (1953)
based on one specimen with gonads extending from thebase of the peduncle almost to the ring canal. Kramp(1965) reported that gonadal length was variable andin Victoria, South Australia specimens extends fromnear the radial ring canal to more than half the distanceto the base of the peduncle. Sugiura (1979) illustrated
and stated, “gonads begin to be formed on the radial

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canals neighboring the basal part of the stomachal
peduncle and with growth of the medusa they graduallyelongate downwards and widen along the radial canal.”
Marginal tentacles typically same length, and about 48
in number, may reach 54 with some small developing
marginal tentacles. Fully formed tentacle number from
Sagami Bay, Japan varies from 16 to 20 in 3.2–5.1 mm
bell diameter and 30 to 36 in 5.2–8.0 mm bell diameter
specimens. Color of tentacle bulb and between bases ofthe four oral “lips” varies from green, reddish brown todark brown. No cirri, excretory papillae or rudimentary
bulbs (warts) are present on ring canal, canal contains
1–3 statocysts between successive tentacles (not visible
after preservation). Reproduction through a polyp andplanula stage. Cnidome composed of elongated fusiform
microbasic mastigophores, measuring 10.5 × 3.0 µm to
9.0 × 2.5 µm. Typically found near surface, often nearrivers or estuaries, down to a depth of 51 m.
Polyps (live and fixed) - Released planula settle
at 24 hours and differentiates in polyp stage after
three days. Attached planula forms stolonal colonies,
arising from creeping hydrorhiza. Hydrorhizal stolons
long, slender with distance between polyps (Fig. 3E),
hydrothecal pedicels with a thin annulated hydrocaulus
that extend in a strong, extensible polyp. The polypgrows erect with a long hydrocaulus, hydranths canbe large or reduced, narrowest at base, gastric region
typically vase-to slightly club-shaped, constrictedshort distance below tentacular whorl, constriction
usually pronounced but sometimes, indistinct, hydranth
expanding again at the distal end, here becoming

Fig. 2. Eirene menoni medusa Australia images. (A) SAMA H3614 (current bell width 14.2 mm) collected 12 Feb 1961 at Aldinga Reef, Victoria,
South Australia and identified by Paul Kramp. (B) SAMA H3615 (current bell width 10.6 mm) collected 1 Jan 1964 between Hopkins and Thistle
Islands, Victoria, South Australia and identified by Ronald V. Southcott. Images courtesy of the South Australian Museum (Shirley Sorokin, Andrea
Crowther and Peter Hunt).
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Fig. 3. Eirene menoni medusae and polyps Japan images. Medusae (A–D) are images from preserved specimens NMST-Co 1808 originally collected
at Shonan Fishing Port, Kanagawa Prefecture, Japan (collected by Gaku Yamamoto). Polyps (E–F) are images of preserved cultured specimens
NMST-Co 1809 from the Kamo Aquarium, Japan (collected by Shuhei Ikeda). Image (E) retracted tentacles on the long hydrocaulus and (F) shape ofhydrotheca buds. Scale bars: A–D = 1 mm; E–F = 0.125 mm.
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subspherical to knob-shaped (Fig. 3E, F) supporting
whorl of tentacles; tentacles filiform, in one whorl, 10
marginal tentacles; their bases with intertentacular web.
Hydroid hydranth tentacles when disturbed or preserved
retract almost completely into the hydrotheca (Fig. 3E).
Gonotheca unknown. Polyps appear as “campanulinida”
type. The polyp cnidome is represented by ellipticalatrichs measuring 6.0 × 2.5 µm to 5.0 × 2.0 µm.
Indo-Pacific Ocean biogeographic distribution
for 21-records of Eirene menoni [alphabetized bygeographic source with map numbers] (Fig. 4).
Australia - (1) Trinity Harbor, Cairns [~16°S,
146°E], (7) Sydney, NSW [~33.8°S, 151.2°E], (11)North Cape, Northern Territory [10°S, 143°E],(12) Moreton Bay, Queensland [27.3°S, 153.3°E],(17) Aldinga Reef, Gulf of Saint Vincent [35.16°S,
138.46°E], (18) Broome, North West Territory [17.96°S,

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122.24°E], (Kramp 1953; Kramp 1965; Gershwin etal. 2010). China - (2) Chengshan [37.2°N, 122.2°E],(as Phortis lactea) (3) Daya Bay [22.4°N, 114.4°E],(4) Xaimen Bay [24°N, 118°E], (5) Zhujiang River
Estuary [36.1°N, 120.3°E], (15) Beibu Gulf [20.85°N,
109.26°E], (Ling 1936–1937; Du et al. 2010; Zheng
et al. 2014; Zhang et al. 2015). India - (19, 20) Puri,
Odisha [19.8°N, 85.5°E], Chennai [Madras] [13.1°N,
80.2°E] (as Phortis sp.), (Menon 1932; Kramp
1955). Japan - (8) Aitsu Marine Biological Station,
Kumamoto Prefecture, Kyushu [32.8°N, 130.7°E]; (9)Shonan Fishing Port, Kanagawa Prefecture, Honshu[35°N, 139.5°E this study], (16) Hamanako, Shizuoka
Prefecture, Honshu [34.4°N, 137.3°E], (21) Northern
Oita Prefecture, Kyushu [~33.3°N, 131.2°E] (Sugiura
1979; Sakiyama and Adachi 2001; Okamoto et al.
2016; Iwai 2021). Papua New Guinea - (10) Laing

Fig. 4. Biogeographical map showing validated wild capture distribution records for Eirene menoni and Eirene lacteoides. Eirene menoni records
that are indicated by red triangle refer to holotype location, green circles refer to localities with COI mtDNA sequences, and yellow squares refer
to localities obtained from published studies with sufficient description detail or documentation. Eirene lacteoides records are indicated by a blue
diamond. The GPS data for these records is located under the specific species in the text. Specimens with public aquaria captive culture locations arereferred to in the manuscript text.
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Island [5.3°S, 147.1°E], (Bouillon 1984). New Zealand- (13) North Island [35.18°S, 174.17°E], (Bouillon
1995). South Korea - (14) Chunjangdae [36.2°N,
126.5°E], Park 1996). Vietnam - (6) Nha Trang [12.3°N,
126.5°E], (Kramp 1962). This species is widespreadthroughout the Indo-Pacific.
Incertae sedis? Eirene menoni from the IndoPacific Basins?Irene ceylonensis Annandale (1907) Port
Channing, Calcutta, India adult medusae 20–25 mm
wide, with 100 marginal tentacles. ?Phortis lactea
Mayer 1910 from Tortugas, Florida, USA. Geographic
distance and width of peduncle base. ?Phortis lactea
var. chiaochowensis n. var. Kao et al. (1958) two
specimens off Tsingtao, China, 15 Jun 1956. Medusae
with 60 or more marginal vesicles. Stomach short,situated on conical peduncle, never extending beyond
umbrella margin. Illustration with wide peduncle base,illustration may be that of Phortis lactea from Mayer(1910) original drawing. ?Eirene menoni Kramp (1955)
from Khal, Dakhnidari Canal, near Calcutta collected
13 May 1926 at 7–8 m depth. Specimens 1–13 mm with
16 to 72 tentacles. ?Eirene menoni Santhakumari and
Vannucci (1971) Cochin Harbor, India no description.
?Eirene menoni Thomas and Chhapgar (1977)
Maharashtra, India 46 marginal tentacles with two
rudimentary bulbs between tentacles. ?Eirene menoni
Santhakumari et al. (1997) Bombay Harbor, India nodescription. ?Eirene menoni SAMA H1605 (non “Eirene
menoni” specimen with no peduncle and more than 4oral “lips”) Moreton Bay, Australia. ?Eirene menoni(sensu Eirene hexanemalis) Buecher et al. (2005) fromeastern South Africa (Algoa Bay to Tugela River mouth,
Indian Ocean), up to 10 mm bell diameter with up to 30
marginal tentacles, and up to 6 radial canals.
Eirene lacteoides Kubota and Horita, 1992
Kobu-eirene-kurage
Type locality: Captive culture-Toba Aquarium,
Toba, Japan. Holotype ZIHU-498 deposited in the
Zoological Institute, Faculty of Science, Hokkaido
University, Sapporo, Japan (not observed).
Material examined: Medusae - preserved
cultured NSMT-Co 1813 (3 specimens) 17 Feb
2002 bell diameter mm (12.5,14.5, 22.3, Fig. 5A–D)
Enoshima Aquarium via Toba Aquarium polyps), Gaku
Yamamoto. NSMT-Co 1814 (2 specimens) 9 Feb 2022
Enoshima Aquarium, Gaku Yamamoto. BPBM-D2777(8 specimens) 6 Aug 2022 Waikiki Aquarium cultured
8- grown out in monoculture tanks, Kelley Niide
collector. Polyps - preserved cultured: NSMT-Co 1815

page 9 of 15colony 4 Mar 2022 Enoshima Aquarium via Toba
Aquarium polyps, G. Yamamoto collector Fig. 5E–F).
BPBM-D2777 colony 6 Aug 2022 Waikiki Aquarium,
Kelley Niide collector. GenBank sequences - for E.
lacteoides (see Table 1).
Morphological description: Amended species
description. After Kubota and Horita (1992); Huang etal. (2009) with polyp description; this study.
Medusae - bell wider than high and the umbrella
apex is as thick as the length of the peduncle, bell
diameter reaches 32 mm with up to 160 marginaltentacle bulbs (fully formed and initial presentation as
small tentacle bulbs), typically with four radial canals,
rarely five, gonads start to develop near center of radial
canals and move distally to the ring canal, but do notreach the ring canal (Fig. 5A, B), never extending to thepeduncle, the peduncle is fairly wide at proximal baseand is somewhat cone shaped towards the distal end (Fig.
5B), with well-developed gastric peduncle, without
marginal and lateral cirri, with adaxial excretory papillae(visible on larger specimens) (Fig. 5C), rudimentary
bulbs (marginal warts) range from 2–7 per quadrant
reaching up to 17 in medusae (Fig. 5D) and statocysts(not visible in preserved specimens); four conicalprojections present in larger specimens (11–31 mm bell
diameter) on the interradial distal end of the peduncle(Fig. 5A), four oral “lips” extend beyond the bell andbecome highly crenulated and folded with maturity, oral
“lips” in preserved larger specimens appear “talon-like”(Fig. 5A, B).
Polyps - colonies stolonal, arising from creepinghydrorhiza, hydrorhizal stolons medium length giving
rise to hydrothecal pedicels of short lengths, hydrothecal
pedicels (Fig. 5E) with hydrocaulus that branch withpedicellate gonotheca forming a single bud (Fig. 5F),
hydranth expands at distal end becoming subspherical toknob shaped, with a whorl of tentacles (6–18), tentacles
filiform, with intertentacular web. Polyps appear“campanulinida” type.
Current validated wild biogeographic distributionof E. lacteoides - Jiaozhou Bay [Yellow Sea] (36.12°N,
120.25°E), and Changjiang River Estuary [East China
Sea] (31.52°N, 122.15°E), China, Pacific Ocean [Zheng
et al. 2014] (Fig. 3). Expected presence in the waters ofJapan and Hawaii.
Molecular phylogenetic results
The COI gene sequences provided consistentspecies separation for all Eirene species within
the phylogenetic tree, including E. menoni and E.
lacteoides (Fig. 6). Eirene menoni COI sequencesfrom Japanese and Chinese formed a well-supportedmonophyletic clade for the nine specimens, comprising© 2023 Academia Sinica, Taiwan

Zoological Studies 62:49 (2023)

seven closely related haplotypes, with three individuals
sharing a single haplotype, and differing by lessthan 1% overall. Likewise, the 11 E. lacteoides COI
sequences from Japan, China and Hawaii formed awell-supported monophyletic clade with extremely
low pairwise divergence, again with a mean of lessthan 1%, and consisted of four very closely related
haplotypes. Interestingly, the GenBank sequences
mislabeled as “Tima formosa” from China matchedpage 10 of 15

E. lacteoides sequences forming a third strongly
supported monophyletic clade. We conducted a pairwise
molecular divergence analysis comparing within
and between clade divergences. As evidenced by the
low bootstrap support in the deeper nodes of the tree
occurring between the six clades, there was no clear
pattern of similarity within the genera Tima and Eirene.
For example, while the pairwise molecular divergence
between the two species of Tima was high, 0.174

Fig. 5. Eirene lacteoides cultured medusae image of NMST-Co 1813 originally captive culture Enoshima Aquarium specimen obtained by Gaku
Yamamoto. Images (A) arrows- indicate conical projections, (C) arrows- adaxial excretory pores, (D) lines- rudimentary bulbs (warts). Images ofpolyps NMST-Co 1815 originally cultured at Enoshima Aquarium (Gaku Yamamoto). Image (F) shows a hydrotheca and a gonotheca. Scale bars: A–
C = 1 mm, check on D–F = 0.25 mm.
© 2023 Academia Sinica, Taiwan

Zoological Studies 62:49 (2023)(17.4%), the mean divergence among the four speciesof Eirene was even higher, 0.220 (22%) (Table 2). By
comparing the divergence of each species of Tima one
at a time to each of the four species of Eirene and taking
the mean value, we found T. bairdii had a divergenceof 0.195 (19.5%) when compared to all Eirene species,
and T. nigroannulata had a divergence of 0.191 (19.1%)
when compared to each of the four Eirene clades. By
calculating the range of divergences from the lowest
to the highest values, we found a minimum valueof 0.144 (14.4%) which was actually between two
China and Japan
GermanyJapan
Sweden and Norway
China
China, Japan (Public Aquarium)
Hawaii (Public Aquarium)

page 11 of 15

species from the two different genera, T. nigroannulata
compared to E. menoni. Likewise, the highest pairwise
value between any two species was 0.247 (24.7%), andthis was found between two species within the same
genus, E. viridula and E. lacteoides. The COI data
for species of Eirene and Tima therefore suggest that
there are substantial evolutionary differences among
species within a genus in the family Eirenidae. Basedon the rule of thumb for the estimated substitution rateof the COI gene of about 2% per million years, these
within genus divergence values and the overall pattern

Eirene menoni

Tima nigroannulata

Eirene viridula
Tima bairdii
Eirene hexanemalis

Eirene lacteoides

LC198742
Outgroups
KM200330

Fig. 6. Phylogram based on mitochondrial cytochrome c oxidase I gene fragments from 30 hydromedusan ingroup members of the family Eirenidae,
with two outgroup taxa. Specimens were collected from the wild and two public Aquaria. The tree was generated with 1000 bootstrap replicates using
the maximum-likelihood optimality criterion with the empirically-determined best-fit substitution model. The optimal model selected was TamuraNei, non-uniformity of evolution rates among sites and this was modeled using a discrete Gamma distribution (+G) with 5 rate categories. Under theassumption that a specified fraction of sites is evolutionary invariable (+l), TN93+G+l. This model had 66 parameters, BIC = 6461.115 (BayesianInformation Criterion), AICc = 6461.12 (Akaike Information Criterion, corrected), maximum likelihood score InL = -2907.703, gamma correction (G)= 1.40, and the proportion of invariant sites (l) = 0.49. Clades are color-coded by species and geographic sampling, including the following general
regions by bar color: Red = Eirene menoni, field sampled offshore of China and Japan; Purple = Tima nigroannulata field sample offshore of Japan;
Blue = Eirene viridula field sampled offshore of Germany; Gray = Eirene bairdii, field collected offshore Norway and Sweden; Orange = Eirene
hexanemalis, field collected offshore China; Green = Eirene lacteoides, field collected offshore of China and captive culture Enoshima Aquarium andWaikiki Aquarium. Outgroup taxa are LC198742 = Cassiopea sp. and KM200330 = Alatina alata (see Table 1).
© 2023 Academia Sinica, Taiwan

Zoological Studies 62:49 (2023)

page 12 of 15

suggest that the time to a most recent common ancestor
within each genus is between 8.7 million years for Tima
(17.4% divergence), and 11 million years for Eirene(22% divergence). Despite the high divergence values
that this study revealed between species within a genus,
sequences from a single species from thousands ofkilometers away showed little or no divergence, suchas a single haplotype shared between specimens of E.
lacteoides from China, Hawaii and Japan.
DISCUSSION
The morphological plasticity present in the
Hydrozoa is manifested in intraspecific variation inlife cycles, population dynamics, meristic characters,
growth, and reproduction (Cunha et al. 2016).
Typically, only a single medusa size class is captured
during plankton tows, which compounds difficultyin identifying the complete species ontogenetic
morphology. As we move forward in classifying thefamily Eirenidae integrative taxonomy is essential todetermine species delineations, boundaries and ranges(Dayrat 2005).
Based on the phylogenetic reconstruction
presented in this study, which was generated usingaligned mtDNA gene fragments, the genera Eirene andTima are sister taxa. In spite of this relatively close
evolutionary relationship, these two groups exhibit key
morphological differences. Divergent morphologicaltraits include size (with adult Tima exceeding 32 mm
in bell width), tentacle bases and proximal portionof tentacles that have a longitudinal furrow, and thepresence of continuous gonads along the radial canal
to stomachal peduncle (Kramp 1936; Bouillon et al.
2006; this study). Upon our examination of the genus
Eirene descriptions, we found that rudimentary bulbs(warts) were present in at least eight species. Therefore,
we have amended the genus description to reflect thismorphological feature. However, more attention needs

to be focused on rudimentary bulbs in species that have
60 or more marginal tentacles, as the tentacles tend to
exhibit continuous growth. This results in a challenge in
differentiating between developing marginal tentacles
and permanent rudimentary bulbs. In addition, some
COI sequences obtained from GenBank labeled as
Clytia sp., matched our Eirene COI data (unpublisheddata). Thus, genus Eirene warrants a careful systematic
re-evaluation.
It should be mentioned that previous phylogenetic
studies based on COI sequence data have shown atendency to lack a sufficient phylogenetic signal toconsistently diagnose generic level designations amongcertain hydromedusan lineages (Zheng et al. 2014). Thispattern is evident in the lack of genus-level monophyly
for Eirene, Clytia, and Tima in COI phylogenies, andto some degree in 16S-based phylogenies for these taxa
(Zheng et al. 2014; Maronna et al. 2016). However,
the COI gene fragment is a proven, powerful andaccurate tool in species identification and taxonomicallymatching specimens with GenBank accessions in cases
where the GenBank sequences are correctly identified.
The COI gene readily separated E. menoni andE. lacteoides samples and showed well supportedmonophyletic clades within our phylogenetic tree.
However, when we examined family and genericrelationships, the COI gene sequences revealed
a general lack of ability to resolve basal clade
relationships, resulting in generic polytomies andpolyphyly with low bootstrap support among thevarious resolved and supported species clades. Zheng etal. (2014) and Maronna et al. (2016) also reported that
COI showed a similar inability to achieve genus levelmonophyly. Currently, neither 16S nor COI appear to beideally suited for higher level taxonomic assessment of
the genera that comprise Eirenidae.
Based on our analyses, E. menoni is a wideranging species in the Indo-Pacific Ocean and likely
has a wider distribution in the Indian Ocean than iscurrently recognized. The E. menoni hydromedusae

Table 2. Pairwise COI mtDNA sequence divergence matrix, based on the Kimura-2 parameter substitution modeland COI gene sequences for family Eirenidae species and two outgroup species. Values in the table are moleculardivergence values, each value represents the difference between two clades in the tree shown in figure 4 (to convert
molecular divergence values to percentages multiply x 100, i.e., 0.196 = 19.6%)
1. Outgroups
2. Tima bairdii
3. Tima nigroannulata
4. Eirene lacteoides
5. Eirene menoni
6. Eirene hexanemalis
7. Eirene viridula© 2023 Academia Sinica, Taiwan

1
0.299
0.301
0.317
0.297
0.312
0.331

2

3

4

5

6

0.174
0.223
0.172
0.182
0.202

0.230
0.144
0.196
0.193

0.208
0.204
0.247

0.185
0.181

0.219

Zoological Studies 62:49 (2023)

from China and Japan formed a well-supported andresolved monophyletic clade (Fig. 6). During thisstudy we encountered multiple misidentifications in
the literature, and therefore urge caution when creating
species checklists. Eirene menoni is a distinct species
that can be identified by COI sequencing and a numberof diagnostic morphological features, including a
relatively narrow tubular peduncle, marginal tentacle
number, and absence of cirri, rudimentary bulbs, andadaxial excretory pores. There is variability in theposition of the gonads on the four radial canals fromdiffering geographic locations, and this also warrants
further investigation.
This study reflects a clear need for critical analysisof species checklists even when combined with DNA
sequence records, which can result in misidentified
GenBank entries that can perpetuate hydrozoan
taxonomic confusion and errors. For example, we wereforced to use incertae sedis for numerous E. menoni
published records, a misidentified museum specimen,
and unverified checklist records, where the description
did not match the original or the amended description of
the species in question. Additionally, there was a serious
lack of formal descriptions.
The polyp of E. menoni has a long, erecthydrocaulus, with strongly retractable tentacles
into the hydranth (Bouillon 1984). An examinationof the cultured polyps revealed no direct presenceof gonotheca or remnants on the hydrocaulus. The
hydrorhiza/stolon also had no presence of gonotheca.
Many hydrocaulus pedicels had a clean distal end withno remnants. It is possible that the mature hydranthbecomes a gonangia and produces a medusa bud. The
absence of obvious gonotheca in E. menoni warrants
additional study.
We have confirmed the presence of E. lacteoides
in the Yellow and East China Seas. This species likely
has a larger range than previously documented, as notedwith regards to the cultured samples that appeared intanks at the Toba Aquarium and the Waikiki Aquarium.
Although hydrozoan specimens found in public aquaria
may not offer direct information about their geographic
distribution, they can serve as indicators of a species’
presence in coastal waters, even if there is no official
documentation. This situation raises the possibility ofa marine biological introduction. The presence of Tima
formosa in the Pacific Ocean cannot be verified withspecimens from Japan identified as T. nigroannulata(Calder et al. 2021) or the samples from China
recognized as E. lacteoides (this study). The genus
Eirene is represented by two readily identified species
in the coastal waters of Japan (E. hexanemalis and E.
menoni) and likely E. lacteoides. Additional study isneeded as 14 Eirene species have been reported frompage 13 of 15

the seas around China (Du et al. 2010; Zheng et al.
2019).
Thorough integrative taxonomic studies focusedon the Pacific Eirenidae are warranted. Furthermore,
documentation of levels of interspecific morphologicalvariation is essential to the understanding anddelineation of biogeographic boundaries anddistributional ranges (Cunha et al. 2016). Small
planktonic marine species, and particularly those withcomplex life cycles that include a sessile life stage, suchas hydrozoans, tend to be predisposed to anthropogenic
introduction via commercial shipping, and longdistance dispersal via rafting on marine debris (Carlton
1987; Choong et al. 2018). The Hawaii specimens
shared a single haplotype with China and Japan in the
North Pacific Ocean. The presence of E. lacteoides
in Hawaiian waters likely represents a previously
undetected biological invasion (see Calder 2020).
E ff e c t i v e m a r i n e c o a s t a l m a n a g e m e n t i senhanced by regular harbor and nearshore surveys,
as early detection of invasive marine species, before
they can spread and become established is essential
to their control. Due to their small size, complex life
histories and poorly resolved taxonomic status, marinehydrozoans are not frequently detected or documented
as introduced species. The integrative approach used inthis study, namely the use of molecular, morphologicaland life history data together provide an unambiguousbasis to identify biological invasions as well as
delineate biogeographic patterns, and ultimately tobetter understand the geographic sources of marine
introductions.
CONCLUSIONS
The description of the genus Eirene was modified
to include the presence of rudimentary bulbs (warts).
Validated distribution records from the Indo-Pacific areprovided for E. menoni. The first field collected records
are provided for E. lacteoides from the Yellow andEast China Seas. The distribution of E. lacteoides may
be more extensive than the original Japan record with
the Waikiki Aquarium record from Hawaii. Using COI
genetic sequencing correctly identifies eirenid species.
However, COI sequencing does not resolve family andgenus relationships within the Eirenidae. Integrative
taxonomy is essential to properly identify Eirene
species.
Acknowledgments: This paper is dedicated to Dale
R. Calder (1941–2022) based on his multiple decades ofinvaluable scientific and taxonomic contributions, andparticularly comprehensive studies on the Hydrozoa.
© 2023 Academia Sinica, Taiwan

Zoological Studies 62:49 (2023)

Dale was much more than just a productive researcher:he was also generous, kind, and a great collaborator.
He regularly made time to help other scientists. He
mentored and taught numerous young researchers andmade everyone he worked with a better scientist. We are
grateful to the staff at the Toba Aquarium for sharing
polyps from E. lacteoides that enabled the genetic
confirmation of taxonomic identity of wild specimensfrom China. We thank the staff of the Enoshima,
Tsuruoka City Kamo and Waikiki Aquarium’s forresearch support. We appreciate the helpful discussion
and comments of Peter Schuchert and are grateful to
Andrea Crowther, Shirley Sorokin, and Peter Hunt of
the South Australian Museum for providing images.
Thanks to Holly Bolick, Bernice P Bishop Museum,
Hawaii and Hiroshi Namikawa, National Museumof Nature and Science, Tokyo, Tsubuka, Japan forarchiving specimens in their collections. GC and
BH acknowledge Ocean Research Explorations forfinancial support, the WoRMS database for maintaining
a comprehensive species list and providing access to
obscure literature, Jennifer Crites for medusae andpolyp figure creation, and Tina Carvalho, University of
Hawaii at Manoa Microscopy Core, supported by NIH
NIGMS grants 139753 and P20GM125508 for technicalmicroscope assistance. Support was provided by theHolland Laboratory and the Oceanic Institute at Hawaii
Pacific University. This is Ocean Research Explorations
publication number 010.
Authors’ contributions: GLC conceived the projectand conducted morphological study. GLC and BSH
wrote drafts of the manuscript. BSH conducted PCR,
gene sequencing and created the phylogenetic tree. GY
SI, AA, and KN collected and supplied specimens. All
authors reviewed drafts of the manuscript and approved
the final manuscript.
Competing interests: The authors declare that they
have no conflict of interest.
Availability of data and materials: DNA sequence
data generated from this study were deposited into the
NCBI GenBank. Specimens from Japan were deposited
into the NSMT, Tsukuba and from Hawaii at the BPBM,
Honolulu.
Consent for publications: All authors agree to the
publication of this work in Zoological Studies.
Ethics approval consent to participate: Not
applicable.

© 2023 Academia Sinica, Taiwan

page 14 of 15

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