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Integrative taxonomy of crustacean y-larvae (Thecostraca: Facetotecta) using laboratory-rearing and molecular analyses of single specimens, with the description of a new vermiform species

May 2022
Zoological Journal of the Linnean Society 196(1)

DOI:10.1093/zoolinnean/zlac020

Authors:

Jørgen Olesen

University of Copenhagen

Niklas Dreyer

CHU Sainte-Justine Research Center & Université de Montréal

Ferran Palero

University of Valencia

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Facetotecta, the taxon established for ‘y-larvae’, is the last major crustacean group for which the adult stage remains unknown. With only 14 described nominal species, all in the genus Hansenocaris, their incompletely known life cycle, small size and dearth of molecular data have hampered assessments of their true species diversity. Based on field studies during which > 11 000 y-larvae were sampled, a new integrative approach for studying the taxonomy of y-larvae is outlined. It focuses on last-stage nauplii and y-cyprids and includes methods for rearing lecithotrophic y-larvae for documenting the morphology of specimens with live photomicroscopy and scanning electron microscopy (SEM) and for obtaining molecular systematic data. This new and integrated approach, whereby each single specimen provides multiple kinds of information, was implemented to describe Hansenocaris demodex sp. nov., a unique y-larval form with semi-vermiform nauplii that occurs in the waters of Okinawa (southern Japan) and Taiwan. A preliminary Facetotecta phylogeny shows remarkable congruence between the morphology of all newly sequenced y-larvae and molecular data (18S rDNA). Four independent clades are formed by H. demodex and three other types/species of y-larvae, together being the sister-group to a smaller clade including H. itoi and unnamed species from GenBank.

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Zoological Journal of the Linnean Society, 2022, XX, 1–44. With 15 figures.

Integrative taxonomy of crustacean y-larvae

(Thecostraca: Facetotecta) using laboratory-rearing

and molecular analyses of single specimens, with the

description of a new vermiform species

JØRGEN OLESEN1,*, NIKLAS DREYER1–4,, FERRAN PALERO5,

DANNY EIBYE-JACOBSEN1, YOSHIHISA FUJITA6, BENNY K. K. CHAN2 and

MARK J. GRYGIER7,8

1Natural History Museum of Denmark, University of Copenhagen, Denmark

2Biodiversity Research Center, Academia Sinica, Taipei, Taiwan

3Department of Life Science, National Taiwan Normal University, Taipei, Taiwan

4Biodiversity Program, Taiwan International Graduate Program, Academia Sinica, Taipei, Taiwan

5Institut Cavanilles de Biodiversitat i Biologia Evolutiva (ICBIBE), Valencia, Spain

6General Education Center, Okinawa Prefectural University of Arts, Naha, Okinawa, Japan

7Center of Excellence for the Oceans, National Taiwan Ocean University, Keelung, Taiwan

8National Museum of Marine Biology & Aquarium, Checheng, Pingtung, Taiwan

Received 5 August 2021; revised 17 January 2022; accepted for publication 16 February 2022

Facetotecta, the taxon established for ‘y-larvae’, is the last major crustacean group for which the adult stage remains

unknown. With only 14 described nominal species, all in the genus Hansenocaris, their incompletely known life

cycle, small size and dearth of molecular data have hampered assessments of their true species diversity. Based on

field studies during which > 11 000 y-larvae were sampled, a new integrative approach for studying the taxonomy

of y-larvae is outlined. It focuses on last-stage nauplii and y-cyprids and includes methods for rearing lecithotrophic

y-larvae for documenting the morphology of specimens with live photomicroscopy and scanning electron microscopy

(SEM) and for obtaining molecular systematic data. This new and integrated approach, whereby each single specimen

provides multiple kinds of information, was implemented to describe Hansenocaris demodex sp. nov., a unique

y-larval form with semi-vermiform nauplii that occurs in the waters of Okinawa (southern Japan) and Taiwan.

A preliminary Facetotecta phylogeny shows remarkable congruence between the morphology of all newly sequenced

y-larvae and molecular data (18S rDNA). Four independent clades are formed by H. demodex and three other types/

species of y-larvae, together being the sister-group to a smaller clade including H. itoi and unnamed species from

GenBank.

ADDITIONAL KEYWORDS: classification – culturing – cyprid – Hansenocaris – larval biology – nauplius –

parasitism – phylogeny – pores – setae – systematics.

INTRODUCTION

Planktonic crustaceans, such as copepods and krill,

dominate the faunal biomass of the oceans of the

world (Schminke, 2007; Atkinson et al., 2012; Bar-On

& Milo, 2019). Larval plankton is no small part of this,

particularly as dispersal stages in coastal waters. Life-

history studies combined with sequencing technologies

have uncovered the true identity of many planktonic

larvae at several taxonomic levels (Palero et al., 2009;

Bracken-Grissom et al., 2012; Torres et al., 2014; De

Grave et al., 2015; Genis-Armero et al., 2020). One

widely distributed group of crustaceans remains an

enigma in marine biology: Facetotecta or y-larvae,

which are still only known from their planktonic larval

*Corresponding author. E-mail: Jolesen@snm.ku.dk.

[Version of record, published online 31 May

2022; http://zoobank.org/ urn:lsid:zoobank.

org:pub:97F607E9-7B62-4087-8D67-2CB9AA3E7B70]

applyparastyle “fig//caption/p[1]” parastyle “FigCapt”

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2 J. OLESEN ET AL.

stages and a putative juvenile stage. Y-larva taxonomy,

with only 14 formally described species based mostly on

incomparable life-cycle stages, is essentially in a state

of confusion. Here an integrative taxonomic approach,

using culturing procedures and molecular methods, is

applied to biodiversity studies of Facetotecta.

Y-larvae occur naturally as nauplius (y-nauplii) or

cyprid (y-cyprids) stages (Grygier, 1996; Kolbasov &

Høeg, 2003; Høeg et al., 2014). Hansen (1899) initiated

a parataxonomy by recognizing five different naupliar

forms, denoted by Roman numerals I–V, that he termed

‘Larven vom Typus y’ (i.e. larvae of type y). These

and similar larvae were later nicknamed Hansen’s

y-larvae and were scatteredly reported worldwide.

The succeeding stage in the life cycle, the cypris y,

was first described from Danish waters (the Sound)

by Bresciani (1965). The most recent addition to the

life cycle, the vermiform ypsigon larva, was induced to

moult from y-cyprids after treating the latter with a

moulting hormone (Glenner et al., 2008). Its discovery

led to suggestions that the unknown y-adults are

endoparasites of yet-to-be-identified marine hosts

(Glenner et al., 2008; Pérez-Losada et al., 2009).

Y-larvae are closely related to barnacles (Pérez-

Losada et al., 2009; Petrunina et al., 2013; Chan et al.,

2021). Their cirripede affinities were discussed upon

their discovery by Hansen (1899), who also suggested

a parasitic nature for y-larvae, but his tentative

classification of them within Darwin’s (1854) cirripede

suborder Apoda proved erroneous when Bocquet-

Védrine (1972, 1979) showed that Darwin had

established this group for a parasitic isopod. Grygier

(1985) resurrected the taxon Thecostraca, originally

proposed by Gruvel (1905), to comprise Ascothoracida

(a group of parasites), Facetotecta (coined by Grygier

himself therein for y-larvae) and Cirripedia, but

subsequently failed to fully resolve the relationships

among these three groups (Grygier, 1987). Bresciani

(1965) had previously pointed out ascothoracidan-

like features of cypris y and Itô (1986b) expressed

skepticism that Facetotecta are truly distinct from

Ascothoracida. Nonetheless, the validity of Thecos-

traca has been supported using larval morphological

characters (Høeg & Kolbasov, 1992; Pérez-Losada

et al., 2012) and molecular data (Pérez-Losada et al.,

2009; Petrunina et al., 2013), with Facetotecta as the

sister-group to either all remaining thecostracans or

just Ascothoracida. Originally proposed as an order

(Grygier, 1985), Facetotecta is currently ranked as

a subclass (Martin et al., 2014; Chan et al., 2021) or

infraclass (Martin & Davis, 2001).

Early significant discoveries related to y-larval

parataxonomy and biology came from Atlantic and

Scandinavian waters (e.g. Hansen, 1899; Bresciani,

1965; Schram, 1970a, b, 1972; Elofsson, 1971). However,

y-larvae are found in all major oceans, although mostly

only in small numbers, such as the 24 specimens

collected in the Baltic Sea and Atlantic Ocean on which

Hansen (1899) based the first significant report on

y-larvae and the 29 specimens reported by McMurrich

(1917) from Passamaquoddy Bay, Canada. The 103

specimens reported from Norway by Schram (1970b,

1972), 102 specimens from the Mediterranean reported

by Belmonte (2005), 103 specimens from Sesoko Island

in the Ryukyu Islands, Japan, examined with SEM by

Grygier (1991a), 150 specimens collected around the

Manazuru Peninsula in Sagami Bay, Japan, by Kikuchi

et al. (1991) and Watanabe et al. (2000), ‘hundreds of

specimens’ reported from off Halley Bay, Antarctica, by

Dahms et al. (1990) but not yet studied, and about 500

specimens collected from the White Sea, Russia, in four

spring/summer seasons and reported by Kolbasov &

Høeg (2003) and Kolbasov et al. (2021a) represent the

large majority of the larvae reported to date. In many

other cases, only a few specimens were caught (e.g.

Bresciani, 1965; Swathi & Mohan, 2019) and even the

groundbreaking taxonomic and morphological works

of Itô (1984, 1985, 1986a, b, 1987a, b, 1989, 1990b,

1991) and Itô & Takenaka (1988) (see summaries by:

Kikuchi et al., 1991; Watanabe et al., 2000; Kolbasov &

Høeg, 2003; Kolbasov et al., 2007; Grygier et al., 2019)

were based on perhaps as few as 35 individual larvae in

total from Tanabe Bay on the Pacific coast of Honshu,

Japan. Additionally, a number of oceanographic works

without specimen counts have provided density data

suggesting an abundance of planktonic y-larvae

in various seas (e.g. Mileykovskiy, 1970; Böttger-

Schnack, 1995; Gallego, 2014; Weydmann et al., 2014).

Nonetheless, in general, the scientific information on

y-larvae worldwide remains sparse and scattered,

limited to some 170 items comprising research and

review papers, meeting abstracts, ‘grey literature’ and

internet records.

The current status of y-larva research is

unsatisfactory, not least in light of the bulk of

undescribed forms. Hansen (1899) considered ten to

12 species to be present in his limited material and

suggested that more than 100 species of y-larvae may

inhabit the world’s oceans. More than 20 undescribed

species have been reported to occur in Tanabe Bay,

Japan (Itô, 1990b) and more than 40 around Sesoko

Island, Japan (Glenner et al., 2008).

Because of the scattered occurrence of y-larvae,

their small size and incompletely understood life-cycle,

knowledge of their diversity has been built up slowly

based on tedious sorting of plankton samples, light-

microscopy-based drawings and photography of single

larval specimens, and attempts to combine plankton-

caught larvae into developmental series. Starting

with Hansen (1899), plankton-caught y-nauplii were

numbered informally as distinct types, creating a

parataxonomy [see summary by Grygier et al. (2019)];

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TAXONOMY OF CRUSTACEAN Y-LARVAE 3

types I–XII and subtypes VIII-a, -b and -c exist, with

duplication of type VI by Steuer (1904) and Grygier

(1987) and also ‘Manazuru Types I and II’ of Watanabe

et al. (2000). Itô (1985) formally proposed a new genus,

Hansenocaris Itô, 1985 (type species: H. pacifica Itô,

1985), to accommodate three species based on y-cyprids

described or mentioned in his previous papers (Itô,

1984; Itô & Ohtsuka, 1984). This could have provided

a basis for a standardised treatment of y-larvae, but

instead a complicated mixture of informal (e.g. Itô,

1986a, 1987a, b) and formal (binomial) nomenclature

ensued. Among the 14 formally described species

of y-larvae proposed by Itô and others (Table 1), six

are based on nauplii alone, six on cyprids alone and

only two, Hansenocaris furcifera Itô, 1989 and H. itoi

Kolbasov & Høeg, 2003, comprise both cyprids and

planktotrophic nauplii. The designated type series of

H. furcifera consisted only of y-cyprids, but nauplius y

type IX of Itô (1987b) and successive instars are known

to be conspecific with it (Itô, 1989, 1990b).

The usefulness of choosing wild-caught nauplii,

representing one or few instars, as the basis for formal

species descriptions, as was done for four species

by Belmonte (2005) and one species by Swathi &

Mohan (2019), is questionable, because it is likely to

be troublesome to link these to the cyprid or other

naupliar stages of the same species. Choosing the

y-cyprid as the name-bearing instar (Itô, 1985, 1986b,

1989; Kolbasov & Høeg, 2003; Kolbasov et al., 2007;

2021b) has some practical value, as there is only one

cyprid instar in the facetotectan life-cycle, facilitating

homology-based comparative analyses between

species. However, a superior strategy for studying the

taxonomy of y-larvae would be to base species

descriptions on a combination of nauplii and cyprids

linked through direct evidence (i.e. individual moult

sequences). Surprisingly, such efforts have been limited

to Hansenocaris furcifera (Itô, 1989, 1990b: fig. 9) and

one unnamed lecithotrophic species partly illustrated

and briefly described by Itô (1991). The only other form

of y-larvae for which both nauplii and the cyprid are

known is H. itoi (Kolbasov et al., 2021a).

The current system for naming y-larvae, combining

a formal taxonomy (with binomial names) that are

often based on incomparable life-history stages with

a parataxonomy based on wild-caught nauplii using

Roman numerals as identifiers, is highly unsatisfactory.

Nicknames for certain undescribed forms also exist

(Grygier et al., 2019). This inconsistent approach,

together with the scarcity of molecular data, has

failed to reflect the true species diversity of y-larvae

and has essentially resulted in parallel nomenclature

systems for their taxonomy, which hinders efficient

progress in exploring that diversity. To remedy this

situation, a novel protocol is suggested herein with a

focus on comparable (homologous) life stages among

facetotectan taxa, namely the last-stage nauplius

(LSN) and the cyprid. The protocol combines culturing

techniques (with an emphasis on individual rearing

of late larval stages), live photography and molecular

techniques (DNA barcoding) based on individual

larval specimens. This approach has proven to be

particularly successful for y-larvae with lecithotrophic

nauplii, which are able to moult without feeding under

laboratory conditions as they pass through the various

stages of development. It is recommended for future

taxonomic work on facetotectans since it provides a

basis for: (1) preparing species descriptions based on

demonstrably conspecific nauplii and cyprids, thereby

avoiding parallel taxonomies; (2) linking the resulting

taxonomic names with adults, whenever these become

known in the future; (3) identifying plankton-caught

y-larvae based on hitherto unused morphological

(e.g. colour patterns) or barcoding-type data; and (4)

allowing comparisons between equivalent stages of

different putative species.

Practical benefits and limitations of individual

larval rearing were explored during fieldwork during

2017–19 at Green Island, Taiwan, and Sesoko Island,

Okinawa, Japan (Fig. 1). Methodological details are

outlined and new data are used to formally describe

one of the most distinctive Facetotecta species from

these two sampling sites. Integration of new culturing

techniques, live photography, microscopical techniques

(SEM and LM) and molecular analyses is essential to

uncover the true species diversity of Facetotecta. Our

integrated taxonomic approach is presented to provide

a baseline for future descriptions of y-larvae with

lecithotrophic nauplii.

MATERIAL AND METHODS

Overview Of methOdOlOgy

Y-larvae (nauplii or cyprids) captured alive off Sesoko

Island are practically unidentifiable based on currently

available knowledge due to their unanticipated diversity.

For example, the catch of one morning (19 October

2018) resulted in 25 y-larvae that possibly represent > 15

different species (Fig. 2, live larvae: https://youtu.be/

seo-63AK10E). To facilitate taxonomic work on such

populations, a novel method is outlined for individual

rearing of lecithotrophic nauplii to the cyprid stage that

produces maximum information from different life-cycle

stages of the same taxon. In many cases, this method

provides both morphological and molecular data for

the same individual at different points in development.

The methodology involves: (1) sampling (Figs 1, 2); (2)

individual rearing to last-stage nauplius (LSN) and

cyprid (Fig. 3); (3) microscopy of live specimens (e.g. for

documenting their colour patterns) (Figs 2, 3, 11); (4)

subsequent fixation and storage of individual larvae

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4 J. OLESEN ET AL.

Table 1. Described species of Facetotecta (‘y-larvae’) and the life stage(s) on which the descriptions were based

Species Described life stage Feeding

strategy*

Type locality

Hansenocaris hanseni (Steuer, 1904) Type IV nauplius P Gulf of Trieste, northern Adriatic

Sea (Italy)

Hansenocaris pacifica Itô, 1985 Cyprid: Itô (1985); Itô & Ohtsuka (1984) L?†Tanabe Bay, Shirahama, Wakayama

Prefecture (Japan)

Hansenocaris rostrata Itô, 1985 Cyprid: Itô (1985); Itô (1984) ? Tanabe Bay, Shirahama, Wakayama

Prefecture (Japan)

Hansenocaris acutifrons Itô, 1985 Cyprid: Itô (1985) ? Tanabe Bay, Shirahama, Wakayama

Prefecture (Japan)

Hansenocaris tentaculata Itô, 1986b Cyprid: Itô (1986b) ? Tanabe Bay, Shirahama, Wakayama

Prefecture (Japan)

Hansenocaris furcifera Itô, 1989 Cyprid: Itô (1989)

Nauplius: Itô (1986a, 1987b, 1990b)

P Tanabe Bay, Shirahama, Wakayama

Prefecture (Japan)

Hansenocaris itoi Kolbasov & Høeg, 2003 Cyprid: Kolbasov & Høeg (2003)

Nauplius: Kolbasov & Høeg (2003); Kolbasov et al. (2021a)

P Kandalaksha Bay, White Sea

(Russia)

Hansenocaris corvinae Belmonte, 2005 Nauplius: Belmonte (2005); Swathi & Mohan (2019)‡P Salento Peninsula (Italy)

Hansenocaris leucadea Belmonte, 2005 Nauplius: Belmonte (2005); Swathi & Mohan (2019)‡P Salento Peninsula (Italy)

Hansenocaris mediterranea Belmonte, 2005 Nauplius: Belmonte (2005) L Salento Peninsula (Italy)

Hansenocaris salentina Belmonte, 2005 Nauplius: Belmonte (2005) P? Salento Peninsula (Italy)

Hansenocaris papillata

Kolbasov & Grygier, 2007

Cyprid: Kolbasov et al. (2007) ? Banggai Archipelago, off Sulawesi

(Indonesia)

Hansenocaris portblairenae Swathi & Mohan, 2019 Nauplius: Swathi & Mohan (2019) P Great Andaman Island (India)

Hansenocaris spiridonovi

Kolbasov et al., 2021b

Cyprid: Kolbasov et al. (2021b) ? Azores Islands (Portugal)

Hansenocaris demodex sp. nov. Nauplius: This work

Cyprid: This work

L Sesoko Island, Okinawa (Japan)

*L, lecithotrophic; P, planktotrophic nauplii. As reported in literature or based on absence/presence of feeding spines as depicted in original illustrations (see more criteria in Materials and Methods).

†Cyprids of H. pacifica first linked by Itô to lecithrotrophic nauplii of Type XI (Itô, 1986a), but this was later dismissed (Itô, 1987b). The real nauplii of H. pacifica have never been explicitly identified

or described, but probably belong to one of Itô’s many unpublished lab-reared forms, in which case they would be lecithotrophic.

‡Swathi & Mohan (2019) identified some wild-caught planktotrophic nauplii from the Andaman Sea as conspecific with the Mediterranean H. leucadea and H. corvinae. However, due to the great

distance between the localities and the large morphological diversity of planktotrophic nauplii (unpublished), these identifications are in need of confirmation with molecular methods.

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TAXONOMY OF CRUSTACEAN Y-LARVAE 5

for different purposes; (5) post-expedition sorting and

digital handling of specimens (Fig. 3); (6) detailed

microscopy (e.g. SEM); and (7) molecular sequencing of

individual larval specimens. The setup for rearing was

modified from Itô (1990a, b, 1991).

More than 11 000 y-larvae were collected and

sorted from live plankton samples during several

field trips to Sesoko Island, Okinawa, Japan and

Green Island, Taiwan between 2017 and 2019 (Table

2). Twenty-seven specimens provide the basis for the

present description of Hansenocaris demodex sp. nov.,

including 22 from Sesoko Island and five from Green

Island (Fig. 3; Table 3), although four specimens from

Sesoko that died soon after capture were discarded

and not processed further. The last-stage nauplius

(LSN) and the cyprid are described in detail, while

only limited information is provided for earlier

naupliar stages.

Sampling and rOugh SOrting

The larval material used in this work was collected

near the Marine Science Research Station of Academia

Sinica on Green Island, Taiwan, in 2017 (1 September

to 1 November) and 2018 (24 August to 6 September)

and the University of the Ryukyus Tropical Biosphere

Research Center Sesoko Station on Sesoko Island,

Okinawa Prefecture, Japan, in 2018 (16 October to

5 November) and 2019 (1 June to 23 June) (Fig. 1).

Plankton samples were collected by handheld conical

plankton nets (20 or 30 cm mouth opening, 65, 95 or

100 μm mesh size) deployed from wharfside at Gonguan

Fishing Harbour on Green Island (22°40’33.1"N,

121°29’37.4"E) at 3–5 m depth or from the end of

the laboratory pier at Sesoko Island (26°38’09.4″N,

127°51’55.2″E) in waters varying in depth from about

0.5 to 2.0 m, depending on the tide. Individual tows

were typically 10–15 m long. Fresh samples were

Figure 1. Facetotecta sampling sites in East Asia, 2017–20 and distribution of Hansenocaris demodex sp. nov. A, East China

Sea showing distance (780 km) between the two main sampling sites, Sesoko Island (Okinawa, Japan) and Green Island

(Taiwan). In 2020, several additional specimens of H. demodex were collected from Xiaoliuqiu Island (MJG, unpublished). B,

Sesoko Island (Japan, Okinawa) with indication of sampling site (Sesoko Station); C, Green Island (Taiwan) with indication

of sampling site (Gonguan Fishing Harbour); D, pier at Sesoko Station; E, Gonguan Fishing Harbour.

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6 J. OLESEN ET AL.

Figure 2. Example of newly collected sample of still living y-larvae collected on 19 October 2018 at Sesoko Island off the

pier shown in Figure 1D, representing a variety of developmental stages. A, 25 y-larvae likely representing > 15 different

species, including 18 early lecithotrophic nauplii (*), five planktotrophic nauplii (†) and two cyprids (§). None of the depicted

specimens can be assigned to already described species, but three of the planktotrophic nauplii are similar to Itô’s (1986a)

Pacific type I and one is a cyprid of the type nicknamed ‘Big brown’, both types of which are sequenced as a part of this

work and belong to the same clade as Hansenocaris demodex sp. nov. (see Fig. 15). B, close-up of selected y-larvae with same

numbers assigned as in A. Live video of H. demodex and other y-larvae can be seen here: https://youtu.be/seo-63AK10E.

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TAXONOMY OF CRUSTACEAN Y-LARVAE 7

Figure 3. Overview of most specimens of Hansenocaris demodex sp. nov. collected at Sesoko Island during fieldwork in

2018 and 2019. A, 19 specimens, four of which reached the cyprid stage, collected in the plankton at the naupliar stage

and reared individually, with length, dish number, sample number, and museum registration number indicated for each

specimen as well as a colour code (red, blue, yellow) showing its ultimate treatment/fixation (respectively, molecular work,

SEM or exuvium on slide); some larvae died before preservation and are only referred to by their dish number; B, overview

of setup for individual rearing of larvae; top view of green tray containing 31 2.5-cm-wide dishes with tracking information

written on lids, each containing one to five live larvae. Live video of H. demodex and other y-larvae can be seen here: https://

youtu.be/seo-63AK10E.

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8 J. OLESEN ET AL.

brought to the lab for sorting of live y-larvae in either

Petri dishes or Bogorov-type zooplankton counting

chambers, using a Pasteur pipette under a dissecting

microscope (Fig. 2). During fieldwork at Sesoko Island

all larvae were counted and roughly categorized into

larval type (Table 2).

rearing

At Sesoko Island, stock seawater for cultures was

prepared by passing pumped seawater from the

laboratory through a 62-μm mesh hand-net, without

sterilization. All y-larvae from a given sample were first

placed in a small glass or plastic Petri dish. A second

round of sorting separated cyprids, planktotrophic

nauplii similar in body plan to Itô’s (1986a) Pacific type

I and supposedly lecithotrophic nauplii. The supposed

lecithotrophic nauplii were usually divided into smaller

groups of four to six individuals, representing either a

single type, several distinctly different types to allow

tracking of individuals or a random selection of the

remaining nauplii in the sample, and maintained in

35 × 10 mm transparent lidded plastic dishes two-third-

filled with stock sea water (Fig. 3B). Dishes at both

sites were maintained on table tops in the laboratory at

25–26 °C, without agitation. The condition of all larvae

in each dish was assessed and recorded generally once

per day under a dissecting microscope. Dead and badly

fouled specimens were discarded, and occasionally the

surviving specimens were transferred to a fresh dish with

fresh stock seawater. As soon as a last-stage nauplius

(LSN) appeared – recognizable by the dark-pigmented

compound eyes of the internally developing cyprid instar

– it was placed in a separate dish (serially lettered) to

await its final moult into a free-swimming y-cyprid. For

example, the ten y-larvae taken at 13:00 on 14 June

2019 were split between the two dishes labelled first as

‘14-VI-19 13:00A’ and ‘14-VI-19 13:00B’ (maintaining

their identity as parts of the same sample), and also

as ‘dish 178’ and ‘dish 179’, respectively (their place in

the entire survey). Each dish contained five supposedly

lecithotrophic nauplii. One nauplius from dish 178

reached its final instar on the evening of 17 June, when

it was separated out into dish 178A (moulted to cyprid

two days later), and two nauplii from dish 179 reached

their final instar in the afternoon of 19 June, when they

were separated out into dishes 179A (moulted to cyprid

three days later) and 179B (died). Survivorship to the

LSN stage was about 10%, to the cyprid stage about 5%.

micrOScOpy Of live SpecimenS during fieldwOrk

To obtain objective and reproducible records of

coloration and degree of transparency in situ and other

features that are typically lost upon fixation, and to

facilitate later grouping of larvae into types based on

Table 2. Numbers of y-larvae collected during fieldwork in 2018 and 2019 at Sesoko Station (Okinawa). During sorting the larvae were registered in four general

categories. See text for more details

Early

nauplii,

planktotrophs

Early nauplii,

lecithotrophs

Last stage

nauplius (LSN)

Cyprids Number of tows*

with plankton

net

Mean number of

larvae per tow

Max number of

larvae in one

tow

Total number

of larvae

2018 654 1515 9 88 3481 0.78 20.32 2710†

2019 2888 3622 ? 401 3540 1.95 31.30 6911

Totals 3542 5137 9 489 7021 1.37 31.30 9621

*Each tow covered a distance of 10–15 m.

†Includes 444 larvae that were not classified as planktotrophic or lecithotrophic.

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TAXONOMY OF CRUSTACEAN Y-LARVAE 9

as many features as possible, a large number of larvae

were digitally photographed and/or videographed at

various magnifications. Such observations were made

with a Nikon ECLIPSE 80i compound microscope

(Sesoko Island) equipped with Nomarsky (DIC) optics

or an Olympus IX70 inverted compound microscope

(Green Island), both fitted with a Canon EOS 5D Mark

IV digital camera. Since last-stage y-nauplii (LSN) with

a y-cyprid developing inside and the moulted y-cyprids

themselves were considered to be the key stages for

taxonomic work on facetotectans (see Introduction), all

LSNs that became available, either directly from the

plankton or by rearing from earlier stages, were digitally

recorded whenever possible. Sometimes there was also

time for photographic/videographic documentation of

earlier instars, which, on account of the dish numbering

system described above, could be matched with their

later counterparts (see Fig. 3). About 1275 selected

specimens out of 9621 specimens collected during our

fieldwork in 2018 and 2019 at Sesoko Island were

photographed/videographed this way.

Each nauplius or cyprid to be recorded was moved

temporarily from its culture dish to a shallow depression

slide and kept nearly immobile in a minimal amount of

seawater without using a coverslip. Videos were taken

in preference to still photographs. Increased depth of

field for photographs was obtained by using the HD

video 50 fps mode of the camera while focusing up

and down a couple of times to produce sets of images

amenable to combination into a single image by

means of image-stacking software (Zerene Stacker,

v.1.04). After recording, the live specimens were either

returned to their culture dishes in case of further

moults (nauplii) or were single-fixed immediately for

SEM or molecular processing (cyprids and moribund

LSNs). In some cases, especially during the later

phases of the fieldwork when there was no time to let

the nauplii develop further, a significant number of

lecithotrophic nauplii, regardless of instar, and even

those from fresh samples, were digitally documented

and subsequently fixed so as to cover as many types as

possible. A large number of planktotrophic y-nauplii

that did not moult while kept in culture, and supposed

lecithotrophs that did not moult, were treated the

same way. Any individual nauplius or cyprid that was

preserved received its own sequential sample number

of the form ‘TA-2018-131’ or ‘JA-2019-290’ (from

Taiwan and Japan, respectively), and detailed entries

were immediately made into an Excel file using these

numbers, with cross-references to the culture dish

numbers. Both sorts of numbers were used on labels

prepared later for slides and specimen vials (see

below). In cases where photographed larvae died before

preservation, and sample numbers were therefore not

assigned, their dish numbers are shown in the figures

for reference.

fixatiOn and StOrage Of material

A large number of LSN exuviae were prepared on

slides as semi-permanent glycerine jelly mounts

shortly after moulting to the cyprid stage (normally

within a day). After several hours in a drop of seawater-

based formalin (to kill adhering bacteria and fungi),

to which a similar amount of anhydrous glycerine

was soon added, each specimen was transferred by a

needle into a small droplet of molten glycerine jelly on

a glass slide, which was then covered by a coverslip

supported by four drops of dried nail varnish to avoid

crushing the specimen. The same nail varnish was

used later to seal the slide. Cyprids corresponding to

the mounted LSN exuviae were either preserved in

seawater-buffered 2–4% formaldehyde for scanning

electron microscopy, or in 95–99% ethanol and kept

in the freezer for subsequent molecular work. The

resulting material included 272 microscopic slides

with LSN exuviae of a large number of facetotectan

types from the 2018 and 2019 expeditions to Sesoko

Island, and 745 microvials with specimens fixed for

either morphological or molecular work. The material

is currently stored under the above-described sample

number system(s) at the Natural History Museum of

Denmark, but will gradually be registered with NHMD

numbers as the taxonomic work progresses.

SOrting and digital handling Of SpecimenS

Further sorting and grouping of y-larvae took place

after fieldwork and was based on the photographic

information obtained for a high number of selected live

specimens (see above). In order to obtain comparable

information for each specimen, selected frames from

the HD video sequences were exported as a stack and

blended in Zerene Stacker v.1.04. These stacked

images, together with their corresponding culture-

dish and fixation data, were then sorted/grouped into

large plates in cOreldraw. Most of the data from

H. demodex, which is one of the more distinct species,

represented in 2018/2019 by 22 collected specimens

(Sesoko), are combined in Figure 3, in which photos

placed in horizontal rows represent different instars

of the same organism. This overview served as a basis

for deciding which specimens to process further for

detailed morphological and molecular work on this

new species, and also which to designate as the name-

bearing type (holotype) in the description (see below).

naupliar develOpment

For H. demodex, in addition to the nine LSN

specimens obtained in 2017–19, either by rearing

or by directly collecting them from the plankton,

some information was obtained on early and mid-

stage nauplii from Sesoko Island. This included

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10 J. OLESEN ET AL.

high-resolution photographs of live individuals or

their exuviae at different stages of development (Figs

3, 11), as well as SEM images of several younger-stage

specimens (Figs 12, 13). An incomplete outline of the

naupliar development of this species can, therefore, be

assembled. As the earliest post-hatching stage of the

naupliar development is not known for H. demodex,

the naupliar sequence was numbered from the LSN

(last-stage nauplius) backwards, with the preceding

stage called LSN − 1 (‘last-stage nauplius minus 1’),

itself being preceded by the earlier stage LSN − 2,

etc. This was inspired by the convention used for

ostracods (e.g. Hiruta & Hiruta, 2014) and avoids

the possible problems associated with Itô’s (1990b:

219) designation of the last five instars as ‘the first

through fifth naupliar stages’ [= nauplius 1–5 of

Kolbasov & Høeg (2003)] despite his suspicion that

there may be another ‘true first stage’. If there are six

naupliar stages altogether, or even seven as suggested

by Kolbasov et al. (2021a) for H. itoi, the earliest would

be called LSN − 5 or LSN − 6, respectively.

advanced micrOScOpy

Formalin-fixed specimens selected for SEM were

prepared by rinsing in distilled water overnight,

dehydration in a graded alcohol series to 100% ethanol

and subsequent critical point drying. Specimens were

mounted on metal stubs on carbon tape, coated with

an alloy of palladium and platinum and observed/

photographed in a JEOL JSM-6335-F (FE) scanning

electron microscope at the Natural History Museum

of Denmark, Copenhagen (Figs 4–10, 12, 13). Prior to

treatment for SEM, cyprids were photographed using

an inverted compound microscope (Olympus, IX83)

using fully automated image-stacking techniques

(Fig. 7A).

terminOlOgy fOr mOrphOlOgical deScriptiOn

Morphological terminology partly follows Itô (1987b,

1990b). The naupliar cuticle consists of two main parts,

the cephalic shield and the faciotruncal integment

(faciotrunk) (yellow and blue overlays in Figs 4, 5, 12).

When an early- or middle-stage nauplius moults, these

two parts of the exuvium usually become separated,

but the exuvium of a last-stage nauplius (LSN)

typically remains entire. An incompletely moulted

LSN specimen with two layers of unshed naupliar

exuviae covering it displays the moulting zone

between the cephalic shield and the faciotrunk (Fig.

13D, yellow stippled line). Figure 13 shows that the

indivisible faciotrunk (Fig. 13D, E) consists of a wide,

ventral faciomarginal area that surrounds the labrum

and the three pairs of naupliar appendages anteriorly

and laterally, but only extends a short distance

posteriorly, and, posterior to this, the trunk (or hind

body) per se, which includes dorsal, lateral and ventral

cuticle. Although planktotrophic y-nauplii have a well-

defined labrum extending posteriorly from the level

of the mouth opening (e.g. Itô, 1990b: Kolbasov &

Høeg, 2003; Høeg et al., 2014; Kolbasov et al., 2021a),

lecithotrophic y-nauplii lack this or have a median

spine in its place. Nonetheless, the area anterior to the

missing labral extension is usually swollen to various

degrees and exists in different shapes (for H. demodex,

see Figs 5K–M, 12E), with pores and a cuticular ridge

pattern and sometimes ends posteriorly in a distinct

declivity. Despite their differences in various types of

y-naupli, these structures have mostly been referred

to as a labrum (e.g. Schram, 1970b, 1972; Itô, 1986a,

1987a, 1990b; Kolbasov & Høeg, 2003; Grygier et al.,

2019), also in cases where a labral extension is missing.

The term labrum is used in this study to denote the

entire complex of generally homologous structures in

the mouth region of y-nauplii, even though a labral

extension overhanging the mouth opening is missing

in H. demodex.

To identify the facets (plates) of the cephalic shield

y-nauplii, a full set of detailed anterior, anterolateral,

lateral, dorsal and, in case the lateral margins were

inturned, also ventral views were obtained for all

individuals examined with SEM. Nonetheless, plate

identification was hindered by three factors: apparent

absence of the instar upon which Itô’s (1987b) basic

system of plate nomenclature was based (thus

making the boundaries between ‘frontal’ plate F-1

and the ‘window’, and frontal plate F-4 and the ‘brim’,

uncertain); absence in all available instars of clear

plate delineations dorsally and dorsolaterally behind

the ‘frontal’ plates and above the areas corresponding

to the ‘marginal’ and ‘polygonal’ plates; and the lack

of any full sets of naupliar exuviae of particular

individuals, which prevented precise tracing of plate

divisions. Itô’s (1990b) expanded system of plate

nomenclature for later instars requires knowledge of

the order of plate divisions; for example, the names of

four plates derived from an earlier single plate by a

meridional (anterior/posterior) division followed by a

latitudinal (central/external) division will be different

from those derived by division in the reverse order.

Without such information, the ‘apostrophe’ system

employed by Kolbasov et al. (2021a) can be employed

if the cluster of plates corresponding to a larger plate

of an earlier instar can be recognized. In the present

case, with a minimum of ambiguity, it was possible

to match the pattern of anterior and anterolateral

plates of two specimens of H. demodex that appear

to represent successive intermediate instars (NHMD-

916635 and NHMD-916638; Fig. 13C, F) with that

described by Itô (1990b: fig. 7) for the supposed third-

stage nauplius of Hansenocaris furcifera. Based on

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TAXONOMY OF CRUSTACEAN Y-LARVAE 11

Figure 4. Hansenocaris demodex sp. nov., paratype, last-stage nauplius (LSN) from Sesoko Island photographed live before

fixation (A) and in SEM after fixation and mounting (B–H). A, ventral view; B, ventral view; C, dorsal view; D, pair of ventral

pores situated in front of labrum; E, close-up views of pores and sensilla of cephalic shield; F, caudal end in dorsal view; G,

caudal end in ventral view; H, caudal end viewed from behind. Abbreviations: a1, first antenna; a2, second antenna; arthro

membr, arthrodial membrane; ce, compound eye; dc sp, dorsocaudal spine; en, endopod; ex, exopod; fur sp, furcal spine; la,

labrum; md, mandible; ne, nauplius eye; rud, rudimentary. Small Arabic numerals, many annotated with l or r for left and

right, respectively, refer to cuticular structures (see Table 4). Yellow overlay, cephalic shield; blue overlay, faciotrunk; red

overlay, anterior field of facets. Square with dotted outline: sample number, museum number and type status.

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12 J. OLESEN ET AL.

Figure 5. Hansenocaris demodex sp. nov., paratype, last-stage nauplius (LSN) from Sesoko Island in SEM. A, lateral view;

B, cephalic shield, anterior view; C–J, close-up views of pores and sensilla of cephalic shield and faciotrunk; K–M, labrum

and naupliar limbs (a1, a2, md). Abbreviations: a1, first antenna; a2, second antenna; md, mandible; la, labrum; scl, sclerite

of a1. Small Arabic numerals, many annotated with l or r for left and right, respectively, refer to cuticular structures (see

Table 4). Yellow overlay, cephalic shield; blue overlay, faciotrunk; red overlay, anterior field of facets. Square with dotted

outline: sample number, museum number and type status.

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TAXONOMY OF CRUSTACEAN Y-LARVAE 13

that, corresponding regions in our earliest available

instar of H. demodex (Fig. 12B, D) could be identified,

as well as in its LSN (Fig. 5B).

Various terms are available for the limbs and caudal

armature of y-nauplii. Terms adopted in this study

include first antenna or a1, second antenna or a2,

Figure 6. Hansenocaris demodex sp. nov., last-stage nauplius (LSN) from Green Island in SEM. A, lateral view; B, dorsal

view. C, ventral view; D, frontal view. Abbreviations: a1, first antenna; a2, second antenna; dc sp, dorsocaudal spine; fur sp,

furcal spine; la, labrum; md, mandible. Arabic numerals, many annotated with l or r for left and right, respectively, refer to

cuticular structures (see Table 4). Square with dotted outline: sample number and museum number.

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14 J. OLESEN ET AL.

Figure 7. Hansenocaris demodex sp. nov., holotype (A, B, D–I) and paratype (C), cyprids from Sesoko Island photographed

before drying (A) and in SEM after drying and mounting (B–I). A, lateral view; B, lateral view; C, cephalic region, lateral

view; D, cephalic region, ventral view; E, bifid appendix (frontal filaments?); F, labrum; G, pores and sensilla; H, close-up of

hook of left first antenna; I, abdomen and telson, dorsal view. Abbreviations: a1, first antenna; a1 hk, first antenna hook;

a2 rud, second antenna rudiment; ae, aesthetasc; en, endopod; ex, exopod; la, labrum; md rud, mandible rudiment; par occ

pro, paraocular process. Roman numerals I–VI – thoracic segments. Large Arabic numerals 1–3 – abdominal segments.

Small Arabic numerals, many annotated with l or r for left and right, respectively, refer to cuticular structures (see Table 5).

Square with dotted outline: sample numbers, museum numbers and type status.

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TAXONOMY OF CRUSTACEAN Y-LARVAE 15

mandible or md, furcal spines (one subterminal ventral

pair) and dorsocaudal spine (terminal and unpaired).

The latter spine is not always situated dorsally in

y-larvae and, therefore, was termed a caudal spine by

Grygier et al. (2019), but since it is homologous to the

dorsocaudal spine of Cambrian Orsten crustacean larvae

Figure 8. Hansenocaris demodex sp. nov., holotype (B, C) and paratypes (A, D), cyprids from Sesoko Island in SEM. A,

live specimen in four different positions; B, cephalic shield, frontal view; C, cephalic shield, dorsal view; D, cephalic shield,

oblique posterior view. Abbreviations: par occ pro, paraocular process. Small Arabic numerals, many annotated with l or r

for left and right, respectively, refer to cuticular structures of the cyprid (see Table 5). Squares with dotted outlines: sample

numbers, museum numbers and type status.

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16 J. OLESEN ET AL.

Figure 9. Hansenocaris demodex sp. nov., holotype (A, B, D, E) and paratype (C) cyprid from Sesoko Island in SEM. A,

posterior thoracic segments and anterior abdominal segments, lateral view; B, thoracopods, ventral view; C, telson, dorsall

view; D, telson, terminal view; E, right furcal ramus, posterior view. Abbreviations: fur ram, furcal ramus; ba, basis; co, coxa;

en, endopod; ex, exopod; pro scle, proximal sclerites of thoracopods; thp 1, thoracopod 1. Roman numerals I–VI – thoracic

segments. Large Arabic numerals 1–3 – abdominal segments 1–3. Small Arabic numerals, many annotated with l or r for left

and right, respectively, refer to cuticular structures (see Table 5). Square with dotted outlines: sample numbers, museum

numbers and type status.

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TAXONOMY OF CRUSTACEAN Y-LARVAE 17

and cirriped nauplii (see, e.g.: Walossek, 1993; Walossek

et al., 1996; Chan et al., 2014), the term dorsocaudal

spine is preferred, not to be confused with the so-called

dorsocaudal organ, an organ of unknown function

situated posterodorsally on the trunk in some y-nauplii

(Elofsson, 1971; Schram, 1972; Høeg et al., 2014).

Figure 10. Hansenocaris demodex sp. nov., cyprid from Green Island in SEM. A, lateral view; B, cephalic shield, frontal

view; C, putative lattice organ, close-up; D, thoracic limbs 1–6; E, thorax, abdomen and telson, dorsal view. Abbreviations:

ba, basis; co, coxa; en, endopod; ex, exopod; la, labrum; pro scle, proximal sclerites of thoracopods; thp 1, thoracopod 1; thp 6,

thoracopod 6. Small Arabic numerals, many annotated with l or r for left and right, respectively, refer to cuticular structures

(see Table 5). Square with dotted outline: sample number and museum numbers. Dotted arrows in D point to long medial

setae of thoracopodal endopods.

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18 J. OLESEN ET AL.

Figure 11. Hansenocaris demodex sp. nov., nauplii from Sesoko Island photographed alive (A–J) or as slide-mounted exuvium

(K, L). A–E, different naupliar instars; F, penultimate naupliar instar with exuviae from two prior moults still attached

posteriorly; G, cyprid with exuviae from three prior naupliar moults still attached posteriorly; H, last-stage nauplius in lateral

view; I, posterior end of cyprid with three prior naupliar moults still attached; J, posterior end of cyprid with three prior naupliar

moults still attached; K, L, exuvium of last-stage nauplius, ventral and lateral views. Abbreviations: a1, first antenna; a2, second

antenna; ce, cypris eye; md, mandible; ne, nauplius eye; LSN, last-stage nauplius; thx, thorax. Squares with dotted outlines: dish

numbers, sample numbers and museum numbers; some larvae died before preservation and are only referred to by their dish

number. Type status in red.

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TAXONOMY OF CRUSTACEAN Y-LARVAE 19

Figure 12. Hansenocaris demodex sp. nov., early nauplius (LSN − 5 or LSN − 6?) from Sesoko Island in SEM. A, ventrolateral

view; B, lateral view; C, dorsal view; D, frontal view; E, labrum and naupliar appendages, right side; F, naupliar appendages,

right side, median view; G, first and second antennae, right side, lateral view, asterisk indicating rudimentary segment of

a2 exopod; H, dorsocaudal spine and furcal spines, terminal view. Abbreviations: a1, first antenna; a2, second antenna; ba,

basis; co, coxa; dc sp, dorsocaudal spine; en, endopod; ex, exopod; fur sp, furcal spine; F-1 to F-3, frontal plates 1–3; la, labrum

complex; md, mandible; rud, rudimentary; W, window plate. Small Arabic numerals, many annotated with l or r for left and

right, respectively, refer to those cuticular structures that could be matched with those of the LSN (see Table 4). Yellow overlay,

cephalic shield; blue overlay, faciotrunk; red overlay, anterior field of facets. Square with dotted outline: museum number.

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20 J. OLESEN ET AL.

Figure 13. Hansenocaris demodex sp. nov., paratypes from Sesoko Island in SEM:, an early nauplius (LSN − 3?) (A–C)

and a later nauplius (LSN − 2) with unmoulted later larval stages still inside. A, ventral view; B, lateral view; C, cephalic

shield, frontal view; D, ventrolateral view; E, lateral view; F, cephalic shield, frontal view; G, cephalic shield, dorsal view.

Abbreviations: a1, first antennae; a2, second antennae; dc sp, dorsocaudal spine; ex, exopod; fur sp, furcal spine; la, labrum;

md, mandible. Small Arabic numerals, many annotated with l or r for left and right, respectively, refer to those surface

structures that could be matched with those of the LSN (see Table 4). Green overlay, LSN; purple overlay, LSN − 1; red

overlay, anterior field of facets. Squares with dotted outline: sample numbers, museum numbers and type status.

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TAXONOMY OF CRUSTACEAN Y-LARVAE 21

There is no existing terminology for the few setae and

many pores on the body surface of nauplius y, although

it has been suggested that those of the cephalic shield

could be named after the facet (plate) in which they are

first seen during the course of development (Kolbasov

et al., 2021a). Here, as a stopgap measure, all have

been mapped and arbitrarily numbered from #1 to

#34, with the annotations ‘r’ and ‘l’ for the right and

left sides, respectively, if the structure is paired (see

detailed description below).

For y-cyprids, the established terminology that is

widely applied to other crustaceans is used for body

regions and appendages (e.g. thorax, abdomen, telson

and thoracopods), but no terminology exists for most

details of the complex cuticular ornamentation,

such as pores and sensilla, and these are, therefore,

here numbered arbitrarily (#1–#64) like those of the

nauplius. Previously, the terms mouth cone, mouth

parts of the piercing type (Bresciani, 1965), oral

pyramid (e.g. Itô, 1985, 1986b) or sometimes just

labrum (Kolbasov & Høeg, 2003; Kolbasov et al, 2007)

have been used for the extended mouth region in

y-cyprids. The term labrum is used here since parts

of this structure can readily be homologized between

the nauplii and the cyprid, based on the presence of

a characteristic similar pore pattern (e.g. #30–#32 in

nauplii, #62–#64 in the cyprid of H. demodex).

mOlecular analySeS

Individually fixed specimens were transported on

ice in tightly sealed boxes from the collection sites to

the molecular laboratory at Academia Sinica (Taipei,

Taiwan). Fixed specimens were transferred in a 0.1–

0.5-μL droplet of ethanol to 200-μL tubes (Gunster

Biotech, New Taipei City, Taiwan) and incubated at

36 °C for 15 min with tube lids open to allow ethanol to

evaporate. DNA extraction of Green Island specimens

(N = 3) followed a modification of the protocol of Schizas

et al. (1997): 1 μL of 10 × PCR buffer was diluted in 9 μL

ddH2O and added to the tubes containing individual

y-larvae. Tubes were incubated at 94 °C for 2 min to

denaturize the protein and then transferred to ice. After

adding 1 μL protein kinase K (QIAGEN, Chatsworth,

CA, USA) and vortexing the tubes, samples were

incubated at 55 °C for 15 min, followed by 70 °C

for 10 min. After returning the tubes to ice, 10 μL of

GeneReleaser (BioVentures, Inc, TN, USA) was added

to the tubes, which were then incubated in the following

thermal cycle: 65 °C for 15 s, 8 °C for 15 s, 65 °C for 45 s,

97 °C for 90 s, 8 °C for 30 s, 65 °C for 90 s, 97 °C for 30 s,

65 °C for 30 s, 80 °C for 3 min and 4 °C for 10 min. Tubes

were centrifuged for 1 min and c. 15 μL supernatant was

transferred from each one to fresh 200-μL PCR tubes.

Finally, 10 μL AE-buffer (QIAGEN, Chatsworth, CA,

USA) was added to stabilize the DNA extract. Sesoko

Island y-larvae (N = 19) were DNA-extracted by adding

40 μL AE-buffer and 4 μL protein kinase K (QIAGEN,

Chatsworth, CA, USA) to 20-μL PCR tubes (Gunster

Biotech, New Taipei City, Taiwan) containing single

y-larvae. The tubes were then incubated at 56 °C for

1 h, and then at 72 °C for 15 min, modifying the protocol

for extracting DNA from formalin-fixed specimens in

Palero et al. (2010). This extraction method is preferred

here over the modified Schizas protocol.

No Facetotecta-specific primers are available

so far. To ensure polymerase chain reaction (PCR)

amplification success, despite the small specimen size

(which implies low quantities of available DNA) and

potential degradation following heat exposure during

transportation from the tropical sampling sites, new

nuclear primers were designed to amplify a partial

region of the 18S ribosomal DNA (rDNA) gene based

on Facetotecta sequences available from GenBank.

Primers spanning highly conserved sites and flanking

hypervariable regions were designed to amplify short

fragments (~300 bp) ensuring high amplification rates

while simultaneously allowing species discrimination.

Polymerase chain reactions, with total reaction volumes of

20 μL, contained ~3 μL genomic DNA, 0.4 μL (10 μmol/L)

of each newly designed primer (18S Face1a: 5′-CTGCG

AATGGCTCATTACATCGGTCAT-3′ and 18S Face1b:

5′-GGTAGTCCAATACACTACCATCGACAGCT-G-3′),

4 μL Fast-Run Taq Master Mix (Protech Technology

Enterprise, Taipei, Taiwan) and 12.2 μL ddH2O (total

reaction volume 20 μL). PCR reactions were carried out

in a DNA Engine thermal cycler (Bio-Rad, Richmond,

California, USA) including a denaturation step of

95 °C for 5 min and then 40 cycles of denaturation at

95 °C for 1 min, primer annealing at 60 °C for 30 s and

extension at 72 °C for 30 s. The reaction was terminated

with a 10-min extension at 72 °C and 20 min at 4 °C.

PCR products were visualized using agarose (1.5%) gel

electrophoresis. Chromatograms were generated with

an ABI 3730XL Genetic Analyser by Genomics BioSci &

Tech. Ltd (Taiwan). Sequences were edited, assembled

and aligned using MAFFT with default parameters

(see https://doi.org/10.6084/m9.figshare.17803628.v1).

Our new 18S rDNA data (N = 22) and seven sequences

from GenBank [Hansenocaris itoi from the White

Sea: AF439393 and six unnamed, unphotographed

specimens from Sesoko Island used in Pérez-

Losada et al. (2009): FJ751877–751882] were used

in subsequent phylogenetic analyses. ModelFinder

as implemented in IQ-TREE was used to find the

best nucleotide substitution model for the alignment

(TNe+R2) and we subsequently inferred maximum

likelihood trees with IQ-TREE (-allnni –B 1000 –m

TNe+R2). Bootstrap support values were estimated

using 1000 ultrafast bootstrap replicates.

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22 J. OLESEN ET AL.

RESULTS

Sampling Of y-larvae

Sampling at Green Island (Taiwan) in 2017 and 2018

resulted in the handling and collection of more than

1000 y-larvae, but rearing to cyprids was mostly

unsuccessful and many individuals were fixed as late

naupliar instars either for morphological or molecular

work (see above). During field trips to Sesoko Island,

Japan, in 2018 and 2019, 9621 y-larvae were collected

(2018: 2710 specimens; 2019: 6,911 specimens) (Table 2).

During sorting, the larvae were counted based on a

rough division into the following four provisional

categories (Fig. 2): planktotrophic early nauplii

(specimens without yolk, with a flattened body and spine

rows along the posterolateral margins); lecithotrophic

early nauplii (specimens with yolk); mostly or

entirely lecithotrophic last-stage nauplii (specimens

with a pair of compound eyes in additional to the

anteromedian nauplius eye common to all y-nauplii);

and free-swimming cyprids. The most common type of

larvae (53.3%) were putative lecithotrophic nauplii,

of which the majority were early instars, followed by

planktotrophic nauplii (37.6%) and considerably fewer

but still numerous cyprids (5.1%) (Fig. 2; Table 2).

The division of nauplii into lecithotrophs and

planktotrophs was probably not entirely accurate, since

some supposed lecithotrophs did not moult in culture,

but the supposed planktotrophs could be further

substantiated later by additional criteria: presence/

absence of moulting in culture (planktotrophs do not

moult), presence/absence of antennal and mandibular

feeding spines and coxal endites (generally absent

in lecithotrophs), and presence/absence of a wide,

posteriorly protruding labral extension (generally

non-protruding or spiniform in lecithotrophs). Table

2 also lists a few other general results from the

collecting events, such as the average number of

larvae collected per tow with the plankton net. A total

of 29 specimens of H. demodex were included in the

study: the 2018 and 2019 fieldwork at Sesoko Island

resulted in 22 specimens, five of which died before they

could be preserved; two specimens from Sesoko came

from earlier (1991 and 2005) sampling; five specimens

came from the 2017 sampling at Green Island

(Taiwan). Live video of H. demodex and other y-larvae

can be seen here: https://youtu.be/seo-63AK10E.

taxOnOmy

pancruStacea ZrZavý & ŠtyS, 1997

thecOStraca gruvel, 1905

facetOtecta grygier, 1985

No family-group taxon has formally been proposed.

genuS Hansenocaris itô, 1985

Hansenocaris demodex OleSen, dreyer, palerO

& grygier [Sentence caSe capS]. sp. nov.

(figS 3–13, 14a)

Zoobank registration: urn:lsid:zoobank.

org:act:9FADB53F-9068-424E-9AC2-223C834E87A5.

Diagnosis: Last-stage nauplius (LSN) with markedly

elongate body tapering gradually towards bluntly

rounded caudal end, with no distinct narrowing behind

posterior end of cephalic shield in dorsal view. Dorsal

region of cephalic shield posterior to poorly-defined

‘window’ plate smooth, wholly unfaceted, and devoid of

ridges; surfaces between ridges on rest of shield also

smooth. Plate I-1 of cephalic shield becoming subdivided

by LSN − 3(?) instar. Anterior part of faciomarginal

area with at least one obvious pair of pores. Labrum

triangular or slightly bell-shaped, longer than wide,

lacking any pattern of ridges; its posterior margin a

shallowly indented declivity lacking any free posterior

plate-like extension or labral spine. First antennae

with four setae. Second antennae and mandibles devoid

of feeding structures (lecithotrophic); natatory setal

formulae of their exopods/endopods 1:1:1:1:2/0:1:1:1:2

and 2/2, respectively. Maxillules absent. Pair of

reduced furcal spines situated ventrally, forward from

base of short and blunt, terminal dorsocaudal spine.

Dorsocaudal organ and lateral trunk spines absent.

Y-cyprid with long, fully faceted cephalic shield with

nearly smooth surfaces between ridges. Small, bifid

appendix in anterior midline between first antennae and

anterior margin of cephalic shield. Labrum extended

as linguiform process with multiple hooks (17 in

holotype) on posterior side, arranged in three irregular

rows. First antenna with gracile, curved hook. Second

antennae and mandibles rudimentary. Thoracopods

with unsegmented exopods. Tergites of thoracomeres

V and VI with free pleural extensions, those of former

with rounded ends, those of latter trapezoidal. Abdomen

three-segmented, all segments short and lacking pleural

extensions. Telson long and lacking serrate spines along

posteroventral margin, with about 19 pores, including

two each on anteriormost plates of upper and lower

lateral rows. Furcal rami short and cylindrical.

Etymology: The specific name, a Latin noun

in apposition to the generic name, refers to the

resemblance of the elongate body form of the naupliar

instars of this species to that of mites of the genus

Demodex Simon, 1842 (Chelicerata: Trombidiformes:

Demodicidae), which are tiny parasites that live in or

near the hair follicles of mammals. The name in turn is

derived from Greek δημός (dēmos), fat, and δήξ (dēx), a

woodworm. The taxonomic description and supporting

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TAXONOMY OF CRUSTACEAN Y-LARVAE 23

molecular diagnosis of this new species were prepared

by JO, ND, FP and MJG, who are thus responsible for

making the specific name demodex available.

Type locality: Japan, Okinawa, Sesoko Island, pier at the

University of the Ryukyus Tropical Biosphere Research

Center, Sesoko Station, 26°38’09.3"N 127°51’55.2"E.

Holotype: Cyprid with exuvium of corresponding

last-stage nauplius (LSN), considered as two separate

parts of same individual specimen (cyprid: Figs 7A, B,

D–I, 8B, C, 9A, B, D, E; LSN exuvium: Fig. 3A, top).

Collected on 15 October 2018 (detailed sampling data

in Table 3), fixed on 21 October 2018, following final

moult after six days in culture. Cyprid stored in dried

condition on SEM stub, LSN exuvium as glycerine jelly

microscope slide, both at the Natural History Museum

of Denmark (NHMD-916629).

Paratypes: Eight larvae from same locality as holotype

but collected on different dates. Two cyprids mounted

for SEM with corresponding LSN exuviae on glycerine

jelly slides (NHMD-916630, NHMD-916632), exuvium

of one LSN on glycerine jelly slide with corresponding

cyprid preserved in ethanol for molecular work (NHMD-

916631), exuvium of one LSN on glycerine jelly slide

(NHMD-916639), one LSN mounted for SEM (NHMD-

916636), one LSN (nested failed moultings, outermost

cuticle is LSN − 2) mounted for SEM (NHMD-916638),

two early nauplii mounted for SEM (NHMD-916633,

NHMD-916635). Detailed sampling data in Table 3.

Other material: Sesoko Island (same locality as

holotype): One early nauplius collected 21 September

1991 mounted for SEM (NHMD-916640); six early

nauplii (JA-2018-111, JA-2019-001, -100, -321, -322,

-378-0), three LSN (JA-2019-165, JA-2019-107, -136)

and one cyprid (JA-2018-108), all preserved in ethanol

for molecular work (no vouchers retained). Green Island

(Taiwan): One LSN (NHMD-916641) and one cyprid

(NHMD-916642) collected early September 2018, and

mounted for SEM; three nauplii preserved in ethanol for

molecular work (TA-2018-066, -101, -166, no vouchers

retained) Detailed sampling data in Table 3.

Description

Last-stage nauplius (LSN) (Figs 3A, 4–6, 11D, E, K, L,

14A): Mainly based on paratype NHMD-916636

(Figs 4, 5). Body markedly elongate, slightly depressed

dorsoventrally. Total length (TL) 380 μm (alive)

or 375 μm (after critical point drying), including

dorsocaudal spine; lengths of other live specimens from

Sesoko Island 352–390 μm (N = 8) (Fig. 3). Greatest

width 140 μm and greatest dorsoventral thickness

100 μm. Cuticle transparent with nearly fully

developed, yellowish/brownish cyprid clearly visible

inside, with median nauplius eye and pair of compound

eyes. In dorsal view, frontal margin evenly rounded,

lateral margins tapering gradually towards bluntly

rounded caudal end, with no sharp border between

cephalic shield and trunk (slight indentation visible

in most other specimens), ending in blunt dorsocaudal

spine. In lateral view, body not entirely straight, but

bent c. 35° ventrally behind naupliar appendages.

Three pairs of naupliar appendages (a1, a2, md)

arising close together on ventral side at about 25% of

body length, flanking triangular, moderately bulbous

labrum. Large parts of ventral side of body (posterior

to labrum) and dorsal side of trunk ornamented with

transverse cuticular ridges. Cephalic shield with

ridge-bounded facets, except for smooth central area

flanking dorsal (but not anterior) midline. Entire body

displaying bilaterally symmetrical pattern of variety

of diverse pores and sensilla, all fully mapped and

numbered herein (see detailed description below).

No ‘ghost-like’ image of part of the cyprid thorax,

particularly the thoracopods and their setae, visible

inside any LSN exuviae (as previously detected in

many types of y-larvae; Grygier et al., 2019).

Anterior part of faciomarginal area almost

featureless except for pair of closely-set pores with

oval openings (#34, Fig. 4B, D) and pair of depressions

(#33), latter resolved as pores with slit-like openings

in Green Island specimen NHMD-916641 (Fig. 6C).

Large, triangular elevation (labrum) present between

naupliar appendages (Figs 4B, 5K–M), slightly bell-

shaped in mounted exuvium of paratype NHMD-

916630 (Fig. 11K), with shallowly indented posterior

margin. Labrum with five pores, including three

unpaired pores in midline (#29* posteriorly, #30* and

#31* near midlength), and one lateral pair positioned

level with these latter two (#32), but no free posterior

labral extension. Openings of pores #30* and #31*

oriented obliquely, but in slightly different ways in

this and the above-mentioned Green Island specimen

(compare Figs 5M, 6C).

Naupliar appendages placed immediately adjacent to

labrum in diagonal rows, with first antennae (a1) closest

to midline, mandibles farthest from it (Figs 4B, 5K).

Each limb arising from separate (a1) or partly

continuous (a2 and md) outpocketings of cephalic

cuticle, these probably serving to enhance appendage

flexibility while swimming. First antennae short and

digitiform (slightly shorter in above-mentioned Green

Island specimen; Fig. 6C), consisting of two distinct

segments and, embedded in cephalic outpocketing,

sclerite possibly representing additional proximal

segment (Fig. 5K). Distal segment twice as long as

proximal segment, bearing two long apical setae, one

apicolateral seta of intermediate length and one small

(rudimentary) medial seta. Second antennae and

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24 J. OLESEN ET AL.

mandibles devoid of any feeding structures (including

gnathobases or naupliar processes) (Figs 4B, 5K, L).

Coxa and basis of second antenna both broad, ring-

like, with rudimentary serration along distal margin;

endopod narrowly inserted on medial part of end of

basis, being small, cylindrical, and undivided with

two distal setae (one long and one short); exopod more

broadly inserted on lateral part of end of basis, being

composed of five successively smaller annuli, the first

bearing one small medial seta, the next three each

carrying one long medial seta, and the small distal

segment bearing two setae of intermediate length

(setal formula 1:1:1:1:2); in Green Island specimen, a

sixth rudimentary segment visible more proximally

in exopod. Segmentation and setation of mandible

similar to those of second antenna, except basis shaped

differently and proximal annulus of exopod smaller

and lacking any setae (setal formula 0:1:1:1:2).

Table 3. Material (24 specimens) of Hansenocaris demodex sp. nov. from Sesoko Island (Japan, Okinawa) and Green

Island (Taiwan) used in the present study. ‘LSN’ refers to ‘last stage nauplius’ before metamorphosis to the y-cyprid.

‘LSN − 2’ and ‘LSN − 3’, respectively, refer to instars two and three molts earlier than the ‘LSN’; see Material & Methods,

‘Naupliar development’, for explanation. Some of the specimens from Sesoko Island shown in Figure 3 are not listed here

as they died and were discarded without being preserved

Sample number Museum number Material Collection data

JA-2018-014 (holotype) NHMD-916629 • LSN* (Fig. 3)

• Cyprid† (Figs 3, 7–9)

15-Oct-2018#,**

JA-2018-013 (paratype) NHMD-916630 • LSN* (Figs 3, 13)

• Cyprid† (Figs 3, 7–9

17-Oct-2018#,**

JA-2018-108 (paratype, exuvium) NHMD-916631 • LSN* (Figs 3, 11)

• Cyprid§ (Fig. 3)

22-Oct-2018#,**

JA-2018-111 No voucher • Nauplius (failed moulting)§ (Figs 3, 11) 22-Oct-2018#,**

JA-2018-248 (paratype) NHMD-916632 • LSN* (Fig. 3)

• Cyprid† (Figs 3, 8)

3-Nov-2018#,**

JA-2018-274 (paratype) NHMD-916633 • Nauplius† (Fig. 3) 3-Nov-2018#,**

JA-2019-001 No voucher • Nauplius§ (Fig. 3) 1-Jun-2019#,††

JA-2019-320 (paratype) NHMD-916635 • Nauplius† (Figs 3, 11, 13) 22-Jun-2019#,††

JA-2019-321 No voucher • Nauplius§ (Fig. 3) 22-Jun-2019#,††

JA-2019-322 No voucher • Nauplius§ (Fig. 3) 22-Jun-2019#,††

JA-2019-165 No voucher • LSN§ (Fig. 3) 10-Jun-2019#,††

JA-2019-048 (paratype) NHMD-916636 • LSN† (Figs 3–5) 10-Jun-2019#,††

JA-2019-099 (paratype) NHMD-916638 • LSN (failed moulting, outermost cuticle is

LSN − 2)† (Figs 3, 11, 13)

11-Jun-2019#,††

No number (paratype) NHMD-916639 • LSN*2005#,‡‡

JA-2019-107 No voucher • LSN (failed moulting)§ (Figs 3, 11) 12-Jun-2019#,††

JA-2019-136 No voucher • LSN (failed moulting)§12-Jun-2019#,††

JA-2019-100 No voucher • Nauplius§14-Jun-2019#,††

JA-2019-378-0 No voucher • Nauplius§20-Jun-2019#,††

No number NHMD-916640 • Early nauplius† (Fig. 12) 21 or

22-Sep-1991#,‡‡

TA-2018-112 NHMD-916641 • LSN† (Fig. 6) 4-Sep-2018¤,§§

TA-2018-152 NHMD-916642 • Cyprid† (Fig. 10) 6-Sep-2018¤,§§

TA-2018-066-101-166 No voucher • 3 nauplii§31-Aug-2018,

2-Sep-2018,

6-Sep-2018¤,§§

#Japan: Okinawa, pier of Tropical Biosphere Research Center Sesoko Station.

¤Taiwan, Green Island, Gongguan Harbour.

*Formalin-fixed exuvium, on glycerine jelly slide.

†Formalin-fixed, on SEM stub.

§Ethanol-fixed and processed for molecular work (resulting in lack of voucher).

**Collected and processed by DEJ, MJG, YF, ND, JO.

††Collected and processed by DEJ, MJG, YF, ND, FP, JO.

‡‡Collected and processed by MJG.

§§Collected and processed by ND, DEJ, JO.

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TAXONOMY OF CRUSTACEAN Y-LARVAE 25

Cephalic shield approximately 200 μm long, 140 μm

wide, its posterior margin not clearly demarcated from

trunk, but indicated indirectly by shift in cuticular

ornamentation from cephalic shield’s longitudinal

ridges to trunk’s transverse striations (Figs 4C, 5A).

Lateral and frontal parts of shield bent ventrad and

bearing cuticular reticulations (facets or plates) and

lineations, while dorsal surface smooth, with weakly

defined mid-dorsal ‘window’. Ridge-defined reticulation

most complicated anteriorly, with large diversity of

small facets, some squarish and others oblong, while

more lateral facets fewer and longer, transitioning into

long, uninterrupted ridge-bounded belts reaching to

shield’s posterior margin. Arrangement of ridges and

facets roughly bilaterally symmetrical, but symmetry

of size, shape and degree of subdivision of contralateral

facets imprecise. Arrangement not described in detail,

but full pattern of two LSN from Sesoko Island and

Green Island as shown in Figures 4C, 5A, B and 6,

respectively, similar in both specimens but with some

differences: (1) arrangement of anterior facets more

strictly symmetrical in Green Island specimen; (2)

some oblong frontal facets of Green Island specimen,

most notably those above central pore #1 (Fig. 6D),

corresponding to pairs of smaller facets in Sesoko

specimen (Fig. 5B); and (3) all facet-bounding ridges

less distinct in Green Island specimen, sometimes

even absent (especially dorsally).

Cephalic shield ornamented with large number of

other types of structures (pores, setae), these being

fully mapped on paratype NHMD-916636 from Sesoko

Island (Table 4; Figs 4, 5): 31 structures in total,

mostly in pairs and concentrated in anterior third of

shield. Most common type consisting of 23 relatively

large (3–4 μm) pores, among which just one unpaired

and situated on midline close to anterior margin

(#1, Fig. 5B), remainder distributed anteriorly and

laterally in pairs (Table 4; Figs 4, 5). A few other kinds

of structures present dorsally: two widely separated

pairs of anterior setae (#7 and #8; Fig. 4E), one mid-

dorsal pair of small, circular pores (#12) and one pair

of small sensilla near posterior margin (#16, Fig. 5F).

Pore/sensilla pattern of specimen NHMD-916641

from Green Island (Fig. 6) the same, except for minor

differences in precise position of some structures (e.g.

pore pair #19, located between different transverse

lines in the two specimens).

Elongate trunk comprising about 45% of TL in dorsal

view, 62% in ventral view. Behind slight indentation

at posterior end of cephalic shield, trunk tapering

smoothly with its lateral margins subtending angle of

c. 20°, then somewhat rounding off at posterior end.

Dorsocaudal spine robust, short, blunt in NHMD-

916636 (Fig. 4), somewhat longer and more pointed

in other LSN specimens (Fig. 6). Pair of reduced

furcal spines situated ventrally, about 30 μm forward

from base of dorsocaudal spine. Lateral flanks and

posterodorsal part of trunk bearing about 25 paired

or dorsally continuous, regularly spaced and annular

cuticular ridges with posteriorly directed, serrate

crests (Figs 4, 5). In specimen NHMD-916641 from

Green Island, these ridges less distinct dorsally (Fig.

6B). In both specimens, nine pairs of large pores

distributed mostly laterally and ventrally on trunk

(#17–#25; Figs 4–6), including one pair (#25) flanking

dorsocaudal spine. In addition, pore pair #13 of cephalic

shield possibly actually belonging to outer border

of faciomarginal area. Pore arrangement generally

bilaterally symmetrical, but precise positions of many

pores far from symmetrical. For example, in NHMD-

916636 left member of pairs #17, #21 and #24 situated

far more anteriorly than their right-side counterparts

(most pronounced for most posterior dorsal pair,

#24), and in both this and Green Island specimen,

one member of pair #24 situated significantly more

dorsally than its contralateral partner (Figs 4F, 6B).

Large, convex, medioventral region of trunk,

reaching from point immediately behind labrum to

point immediately anterior to base of dorsocaudal

spine, this region being broadest anteriorly, reaching

posterolateral margins of cephalic shield on both

sides, then gradually tapering posteriorly to median

pore (#26*) between furcal spines. In NHMD-916636

this region crossed by transverse cuticular ridges with

posteriorly directed, serrate crests (Fig. 4B), these

ridges being fewer and less distinct in Green Island

specimen NHMD-916641 (Fig. 6C)

Cyprid (Figs 3, 7–10): Mainly based on holotype

(NHMD-916629); minor variation found in a few

other examined cyprids from Sesoko Island and Green

Island (Taiwan) mentioned directly in description with

indication of specimen identity (museum number).

Body elongated, consisting of head with large but not

all-enclosing cephalic shield (carapace), six-segmented

thorax, three-segmented abdomen and telson. Three

specimens measured in life 283–305 μm long, two

measured after fixation in ethanol both 360 μm long

and four measured after critical point drying 280–

320 μm long. Total length of individual specimens

greatly different when measured in life, in preservative

(longer, perhaps due to relaxation of musculature) and

after critical point drying (8–20% shrinkage).

Cephalic shield (or carapace) covering head

anteriorly, dorsally and laterally and also covering

anterior part of thorax dorsally and especially

laterally. Small nauplius eye lying anterodorsally

to pair of large compound eyes. Labrum and first

antennae situated on ventral side of head, beneath

compound eyes. Each of six thoracomeres bearing

a pair of biramous thoracopods, unclear whether

tergites of thoracomeres 1 and 2 separate dorsally

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26 J. OLESEN ET AL.

(this area not visible by SEM). In live specimens, main

body and anterior part of cephalic shield deep brown,

lateral parts of cephalic shield largely transparent,

non-functional gut orange (yolk?), and, in both fixed

and living specimens, area beneath cephalic shield

approximately at point of separation from main body

with dorsolateral concentration of orange-coloured

droplets (oil, yolk?) (Figs 7A, 8A).

Cephalic shield relatively long, univalved, only

partially covering dorsal and lateral sides of main

body of larva, with only thoracomeres V and VI being

exposed dorsally and with lateral margins of shield

reaching telson in live specimens (Fig. 8A). Shield

resembling inverted boat with posterolateral parts

much produced, altogether about 175 μm long along

mid-dorsal line and 210 μm along lateral margins.

Long longitudinal and short transverse and oblique

cuticular ridges present, outlining plates (or facets)

and longitudinal bands, these together occupying

entire shield surface but being most distinct anteriorly

and laterally; more anterior facets generally smaller.

Arrangement of facets and bands not strictly

bilaterally symmetrical; overall pattern in dorsal

view almost so (see holotype, Fig. 8C), but significant

asymmetry apparent in anterior view (Fig. 8B), with no

clear left–right correspondence of facets around pore/

sensilla pairs #9 and #10. In another paratype cyprid

from Sesoko Island (NHMD-916630; not shown) and a

cyprid from Green Island (NHMD-916642; Fig. 10B),

anterior face of shield showing more symmetry than

in holotype.

Surface of cephalic shield with numerous pores,

seta-bearing pits and other cuticular structures

(total number 84, counting both members of pairs)

in semi-symmetrical pattern (Fig. 8B, C) comprising

five distinct types of structures (Table 5). First type

(19 in number) with slit-like pore surrounded by

conspicuous circular rim. Except in one case (pore

#1* on midline), these pores always paired, being

concentrated anteriorly and laterally (Figs 7B, 8B,

C). Oblique opening of pore #1* oriented differently

in specimens from Sesoko Island (holotype, Fig. 8B)

and Green Island (Fig. 10B). Second type (28 in 14

pairs) a deep pit with round mouth and single short,

protruding seta (Fig. 8B, C); these pits scattered all

over shield surface, but more highly concentrated

anteriorly. Third type, all with round mouths and

neither cuticular rim nor seta (Fig. 8B, C), including

eight pairs of small pores and three larger, unpaired,

so-called central pores sensu Kolbasov & Høeg (2003),

two situated anteriorly and one posteriorly (#14*, #20*

and #39*; Fig. 8C). Fourth type comprising four pairs

of small groups of micropores (two or five per group),

all situated anteriorly on cephalic shield (Fig. 7D, G).

Fifth type of structure on cephalic shield identified

as lattice organs, grouped into two anterior and three

posterior pairs flanking dorsal midline (Figs 8B, C,

10B, C). Anterior pairs (#13 and #15) distinguished

from general cuticle by their situation 50–60 μm

from anterior end of shield in four weak depressions

surrounding most anterior of unpaired central pores

(#14*). Their cuticle smooth and lacking any small

pores (thus no pore field). First pair (#13) teardrop-

like, about 10 μm long and 7 μm wide, strongly

converging anteriorly and each narrowing posteriorly

towards small terminal pore. Second pair (#15)

elongate, 12 μm long and about 2.5 μm wide, slightly

converging anteriorly and weakly narrowing towards

tiny, posterior terminal pore. Third pair of lattice

organs (#38) situated in front of posterior unpaired

central pore (#39*), almost round with diameter of

about 3 μm and with barely visible posterior terminal

pore. Final two posterior pairs of lattice organs (#40

and #41) situated behind unpaired pore #39*, #41 in

flattened posterior marginal area of shield. These two

pairs, respectively, 10 μm long and about 1.5 μm wide

and about 7.5 μm long and 2 μm wide, lack visible

terminal pores. Lattice organs largely organized the

same way in above-mentioned Green Island specimen

(NHMD-916642), but additional rudimentary pair

possibly present anterior to counterparts of above-

described two anterior pairs (Fig. 10B, C).

Proximal parts of first antennae not visible in

SEM preparations. Segmentation of distal parts also

unclear, but distal armament consisting of conspicuous

curved hook (claw), large aesthetasc and, between

these structures, one short seta with scattered

setules and one smaller simple seta (Fig. 7B–D,

H). Claw remarkably gracile, semicircular. Small,

bifid, thin-walled appendix, possibly homologous

to frontal filaments in nauplii, present in anterior

midline between first antennae and anterior margin

of cephalic shield (Fig. 7E). Labrum extended as

linguiform process with 17 hooks on posterior side,

arranged in three irregular rows of five, six and six

hooks (Fig. 7C, D, F). Posterobasal part of labrum with

five pores: one lateral pair with slit-like openings (#64)

and three in midline, among which two with oblique

slit-like openings (#62, #63) and one partly hidden

proximally (#61; vestigial mouth opening?). Vestiges

of second antennae and mandibles present lateral to

labrum, showing remains of exopods and endopods

in one specimen (NHMD-916630, Fig. 7C), but small,

rounded and wrinkled in another (NHMD-916629,

Fig. 7B). Small pair of so-called bifurcate paraocular

processes present anterior to these, with anterior and

slightly thicker posterior branches both 15 μm long

and situated parallel and adjacent to lateral margin

of cephalic shield (Fig. 7B–D). No pair of postocular

filamentary tufts seen in these preparations.

Among thoracomeres I–VI (Figs 7B, 9A, 10A, E),

posterodorsal margins of tergites V and VI serrate (Figs

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TAXONOMY OF CRUSTACEAN Y-LARVAE 27

7I, 10E). Tergite VI also equipped with two or three

other transverse, serrate cuticular ridges and bearing

widely spaced pair of setae close to posterior margin

(#45; Fig. 7I). Cuticular ridges less distinct on Green

Island specimen (Fig. 10E). Tergites V and VI with free

pleural extensions, those of former with rounded ends,

those of latter trapezoidal with sharp posteriolateral

ends (less trapezoidal in Green Island specimen) (Figs

9A, 10A).

Each thoracomere bearing pair of biramous

thoracopods (Figs 9A, B, 10A, D). Each thoracopod

consisting of lateral basal array of sclerites, coxa

(distinct only in thoracopods 1–5), basis and pair of

rami (exopod and endopod). Proximal sclerites not

described further as this flexing zone appears variable

between specimens. Two-segmented endopod of first

thoracopod with short proximal segment and elongate

(three times longer than wide) distal segment with two

long apical setae; endopod slighty shorter in Green

Island specimen (Fig. 10D). Unsegmented exopod

slightly shorter than endopod, significantly wider

proximally than distally, and bearing two apical setae:

short outer one and long inner one. Thoracopods 2–5

composed of the same elements as thoracopod 1, but

with distal segment of endopod generally longer and

carrying long medial seta (dotted arrows on Figs 9B,

10D) in addition to two terminal setae; and with exopod

larger and bearing three terminal setae: short outer

one and two long inner ones. Thoracopod 6 generally

similar to preceding thoracopods but shorter, and with

unsegmented protopod (coxa and basis fused?).

Abdomen consisting of three short segments, all

lacking pleural extensions but possessing serrate

transverse cuticular ridges and posterior margins; first

segment with dorsolateral pair of setae (#46), third

segment shortest, tapering ventrally and sometimes

strongly intercalated between second segment and

telson (7B, I, 9A, 10A, E). Telson long with dimensions

varying somewhat among specimens, 1.5–1.6 times

as long as greatest width in a specimen from Sesoko

Island (Figs 7E, 9C), but 1.3 times as long as wide in

one from Green Island (Fig. 10E). Telson with dense

reticulation of serrate ridges roughly outlining two

dorsal longitudinal rows of broad plates (Figs 7I, 10C)

(anterior two or three pairs only weakly or not divided

at midline), two lateral rows on each side and five

ventral rows (Figs 7B, 9B–D, 10A). Number of plates

in each dorsal row approximately 13 (11 in Green

Island specimen), 11 in each lateral row and ten in

each ventral row, with those of mid-ventral row set off

half a step from those of other rows.

Total of 19 pores and setae present on telson (Table 5).

Four pairs (#48–#51) placed in characteristic pattern

anterolaterally (Figs 9A, 10A). Pair of pores with slit-like

opening (#52) present in third plate from front in upper

row of lateral plates (Figs 7B, 9C, 10A, E); similar pore

(#53) in fourth plate from rear in lower row of lateral

plates (Figs 7B, 9C, D, 10A). Another similar pair of

pores and pair of setae in pits (#54 and #55, respectively)

situated either in same contralateral pair of posterior

dorsal plates (Fig. 7I) or with pores and setae in

successive pairs of plates (Fig. 9C, D) or, in Green Island

specimen, pore pair #54 absent (Fig. 10E). Terminally,

one pair of dorsal pores (#56) and one ventral central pore

(#57*). Three pairs of ventral pores (Figs 9B, D) located

far anteriorly in outer row of ventral plates (#60) and in

two adjacent posterior plates in same row (#58 and #59).

Furcal rami short and cylindrical, perhaps even disc-

like, with three setae each: two unequal lanceolate setae

with serrate margins, and one irregularly denticulate

short seta (Fig. 9D, E).

Earlier naupliar stages

Number of naupliar stages: An outline of the naupliar

development of H. demodex is provided herein based

on a combination of high-resolution photographs in

life of different instars of the same individual (Figs 3,

11) and SEM images (Figs 12, 13) of several specimens

representing three distinct instars younger than the last-

stage nauplius (LSN). Four nauplii developed to cyprids

while in culture (NHMD-916629, NHMD-916630,

NHMD-916631, NHMD-916632), transitioning 3–6 days

after the date of sampling (Table 6). Of particular

importance are four specimens – three of them LSN as

shown by presence of the developing cyprid’s compound

eyes within – that failed to moult properly, resulting

in a nested set of unshed exuviae with the most recent

stage innermost (Fig. 11F, G, I, J). These specimens

provide direct evidence of the last four stages of naupliar

development, LSN, LSN − 1, LSN − 2 and LSN − 3. In

each series, the dorsocaudal spine generally becomes

thinner and less blunt as development progresses,

with the thinnest spines being found in the LSN. There

appear to be even earlier instars in our material. One

of these, NHMD-916635 (Fig. 13A, B), has a dorsocaudal

spine that is blunter than that of LSN − 3 (cf. Fig. 11F); it

most likely belongs to an earlier instar, perhaps LSN − 4.

Another specimen (Fig. 12), the earliest-stage specimen

examined during this study, has much weaker developed

cuticular reticulation and other cuticular armature than

the specimen mentioned above and appears to be one,

or more likely two, instars earlier than it, i.e. LSN − 5 or

LSN − 6. The naupliar development of H. demodex thus

comprises at least five to six instars, more if instars have

been missed (see Discussion).

Colour, yolk and cyprid morphogenesis during the

naupliar phase: Coloration clearly shown and yolk

boundaries distinct in our photographs of living nauplii

at various moult stages (Figs 3, 11). Anterior and

posterior parts of body brownish in earliest stages (Fig.

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28 J. OLESEN ET AL.

11A, B). Bright-coloured cylindrical region – orange in

specimens from Sesoko Island but yellow in those from

Green Island – extending from behind nauplius eye to

dorsocaudal spine, partly subdivided into droplets or

granules and assumed to be at least partly made of yolk.

Yolky region dividing into two parts early in naupliar

development, these corresponding to future thorax and

abdomen of the cyprid (Fig. 11B). Cyprid achieving its

final form within last-stage nauplius (LSN) and capable

of movement (e.g. thoracopodal beating, abdominal

dorsoventral flection), while still inside naupliar

cuticle. Before final moult, abdomen of cyprid becoming

gradually thinner and yolk becoming concentrated in

dorsal midline, as also seen in free-swimming newly

moulted cyprids (orange area in Figs 7A, 8A).

Naupliar stage LSN − 5 or LSN − 6? (Fig. 12): Earliest

stage among all nauplii of H. demodex examined here

with SEM. Body short (345 μm long), dorsocaudal spine

blunt. Overall body shape much like that of LSN, but

markedly more compact, less elongate and distinctly

inflated (due to greater quantity of yolk?). Large facets

(plates) on anterior and lateral parts of cephalic shield

few in number and separated by faint ridges. Anterior

field of large facets marked with red overlay in Fig.

12A–C in order to trace their fate in following instars.

Assuming only one row of ‘brim’ plates anteriorly, red

overlay encompassing all axial ‘frontal’ plates (F-1 to

F-4; definitive boundaries uncertain, so labelled as

F*) in addition to small parts of ‘elongate’ plate pair

E-1 (i.e. E-1*) in upper corners and both members of

‘intercalary’ plate pair I-1 and ‘polygonal’ plate pair

P-1 in lateral areas, none of these being yet delineated

from their adjoining ‘frontal’ plates (cf. fully delineated

state in later instars). Overlay area flanked on each

side from top to bottom by four plates identifiable under

Itô’s (1990b) and Kolbasov et al.’s (2021a) expanded

systems as ‘intercalary’, ‘elongate’, ‘polygonal’ and

‘marginal’ plates E-1* + I-3(a), I-2, P-2 (bordering

pore #4 posteriorly) and M-1. ‘Marginal’ plate M-2 and

‘superlateral’ plate S, adjoining M-1 posteriorly and

posteroventrally, with S bordering pore #2 anteriorly

and adjoining plate M-3(e) posteriorly. Other plates,

especially dorsally, too poorly delineated to identify

with confidence except for M-2 + M-3(c) behind M-1

and lateral band behind S consisting of M-3(e) and

combined M-4 to M-7.

Labrum similar to that of LSN in general form

and pores, but preceded by distinct median elevation

reaching to pore pair #34, and pore #29 relatively

larger than in LSN and positioned significantly

farther forward, away from posterior margin. Naupliar

appendages differing from those of LSN in minor

ways: all limbs relatively shorter and more robust

than in LSN; exopod of second antenna six-segmented

owing to presence of rudimentary proximal segment

(Fig. 12G, asterisk). Setation of appendages as in

LSN, but medial seta of first antenna longer. Trunk

long and gradually tapering, but relatively shorter

than in LSN and with sides more parallel; many

fewer rows of serrate vertical cuticular ridges/scales

laterally than in LSN and even fewer transverse

ridges dorsally. Both cephalic shield and trunk with

pores and setae of essentially same kind as in LSN,

but fewer in number. Most such structures on cephalic

shield identifiable with counterparts in LSN by form

and position, and thus numbered the same in Figure

12, but a few pores of unclear identity on trunk

labelled only as ‘?’. On cephalic shield, pore pairs #2,

#10 and #14 or #15 present anterolaterally, laterally

and posterolaterally near margin; pore and seta pairs

#4–#8 present anterodorsally (#8r and #8l flanking

Table 4. Overview of distribution of 63 cuticular surface structures (pores, sensilla, etc.) on different body regions of last-

stage nauplius (LSN) of Hansenocaris demodex sp. nov., paratype (NHMD-916636) from Sesoko Island (Japan: Okinawa).

Numbers refer to individual structures as indicated in Figures 4 and 5 and in other depicted nauplii. All structures paired

except asterisks (*) indicate unpaired structures found only in midline.

Large slit-like

pore (3–4 μm

diam.)

Pore

with large

seta

Small circular

pore (1 μm

diam.)

Pore with

small

sensillum

Intermediate-

sized or small

slit-like pore

Distinct

cuticular

depression

Total

Cephalic shield 1*–6,9–11,13–15 7, 8 12 16 31

Trunk, dorsal

and lateral

17–25 18

Trunk, medial

ventral region

26* 27 3

Cephalic part of

faciotrunk,

ventral

28, 31*, 32 29*, 30*, 34

(frontal

filaments?)

33 11

Total 47 4 4 2 4 2 63

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TAXONOMY OF CRUSTACEAN Y-LARVAE 29

plate W), and pore pair #9 present dorsally posterior to

midlength of shield. On trunk, dorsolateral pore pairs

#17, #19, #21 and #24 more-or-less identifiable, but

interpretations complicated by left/right asymmetry in

pore distribution. Pore pair #25 flanking dorsocaudal

spine at posterior end of body and single mesial pore

#26 situated between blunt, rudimentary furcal spines,

these spines being closer to terminal end than in LSN.

Anterior central pore of cephalic shield (#1) and pore

pair #3 flanking it not yet apparent. Posteriorly on

Figure 14. Morphology of four types of y-larvae from Sesoko Island used for molecular analyses (see overview in Table

6), all photographed in life. A, Hansenocaris demodex sp. nov., lecithotrophic LSN (last-stage nauplius) with cyprid inside.

B, ‘Bumblebee’ type, lecithotrophic LSN with cyprid inside. C, ‘Big brown’ type, lecithotrophic LSN with cyprid inside. D,

planktotrophic nauplius, identified as Itô’s (1986a) Pacific type I. Squares with dotted outline: sample numbers.

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30 J. OLESEN ET AL.

shield, seta pair #16 and pore pairs #11, #12 and either

#14 or #15 not yet present. Ventrolateral pore pair #13

also not seen.

Naupliar stage LSN − 3? (Figs 11B, 13A–C): This

stage at least one and probably two instars later

than preceding specimen and probably one instar

earlier than LSN − 2 specimen described below. Facet

arrangement of cephalic shield more complicated

than in preceding specimen, with greater number of

longitudinal bands and division of anterior region into

smaller facets. For example, anterior face of shield

(Fig. 13C, red overlay) now subdivided into three rows

of clearly delineated, ridge-bounded facets in three

rows: axial row extending from anterior margin of

shield to approximately pore pair #6, comprising part

of ‘brim’ and all derivatives of ‘frontal’ (F-1 to F-4)

plates, and pair of shorter, eight-facet rows flanking

it to left and right, consist of derivatives of certain

‘polygonal’ (P-1), ‘intercalary’ (I-1) and ‘elongate’ (E-1)

plates. Unpaired far-anterior pore (#1) and a few other

pores (e.g. pair #3 on anterior face of shield and pair

#23 on ventral side of trunk) now present. In general,

pores more distinctly delineated and with more

pronounced slit-like openings than in above-described

earlier stage. Segmentation and setation of naupliar

appendages identical to those of that specimen, but

trunk with greater number of more distinct vertical

and transverse cuticular ridges, including narrow row

of such ridges along ventral midline (Fig. 13A).

Naupliar stage LSN − 2 (Figs 11G, 13D–G):

Incompletely moulted LSN/cyprid specimen with

cephalic shields and faciotrunk integuments of two

previous instars still attached in nested fashion

(LSN − 1, yellow overlay, and LSN, green overlay).

Moulting zone of both LSN − 1 and LSN − 2 cuticles

running along dorsal transverse seam between rear

margin of shield and trunk dorsum, then continuing

ventrally around border between faciomarginal area

and lateral and anterior margins of cephalic shield.

Mainly outermost LSN − 2 stage observed by SEM

(Fig. 13D–F), but unmoulted cyprid y observed and

photographed in life within three surrounding layers

of naupliar cuticle (Fig. 11G). LSN − 2 differing from

stage described above (LSN − 3?) in having about

twice as many and narrower elongate, band-like facets

posterolaterally on cephalic shield and generally more

complex facet arrangement anteriorly. For example,

brim plates crossing and flanking anterior midline of

shield margin in previous larva (Fig. 13C, arrow), and

there comprising three facets in total, now comprising

two transverse rows of four smaller facets each

(Fig. 13D, F, arrows). Pores and setae of this nested-

cuticle specimen not examined in detail, but

evidently not appreciably different from those of LSN.

Segmentation and setation of naupliar appendages

apparently identical to those of earlier stages. Trunk

more slender than in previously described stages,

weakly tapering terminally with longer, thinner

dorsocaudal spine and with less distinct vertical and

transverse cuticular ridges.

mOlecular analySeS

Twenty-two larval specimens were sequenced,

together representing one feeding (planktotrophic)

and three non-feeding (lecithotrophic) types of nauplii

from Sesoko Island (N = 19 specimens) and Green

Island (N = 3 specimens) (Fig. 14; Table 6). New 18S

sequences for the 22 specimens in the phylogenetic

analyses have been uploaded to GenBank (Accession

codes: OM135272–OM135293). Images of all sequenced

specimens (except some H. demodex), photographed

while still alive, were mapped to the phylogeny (Fig. 15).

Because most Facetotecta nucleotide sequences

previously registered in GenBank are based on

larvae with no morphology recorded, the true nature

of those larvae can only be inferred. Congruence

between molecular (18S rDNA) and morphological

data is nonetheless evident in Figure 15, with similar

specimens grouped together into two main clades (A

and B in Fig. 15). Clade A contains Hansenocaris itoi

(AF439393) and two unnamed taxa from GenBank

(FJ751879, FJ751881). Clade B is larger and includes

four subclades, each represented by several specimens

with mutually identical haplotypes. The first subclade

is composed of four specimens of Itô’s (1986a) Pacific

type I (planktotrophic) and three unnamed taxa

from GenBank (FJ751877, FJ751878, FJ751882).

The second subclade is composed of nine H. demodex

specimens. The third subclade is composed of two

specimens of a type nicknamed ‘Big brown’ and one

unnamed taxon from GenBank (FJ751880). Finally,

the fourth subclade is composed of seven specimens of

a type nicknamed ‘Bumblebee’.

DISCUSSION

y-larvae: individual rearing fOr SpecieS

deScriptiOnS

The challenge of matching wild marine invertebrate

larvae with their adult counterparts dates back to the

19th century (Thomson, 1830; Müller, 1864; Garstang,

1951). In the modern era, efficient matching of different

life-stages in invertebrate life-cycles has been greatly

facilitated by the advancement of culturing procedures,

more recently supplemented by molecular methods

(Pardo et al., 2009; Tang et al. 2010; Feller et al., 2013;

Carreton et al., 2019). Both methods were applied here

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TAXONOMY OF CRUSTACEAN Y-LARVAE 31

to the Facetotecta, the last major invertebrate group

for which the adult stage remains unknown. Some

evidence suggests that the adults are endoparasites

in undiscovered host organisms. Most significantly,

Glenner et al. (2008) found a new life-stage in the

facetotectan life-cycle, the ypsigon, a slug-shaped stage

that emerged from y-cyprids that had been exposed

to a moulting hormone in the laboratory. Similarities

of the ypsigon to the vermigon, the infective stage of

certain parasitic barnacles (Rhizocephala), led Glenner

et al. (2008) to infer endoparasitism of the unknown

y-adults.

As a source of individual y-larvae for laboratory

culturing, the present study primarily relied on a

shallow-water site at Sesoko Island (Japan: Okinawa)

that has repeatedly been suggested to show a

remarkable abundance and diversity of y-larvae

(Grygier, 1991a; Glenner et al., 2008; Grygier et al.,

2019) (Fig. 2). The 9621 y-larvae collected and handled

during more than six weeks of intensive fieldwork

there in 2018 and 2019 amount to more than 25

times the greatest number of y-larvae collected in any

single previous study, viz., the 350 or so specimens

of Hansenocaris itoi from the White Sea reported by

Kolbasov & Høeg (2003). On average, more than one

y-larva was collected in each of the 7021 plankton tows

made during these Sesoko surveys. In a few cases, more

than 30 y-larvae were present in a single 10-15 m tow

(Table 2) and, on these occasions, y-larvae were among

the dominant members of the smaller-sized crustacean

plankton. No other studies at the same scale are

found in the literature, so it remains to be seen how

widespread the mass occurrence of y-larvae actually

is, either along Japanese coastlines or elsewhere. The

full implications of this dataset, including the results

from bi-hourly monitoring of y-larva occurrence, will

be presented elsewhere.

None of these almost 10 000 wild-caught y-larvae

could be convincingly identified to any of the currently

described species, but a few seemed to conform to

previously reported larval types such as Itô’s (1986a)

Pacific type I, which can readily be identified (Fig. 2).

Even a quick glance at a fraction of a given sample (Fig.

2) suggests that numerous types/species are involved.

Y-larva taxonomy is in a highly unsatisfactory state,

when considering the high local abundance of these

larval types and their likely ecological importance.

To remedy this situation, a new approach to species

descriptions is required. For the sake of standardization,

it is here suggested that future species descriptions of

y-larvae focus on a combination of last-stage nauplii

and cyprids, since each of these stages are homologous

to their counterparts across y-larvae and can be

readily identified. For y-larvae with lecithotrophic

nauplii, building on previous results (Itô & Takenaka,

1988; Glenner et al., 2008; Grygier et al., 2019), a

novel and larger scale integrated taxonomic protocol

has been outlined and employed herein to describe

Hansenocaris demodex, a unique form of y-larva with

semi-vermiform nauplii that occurs in the waters of

Japan and Taiwan.

Hansenocaris demodex – a rare but diStinctive

y-larva frOm Japan and taiwan

Hansenocaris demodex is a fairly rare species in

our samples, with only 22 specimens (0.2%) out of

9621 y-larvae collected at Sesoko (Okinawa) in 2018

and 2019. Sampling at Green Island (Taiwan) was

more haphazard, with considerably less quantitative

recording of the take than was done later at Sesoko

Island, but the species is clearly rare at both sites.

Hansenocaris demodex is the first species of Facetotecta

for which several stages of lecithotrophic nauplii, as

well as the cyprid, have been described in full detail,

something not accomplished by Itô (1991), and with

the nauplii and cyprid confirmed to be conspecific

based on individual moult sequences and molecular

data. Its nauplius and cyprid stages all present unique

characteristics. The extraordinarily elongate, semi-

vermiform shape of the nauplii of all known stages

and the distinctive, multihamulate labrum of the

cyprids make this species easily recognizable among

all y-larvae described so far, and also among all other

y-larvae handled during the present fieldwork at

Sesoko and Green Island.

In this paper, H. demodex is documented from

Green Island, Taiwan, and Sesoko Island, Japan,

which are separated by c. 780 km (Fig. 1). 18S rDNA

nucleotide sequences unequivocally demonstrate that

the populations at the two sites are conspecific (Fig.

15), which is in accordance with the morphological

results, except for coloration, with specimens from

Green Island being distinctly yellower (not shown

in the figures). The distribution of this species is

probably linked to the Kuroshio Current, a warm

oceanic current that originates east of the Philippines

and flows in a north-eastward direction past Taiwan,

Okinawa and Japan at a velocity of up to 4 m/s (345

km/day) (Jayne et al., 2009). Intertidal communities of

free-living and parasitic marine organisms are known

to experience a large gene flow along the Kuroshio,

resulting in a lack of genetic differentiation between

populations, even over long distances (Chan et al.,

2007; Chang et al., 2017; Jung et al., 2019; Ma et al.,

2019). This probably explains the great molecular

similarity between the Green Island and Sesoko Island

populations of H. demodex. Indeed, this and other

species of y-larvae may be distributed throughout

the entire Kuroshio region, something that can only

be confirmed by further sampling. One of us (MJG,

unpublished) collected several additional specimens of

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32 J. OLESEN ET AL.

H. demodex in 2020 at the island of Xiaoliuqiu off the

south-west coast of Taiwan. Water masses move from

Taiwan to Okinawa in 2–3 days, which is fast enough

to transport young nauplii of H. demodex between

these two places before they moult to the cyprid, a

transition that our data showed to require 3–6 days

Figure 15. Molecular phylogeny of y-larvae (Facetotecta) based on 18S data combining sequences of nine specimens of

Hansenocaris demodex sp. nov. and 13 other specimens from Sesoko Island with sequences of Hansenocaris itoi and other

limited information from GenBank. For more information on specimens and types, see Table 6 and Figure 14.

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TAXONOMY OF CRUSTACEAN Y-LARVAE 33

in the lab. However, since the majority of the sampled

nauplii of H. demodex at Sesoko Island, as well as the

other forms of lecithotrophic nauplii collected there

(Fig. 2; unpublished data), were young instars, nearby

sources (i.e. spawning y-adults) appear necessary to

sustain the large populations there, regardless of any

Kuroshio-mediated long-distance transport that may

occur.

naupliar mOrphOlOgy Of Hansenocaris demodex

cOmpared tO Other y-nauplii

The nauplii of H. demodex are lecithotrophic, as shown

by the large amount of internal yolk and the lack of

antennal and mandibular feeding structures. Many

types (likely species) of lecithotrophic y-nauplii are

known to exist at Sesoko Island (Glenner et al., 2008;

Grygier et al., 2019; unpublished data) and elsewhere

in Japan (Itô, 1990b; Watanabe et al., 2000), but limited

detailed information is available for comparison. At

least 15 different types (putative separate species)

of lecithotrophic y-nauplii have been reported, all

of which are different from the present specimens.

Among the three types of y-nauplii described by Itô

(1986a), two lacked endites of the second antennae and

were, therefore, the first lecithotrophs reported in the

literature (but Itô did not use the term ‘lecithotrophic’).

Besides lacking feeding armature, these two naupliar

types have a swollen body like H. demodex but in other

ways are clearly different from it, being robust and

having long, pointed dorsocaudal spines. Later, Itô

(1987a) described three forms of large, disc-shaped,

dorsoventrally flattened y-nauplii (‘type VIII’) that

lacked the well-developed feeding armature of the

second antennae and mandibles and were, therefore,

also lecithotrophic, but different from the nauplii of

H. demodex. Itô (1991) illustrated and briefly described

(but did not name) yet another lecithotrophic form of

y-larvae with a large dorsocaudal spine, based on a

possibly complete larval series obtained by laboratory-

rearing, the first of its kind. Belmonte (2005) based

his description of H. mediterranea Belmonte, 2005 on

a few dozen lecithotrophic y-nauplii (see also Høeg

et al., 2014: fig. 18.1H, I). Also, Grygier et al. (2019)

presented photographs, but no descriptions, of eight

clearly mutually distinct forms of lecithotrophic last-

stage nauplii, all of which are different in general body

form from H. demodex.

Y-larvae are well-known for their signature feature,

a complex arrangement of cuticular facets (or ‘plates’)

on the surface of the cephalic shield of both nauplii

and cyprids, the function of which is unknown

(Schram, 1972; Itô 1987b, 1990b; Kolbasov & Høeg,

2003; Kolbasov et al., 2021a). Most information on

y-naupliar facet patterns and their development comes

from planktotrophic y-nauplii, while almost nothing

has been published on lecithotrophs. Schram (1972)

proposed a facet nomenclature, which he based on an

early-stage planktotrophic nauplius with a simple and

symmetrical facet arrangement. Itô (1987b) proposed

a modification of Schram’s (1972) system to encompass

different types of early-stage y-nauplii, including one

lecithotroph (type VII). Efforts were later made, with

only partial success, to extend Itô’s nomenclature

system to later naupliar stages of H. furcifera (see:

Itô, 1990b) and, with modification, also to H. itoi

(see: Kolbasov et al., 2021a), both of which have

planktotrophic nauplii. The present paper includes

the first SEM study of the cephalic shield (and other

body parts) of a lecithotrophic species of y-nauplius,

at a level of detail surpassing the few available SEM

habitus views.

A major difference between H. demodex and

H. furcifera is that, while facets are found on the entire

cephalic shield (and even on the trunk dorsally) in all

stages of the latter, the entire dorsomedian part of the

shield is smooth in all known stages of H. demodex. This

makes a full comparison between the facet patterns in

these two species difficult, but from what can be seen,

it appears that the development of the facets in the two

species follows the same overall pattern. This includes

the presence of essentially only one row of central

longitudinal facets (Fig. 12, red overlay) and a few rows

of long rectangular facets laterally. In both H. demodex

and H. furcifera the facet pattern gradually gets

more complicated in later stages, typically by further

subdivision of already existing facets, the details of

which could not be followed in the new species due to

missing instars. However, one important difference is

evident: in H. furcifera and also H. itoi (see: Kolbasov

et al., 2021a) up through LSN, and in Itô’s (1991)

undescribed species at least up through LSN−1, facet

I-1 never divides. In contrast, in H. demodex facet

I-1, while initially joined with some of the F-facets,

becomes discrete and subdivided into four subfacets no

later than LSN − 3(?) (Fig. 13C; divided differently on

the left and right sides). This subdivision is tentatively

regarded as a diagnostic feature of the species herein.

Despite the role of the facets in defining Facetotecta,

and the occasional use of their arrangement to

distinguish various types of y-nauplii (e.g. Watanabe

et al., 2000), not enough comparative information is

available to apply such data to larger questions. It would

be fascinating to know whether a general pattern of

facets is to be found in all y-nauplii, besides the early-

instar (N-V or N-VI) pattern recognized by, for example,

Itô (1987b). If not, what sort of evolutionary divergence

among facet patterns has taken place? Are the facets

on the cephalic shield of certain ascothoracidan

nauplii, for example, Sessilogoga captiva Kolbasov &

Grygier, 2020 (Kolbasov et al., 2020), homologous to

those of Facetotecta, as assumed by Chan et al. (2021)?

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34 J. OLESEN ET AL.

It is conceivable that heterochrony may have played a

role in the evolution of the facet patterns of y-larvae.

Even within the single species H. demodex, the facet

pattern of the frontal part of the cephalic shield of the

Green Island LSN specimen (Fig. 6D) is distinctly more

symmetrical than that of an LSN specimen from Sesoko

Island (Fig. 5B), and in this respect is more similar to

earlier stages from Sesoko Island (Fig. 13C, F).

The pattern of cuticular surface structures (pores

and setae) has been fully mapped for the LSN of

H. demodex and partly for some selected earlier

stages. Because this is the first such study for any

lecithotrophic y-larva, these data provide a solid basis

for comparison with other described species and forms.

In total, the LSN has 63 cuticular pores and setae

(Figs 4, 5; Table 4), most of which are paired while five

pores on the midline are unpaired, including a frontal

pore on the cephalic shield (#1*), a pore between the

rudimentary furcal spines (#26*) and three pores

of differing morphology on the labrum (#29*, #30*,

#31*). The only species for which comparable data

are available are the planktotrophic H. furcifera (see:

Itô, 1989) and H. itoi (see: Kolbasov et al., 2021a). The

pattern of two pairs of large setae (Fig. 4C, E) flanking

the poorly defined ‘window’ on the cephalic shield in

H. demodex is also present in H. furcifera. While not

explored in detail here, it seems possible to homologize

a number of the pore pairs on the cephalic shield of

H. demodex with those of H. furcifera, while the pattern

depicted for H. itoi seems more remote. Hansenocaris

furcifera appears to lack many pores that were

described for H. demodex, not least on the trunk. For

example, H. demodex has a characteristically arranged

row of four lateral pores on each side of its long trunk

(total of eight: Fig. 5A, #17, #19, #21 and #24) while

H. furcifera has just two pairs of pores (total of four)

in a comparable position on its short trunk. While it is

not possible to say whether or how these sets of pores

actually correspond, serial duplication of such pores as

an autapomorphy of H. demodex related to the extreme

elongation of its trunk region might be an explanation.

Among the ventral cuticular surface structures,

the anterior ones are generally easier to homologize

between H. demodex, H. furcifera and H. itoi, while

this is more difficult for the posterior structures.

For example, equivalents of the frontal filaments in

H. demodex (Fig. 4D, #34) also appear to be present

in H. furcifera and H. itoi. The two lateral pores of the

labrum (Fig. 5K, M, #32) are also present in all three

species. The homologues of the three characteristic

central pores of the labrum in H. demodex (#29*, #30*,

#31*) are more uncertain. Itô (1989) drew only one

central pore in H. furcifera, but its position relative

to the lateral pores suggests that it is homologous

to either pore #30* or #31* in H. demodex, while the

equivalent to pore #29* (vestigial mouth opening?)

in H. demodex may be hidden under the labral

extension in H. furcifera, as is clearly shown in H. itoi.

A comparison with our unpublished SEM photos of a

suite of y-nauplii from Sesoko Island, representing a

diversity of different species (both with planktotrophic

and lecithotrophic nauplii), showed that the presence of

two central pores (#30 and #31) in H. demodex may be

unique to this species. The posteroventral pore pattern

of the trunk in H. demodex is difficult to homologize

precisely with that of H. furcifera and H. itoi. Itô

(1990b) drew three pore pairs in this region in later

stages of H. furcifera, and the same number is seen

in our earliest stage of H. demodex (Fig. 12A, B, #18,

#20, #22), which may indicate homology, but twice this

number of ventral pores is seen in the LSN (Fig. 4B).

In H. demodex, the medial pore (#26*), possibly the

vestigial anus, between the rudimentary furcal spines

is not represented by any comparable structure in

H. furcifera or H. itoi.

Pore patterns in crustaceans often provide

information that is either species-specific or important

at higher taxonomic levels (Mauchline, 1988; Olesen,

1996; Ozawa, 2013; Karanovic & Kim, 2014), but in

H. demodex, the number of pores and their arrangement

change as naupliar development progresses. Therefore,

it may be misleading to compare non-equivalent stages

across taxa. With this in mind, in Facetotecta it will be

most useful to choose the last-stage nauplius (LSN)

for comparison, as this stage is certainly homologous

between species (see above). Supplementary data from

earlier stages should be added whenever possible (as

in this study) to check the degree of stability of pore

patterns during development.

The naupliar appendages in H. demodex,

including second antennae and mandibles that lack

feeding armature on the coxa and basis and have

an unsegmented endopod, are simple in form and,

consequently, unlikely to display any unique features.

However, although the proximal segment of the second

antennal exopod is either lacking or rudimentary

in the LSN (Fig. 5K), it is clearly present in the

earliest examined stage (Fig. 12G). Practically the

same morphology is observed in other lecithotrophic

y-nauplii, such as types VII and VIII (Itô, 1986a,

1987a), and in a variety of other lecithotrophic

y-nauplii from Sesoko and Green Islands studied with

SEM (unpublished). In contrast, the coxa and basis

of the naupliar second antennae and mandibles of

H. furcifera and H. itoi bear median spines, presumably

used in feeding, and both limbs have a two-segmented

endopod (Itô, 1990b; Kolbasov et al., 2021a). The

antennal and mandibular morphology of nauplii

of H. demodex and other lecithotrophs is probably

derived (assuming the loss of planktotrophy), but

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TAXONOMY OF CRUSTACEAN Y-LARVAE 35

this needs to be evaluated in a broader phylogenetic

context.

naupliar develOpment Of Hansenocaris

demodex cOmpared tO Other y-larvae

The number of naupliar instars is important for

inferring higher level relationships in crustaceans.

One currently popular hypothesis is that Thecostraca

is a sister-taxon of Copepoda, together forming a

clade that is sometimes referred to as Hexanauplia

(Oakley et al., 2013; Schwentner et al., 2017; Lozano-

Fernandez et al., 2019). As the name suggests, this

hypothesis assumes that the presence of six naupliar

instars in the life cycle is a synapomorphy for uniting

the barnacles and copepods. However, this scenario

is based on the condition usually seen in Cirripedia

and exceptionally recorded in Ascothoracida (Itô &

Grygier, 1990), but not in Thecostraca as a whole.

For some time, only five naupliar instars had been

documented for facetotectans, including two species

with planktotrophic nauplii (H. furcifera and H. itoi;

see: Itô, 1990b; Kolbasov & Høeg, 2003) and one

unidentified species with lecithotrophic nauplii (Itô,

1991). However, recently, H. itoi has been shown to have

more, supposedly seven, naupliar instars. Whether five

or seven, such numbers could challenge ‘six naupliar

instars’ as a synapomorphy defining Hexanauplia

(Kolbasov et al., 2021a). Based on a detailed analysis

of the naupliar sequences in Copepoda and Cirripedia,

Haug & Haug (2015) have already suggested that the

‘six nauplii’ in the two taxa do not correspond. In light of

the great diversity of y-larvae and their supposed basal

position in the phylogeny of Thecostraca (Pérez-Losada

et al., 2009), the naupliar development of more species

requires study.

Hansenocaris demodex is the first lecithotrophic

species of y-larvae for which a suite of naupliar stages

has been examined in detail. As in Itô’s (1990b) study,

early larvae were sampled in the plankton and reared

in the lab. Y-larvae cannot be kept in reproducing

cultures as no y-adults are known, so it is uncertain

whether the most frequently caught, early-looking

y-nauplii represent the true nauplius 1 (the hatching

stage) or a later stage. Itô (1990b) discussed this

point as it applies to H. furcifera, but nonetheless

referred to the stage with the simplest ‘turtle-shell’

ornamentation of the cephalic shield as nauplius stage

1 because no earlier stage could be demonstrated. No

final conclusion can be reached concerning the total

number of naupliar stages in the development of

H. demodex, again due to the limited material, but

our data suggest at least five or six naupliar instars.

Largely because our SEM material does not include

a stage with the same (or even a truncated) clearly

defined pattern of ridges corresponding to that of Itô’s

stage 1, the earliest specimen examined by us (Fig. 12)

is inferred to be earlier than that. It possibly represents

an LSN − 5 or even an LSN − 6, assuming the seven-

instar sequence inferred by Kolbasov et al. (2021a)

for H. itoi is correct and common to other forms of

y-larvae. It might be the ephemeral ‘true instar 1’ first

hypothesized by Itô (1990b), with the ‘standard’ plate

pattern not yet established, but the fully developed

limb armature with apparently functional natatory

setae tends to argue against this.

Fouling might have been reduced, and survivorship

higher, if the rearing dishes had been constantly

agitated. The same may be true if the protocol

recommended by Itô (1990a) had been fully adopted.

Based on his experience of rearing about 20 putative

species of lecithotrophic y-nauplii to the cyprid stage,

Table 5. Overview of distribution of 120 cuticular surface structures (pores, sensilla, lattice organs, etc.) on different

body parts of cyprid y of Hansenocaris demodex sp. nov., all numbered as indicated in Figures 7–9 and based primarily

on the holotype (NHMD-916629) from Sesoko Island (Japan: Okinawa). Most structures paired, but asterisks (*) indicate

unpaired structures in midline.

Large pore with

slit-like opening

(3–4 μm)

Pore with seta Circular pore

(0.5–1 μm diam.)

Group of

micro-pores

(each 0.5 μm)

Lattice

organs

Total

Cephalic shield

(carapace)

1*, 2, 3, 17, 18, 23, 26,

28, 29(?), 37

4, 6, 8, 10, 12, 16,

21, 25, 30, 35, 36,

42–44

11, 14*, 19, 20*, 22,

27(?), 31–34, 39*

5, 7, 9, 24 13, 15, 38,

40, 41

Thoracomeres Absent Absent Absent Absent Absent 0

Abdominal

segments 1–3

45, 46 47 6

Telson 48–54, 56, 57*, 58, 60 55 59 25

Cephalic region,

ventral

62*, 63*, 64 61* 5

Total 44 34 24 8 10 120

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36 J. OLESEN ET AL.

he reported that survivorship of up to 90% or more

(compared to about 5% in the present study) can be

achieved if nauplii are transferred twice daily to fresh

filtered seawater in sterilized dishes. This protocol

can be used in future efforts to obtain complete moult

series based on fairly small numbers of individuals

of selected target species, but the intensive labour

involved made it impractical while maintaining

several hundred specimens simultaneously, as was

typical in the present study.

cyprid mOrphOlOgy Of Hansenocaris demodex

cOmpared tO Other y-cypridS

Hansenocaris demodex is the first formally named

species of y-larva with lecithotrophic nauplii for

which nauplii and the cyprid have been described

simultaneously. It is also only the third form of y-cyprid

to have been extensively described using SEM, after

H. papillata Kolbasov & Grygier in Kolbasov et al.,

2007 and H. spiridonovi Kolbasov et al., 2021b (see:

Kolbasov et al., 2007, 2021b). Among y-larvae in general

(Table 1), matching nauplii and cyprids has been done

with confidence only for H. furcifera and H. itoi, both

of which have planktotrophic nauplii. Itô (1986a)

identified an early nauplius as H. pacifica and noted

certain similarities, but more differences, between it

and nauplius y type IV from European seas. Itô (1987b)

then recanted this identification and designated the

nauplius in question as ‘type XI’, while noting that its

cyprid, although resembling that of H. pacifica, was

smaller (see also: Itô & Takenaka, 1988). Kolbasov

et al. (2007) summarized morphological information

and provided a key to the seven nominal species

of Hansenocaris that have described cyprids. They

suggested informal groupings of those with a long

cephalic shield and those with a short shield, supported

by other characters. However, they admitted that these

groupings are of hardly any taxonomic value, while a

cladistic analysis employing a larger set of characters

would be premature due the limited number of species.

The cyprid of H. demodex is in some respects different

from all formerly described y-cyprids. All of these,

except for that of H. acutifrons (see Itô, 1985), have a

large, protruding process on the labrum that usually

bears about five hooks (typically one apical and two

subapical pairs posteriorly), which may be indicative

of parasitism (attachment to host). In H. demodex

this process is particularly extended and carries

multiple rows of hooked spines that little resemble the

arrangement in other species unless each cluster of

five distal and three more proximal hooks corresponds

to a single usual hook. The pair of small appendices

in the midline anterior to the first antennae (Fig.

7E), interpreted here as the frontal filaments, have

as far as is known not been documented in any other

y-cyprid. The cephalic shield, as well as the entire body,

especially the telson, of H. demodex is significantly

more elongated than those of most other species, except

for the even more elongated cephalic shield of H. itoi

(Kolbasov & Høeg, 2003; Kolbasov et al., 2021a). Among

the described species, the cephalic shield of H. demodex

most resembles that of H. furcifera in overall form

(see: Itô, 1985), but the similarity is too general to be

an indication of close relationship. The paraocular

processes are small in the new species, but similar in

size to those of, for example, H. itoi. Clear vestiges of the

naupliar second antennae and mandibles are present in

the examined cyprids (Fig. 7B–D) with an indication of

biramosity, but such structures have also been reported

in H. furcifera (Itô, 1989) and H. itoi (Kolbasov & Høeg,

2003), as well as in certain ascothoracidan ‘cyprids’ (e.g.

Grygier, 1988, 1991b).

The thoracopodal protopod of H. demodex follows the

general pattern for y-cyprids in being two-segmented

(coxa and basis) in thoracopods 1–5. Based on our

data, the unsegmented protopod of thoracopod 6 in all

known y-cyprids originated by fusion of the coxa and

basis (Fig. 10D). Uniquely among described y-cyprids,

the exopods of all thoracopods in H. demodex are

also unsegmented, or, if a rudimentary proximal

segment is present, it is so tiny that it is concealed

in the articulation zone between the basis and exopod

(Figs 9A, B, 10D). In other y-cyprids the thoracopodal

exopods are all two- or three-segmented. Endopodal

segmentation of y-cyprids broadly defines two groups

and may be of phylogenetic significance: either

two-segmented in all limbs, with a small proximal

and a long distal segment, or three-segmented in

thoracopods 2–6 but two-segmented in thoracopod

1. Besides H. demodex, the first pattern, which must

be a derived state arising from the fusion of the two

distal segments in the second pattern, is displayed

by H. acutifrons Itô, 1985, H. pacifica Itô, 1985,

H. rostrata Itô, 1985, H. spiridonovi and, except for

an unsegmented thoracopod 1, also by H. tentaculata

Itô, 1986. In these species, segmental fusion in these

endopods is indicated not only by a comparison of

segment length, but also by the presence of an inner

seta midway along the distal segment (Figs 9B, 10D,

white arrows). In accordance with Schram’s (1970a)

interpretation, this armature element is positionally

homologous to the seta in the remaining three species,

H. furcifera, H. itoi and H. papillata, that is located

between the discrete and fully articulated second and

third endopodal segments. Our data are limited, but

a tentative pattern has emerged. The cyprids of at

least some facetotectan species with planktotrophic

nauplii (H. furcifera and H. itoi) have three-segmented

thoracopodal exopods (thoracopods 2–6), while those of

species with lecithotrophic nauplii (H. demodex) have

only two segments. If this distinction applies generally,

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TAXONOMY OF CRUSTACEAN Y-LARVAE 37

then the currently unknown nauplii of H. acutifrons,

H. rostrata, H. spiridonovi and H. tentaculata ought

to be lecithotrophic, since their cyprids have two-

segmented thoracopodal exopods.

Among the other distinctive features of H. demodex

are the rounded, free pleural extensions of thoracic

segment V (Fig. 9A), which are pointed in cyprids of

most other species and quadrate in H. spiridonovi, and

the unusually small, short segments of the abdomen

(Figs 7I, 9A, 10A, E). The telson appears relatively

longer than in other species and lacks the serrate

spines along the posteroventral margin displayed

by all of them, except for H. tentaculata. Finally, the

furcal rami in H. demodex are shorter (perhaps even

disc-like) than in any other species.

All cuticular surface structures (pores, setae,

lattice organs, etc.) of the cyprid of H. demodex have

been mapped using SEM (Table 5), the second time

this has been attempted for any y-cyprid and the first

with complete labelling, thus being able to serve as a

baseline for future studies of y-cyprids. In total, 120

surface structures have been traced and are numbered

in Figures 7–10. The pore patterns of y-cyprids may

prove useful for the taxonomy of y-larvae at the

species level, as demonstrated for the LSN mentioned

above, but the full extent of this is as yet unclear.

For example, 56 pores and seta-bearing pits, mostly

arranged in pairs, are present on the cephalic shield

of H. demodex, excluding lattice organs and micropore

fields. This is somewhat fewer than the 74 or more

such structures that are reportedly present on the

cephalic shield of H. spiridonovi (Kolbasov et al.,

2021b); unfortunately, the latter structures were

not fully mapped, precluding a detailed comparison.

Due to the large quantity of pores and the lack of

established landmarks on the cephalic shield, it is

also practically impossible to homologize pores of the

cephalic shield with any degree of certainty between

the present cyprids of H. demodex and those of

H. furcifera or H. itoi, two of the best-known species

described by light microscopy.

For taxonomy, it may be more useful to compare

‘nose-prints’ based on SEM photos of the anterior face

of the shield (e.g. Fig. 8B) or to search for characteristic

pore patterns of smaller body parts other than the

shield. An example of the latter may be the telson,

which in H. spiridonovi has an unpaired posterior

dorsal pore, two anterior dorsolateral pairs and two

posterior ventrolateral pairs (Kolbasov et al., 2021b:

fig. 5). In contrast, on the same surfaces of the telson,

H. demodex has an unpaired anterior dorsal pore, two

dorsal pairs along the dorsal-dorsolateral boundary

ridge, three pairs in the upper row of lateral plates and

four pairs in the lower row of lateral plates (Figs 9C,

E, 10A, E). In particular, on each side of the telson the

double-dyad anterior arrangement of four dorso- and

ventrolateral pores (#48–#51) appears unique to the

new species (Fig. 9A)

mOlecular diverSity, taxOnOmy, phylOgeny and

the future Of y-larva SyStematicS

DNA barcoding and integrative taxonomic approaches

have been applied in crustacean studies and larval

systematics for over a decade (e.g. Palero et al., 2009,

2014; Tang et al., 2010; Raupach & Radulovici, 2015;

Jakiel et al., 2020), but there have been practically no

prior attempts to address the diversity or systematics

of y-larvae from a molecular perspective. Pérez-Losada

et al. (2009) sequenced three nuclear genes (Histone-3,

18S and 28S rDNA) of six unnamed taxa from Sesoko

Island and of Hansenocaris itoi from the White Sea, but

this was part of an effort to demonstrate the monophyly

of Facetotecta and its position in the Thecostraca, not

alpha-taxonomy. In fact, except for H. itoi, the precise

taxonomic identity of the specimens they used remains

unknown. Sampling of molecular data for y-larvae

has been significantly increased by the present study,

adding nucleotide sequences (partial 18S rDNA) for 22

y-larval specimens, mainly from Sesoko Island. These

data grouped the specimens into four clades, thereby

supporting the monophyly of Hansenocaris demodex,

Itô’s (1986a) ‘Pacific type I’ and two undescribed types/

species of y-larvae nicknamed by us as ‘Big brown’

and ‘Bumblebee’ (Figs 14, 15). 18S rDNA thus appears

to provide a useful supplement to morphological

characterization, although it must be emphasized that

Facetotecta-specific primers targeting mitochondrial

markers are greatly needed. In several invertebrate

taxa, hypervariable regions of the 18S rDNA gene have

been used to distinguish between genera and species

(Wu et al., 2015), even though this gene is traditionally

used to infer higher level phylogenies (Pérez-Losada

et al. 2009; Wilson, 2009; Kjer, 2004).

Some of the unnamed specimens sequenced by

Pérez-Losada et al. (2009) appear to belong to the

same type as some of our specimens photographed

in life. Their ‘Facetotecta sp. 4’ (FJ751880) has the

same nucleotide sequence as the above-mentioned

‘Big brown’ and probably represents the same

species. Three of their taxa, ‘Facetotecta sp. 1, 2 and

6’ (FJ751877, FJ751878, FJ751882), were almost

identical (> 99.8%), thus representing the same

species, and are conspecific with four newly sequenced

planktotrophic y-nauplii that were identified in this

study as Itô’s (1986a) ‘Pacific type I’. However, two

of Pérez-Losada et al.’s (2009) specimens, ‘Facetotecta

sp. 3’ (FJ751879) and ‘Facetotecta sp. 5’ (FJ751881),

did not match any of the newly sequenced specimens,

which highlights the need to associate molecular data

with voucher specimens or photos when sequences

are deposited in GenBank.

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38 J. OLESEN ET AL.

Table 6. Overview of naupliar characteristics of four types of y-larvae from Sesoko Island (Japan) and Green Island (Taiwan) sequenced for molecular analysis

as a part of this paper (see Figs 14, 15)

Name and number of

sequenced specimens

Sample numbers

and collection data

Size‡ General morphology§ Feeding

strategy**

Naupliar

developmental time††

in dish until cyprid

Previous reports

Hansenocaris demodex

sp. nov.

9 specimens

Sesoko Isl.*

JA-2018-108-111-

JA-2019-321-322-107-136

Green Isl. †

TA-2018-066-101-166

See above

LSN:

Length 352–

390 μm

Width c. 130 μm

LSN: Elongate, tapering

posteriorly; orange/

brownish appearance of

cyprid inside LSN; blunt

dorso-caudal spine; furcal

spines reduced and placed

ventrally.

L3–6 days. Cyprid

known, described

herein.

Unreported

‘Big brown’ (nick-

name due to size

and colour)

2 specimens

Sesoko Isl. *

JA-2018-154: 25-Oct-2018

JA-2019-181: 11-Jun-2019

LSN:

Length 320 μm

Width c. 155 μm

LSN: Elongate, tapering

posteriorly; brownish

appearance of cyprid in-

side LSN; long, pointed

dorso-caudal spine; furcal

spines distinct

L6–8 days. Cyprid

known,

undescribed.

Grygier et al. (2019)

‘Bumblebee’ (nickname

after bumblebee-like

brown/orange color-

ation of nauplius)

7 specimens

Sesoko Isl. *

JA-2018-076-077: 18-Oct-

2018

JA-2019-177: 9-Jun-2019

JA-2019-192-207: 11-Jun-

2019

JA-2019-290-293: 14-Jun-

2019

LSN:

Length c. 250 μm

Width c. 135 μm

LSN: Relatively short; cyprid

inside LSN distinctly

coloured, cephalon red-

dish/yellowish, abdomen

brown; caudal spine short

and blunt; furcal spines

short and triangular

L6–9 days.

Cyprid known,

undescribed

Unreported

Itô’s (1986) ‘Pacific

type I’

4 specimens

Sesoko Isl. *

JA-01-B3: 17-Jun-2019

JA-2019-031: 17-Jun-2019

JA-2019-102-104: 15-Jun-

2019

Length 160–

190 μm

Width 110–135 μm

(more instars may

be involved)

Nauplius: truncate

egg-shaped; transparent

with orange-coloured gut;

posteriolateral margins

of trunk region lined with

three prominent spines,

the middle being the most

robust; DC organ present;

short, upturned DC spine;

furcal spines distinct

P No moulting

observed.

Cyprid unknown

Similar to Itô’s

(1986) ‘Pacific

type I’

*Collected and processed by DEJ, MJG, YF, ND, JO.

†Collected and processed by ND, DEJ, JO.

‡Length without posterior spines. Width at broadest point.

§Based on last-stage nauplius if known, otherwise on earlier nauplii. See Figure 14. LSN, last-stage nauplius; DC, dorso-caudal.

**L, lecithotrophic; P, planktotrophic nauplii (based on absence/presence of feeding spines and labral extension, see more criteria in Results).

††Naupliar developmental time after sampling until moulting to cyprid. Hansenocaris demodex sp. nov. based on four specimens, ‘Big brown’ based on five specimens, ‘Bumblebee’ based on 20

specimens. The true developmental time from hatching (which has never been observed for y-larvae) until appearance of the cyprid may be longer.

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TAXONOMY OF CRUSTACEAN Y-LARVAE 39

The new phylogeny of Facetotecta presented here

(Fig. 15) is the most comprehensive to date, but it is

still provisional as it is based on only partial 18S rDNA

data and includes less than 10% of the form variation

sampled at Okinawa during this work. Nevertheless,

the strong congruence with general larval morphology

justifies a brief discussion of the phylogenetic

implications. In the presented phylogeny, Facetotecta

is split basally into two clades. Clade A is represented

only by Hansenocaris itoi and two unnamed taxa from

GenBank. Clade B is represented by H. demodex,

two types of y-larvae with lecithotrophic nauplii

(‘Bumblebee’ and ‘Big brown’) and one type with

planktotrophic nauplii (‘Pacific type I’). These four

types appear to be closely related, but the morphological

disparity among the members of Clade B is significant.

The nauplii of H. demodex, ‘Bumblebee’ and ‘Big brown’

are all elongate, cylindrical, posteriorly tapered larvae

with no clear demarcation between the cephalic shield

and the rest of the body, in contrast to many other

lecithotrophic y-nauplii that have a rounded ‘belly’,

a distinct and often upturned dorsocaudal spine, and

a clearer delineation between the cephalic region

and the trunk as seen in dorsal view (e.g. Itô, 1991;

Belmonte, 2005; Høeg et al., 2014). More particularly,

owing to the reduction of the dorsocaudal and furcal

spines, the nauplii of H. demodex and ‘Bumblebee’ both

display a blunt terminal end of the trunk (Fig. 14A, B).

Confirmation of a close relationship among the

lecithotrophic species in clade B will require detailed

studies of both nauplii and cyprids, which are pending.

Among the four types of y-larvae in clade B, the

one we have identified as Itô’s (1986a) Pacific type

I deviates the most. The morphological differences

between the lecithotrophic nauplii of H. demodex, ‘Big

brown’ and ‘Bumblebee’, and the nauplii of their tiny

planktotrophic relative, Pacific Type I (Fig. 14; Table

4), are remarkable compared to, for example, barnacle

larvae. In the Cirripedia, nauplii of closely related

species mostly resemble each other, irrespective of

the different habitats inhabited by adult barnacles

(Chan et al., 2014). Planktotrophy in marine larvae is

often, but not universally, considered plesiomorphic,

and lecithotrophy derived (Rouse, 2000; Nielsen, 2007;

Collin & Moran, 2018). In y-larvae, plesiomorphic

planktotrophy of nauplii is congruent with the

phylogeny in Figure 15, as the planktotrophic Pacific

type I is the sister-group to the three lecithotrophic

types and another planktotroph, H. itoi, appears in

clade A. The evolutionary polarity of planktotrophic

vs. lecithotrophic feeding in y-nauplii, as well as the

apparently huge morphological diversity of y-larva (c.f.

Figs 2, 14, 15; Grygier et al., 2019), clearly need to be

assessed in a sequence-based multilocus phylogeny

based on broader (more taxa) and more robust (more

genes) data.

CONCLUSIONS

• Y-larvae occur locally in large quantities and with

considerable diversity; y-larvae (both nauplii

and cyprids) caught inshore at, e.g. Sesoko Island

(Japan), are practically unidentifiable, mostly

not corresponding either to previously described

nominal species or to currently recognized ‘types’.

• The current taxonomic approach involves parallel

systems of nomenclature, with formally described

nominal species being based on incomparable life-

history stages and many naupliar types being

included in a Roman-numeral-based parataxonomy;

it fails to reflect the true diversity of y-larvae.

• An integrated taxonomical approach is presented

that combines rearing through several moult

stages, live photography, detailed microscopy of

selected specimens and molecular techniques

(DNA barcoding), in order to establish a reliable

standard for future species descriptions (at least

for lecithotrophs). Future species descriptions

of y-larvae with lecithotrophic nauplii should be

based on a combination of last-stage nauplii and

cyprids.

• A more complete assessment of y-larval diversity

in any given region is needed in order to: (1)

provide identification keys; (2) match y-nauplii

and y-cyprids of the same species; and (3) not least,

assign via molecular data already available species

names for larvae to the corresponding y-adults once

the nature of these (parasitic?) becomes known.

• During fieldwork at Green Island (Taiwan) and

Sesoko Island (Japan) in 2017–19, about 11 000

y-larvae were sampled and handled, more than 25

times the number reported in any previous study.

Extensive sampling and sorting were fundamental

to the development of the novel methodology

presented in this paper.

• To demonstrate the proposed methodology,

a morphologically unique form of

y-larva, Hansenocaris demodex, is described based

on material from Sesoko Island (Japan) and Green

Island (Taiwan). Its life cycle displays a naupliar

phase with elongate, yellow/orange-coloured stages

and a cyprid bearing an unprecedentedly large

number of hooks in a particular pattern on its

labrum.

• Specimens of H. demodex from both localities

exhibit only minor differences, for example, in the

size of the cyprid’s telson (relatively shorter at

Green Island).

• Hansenocaris demodex is the first formally

described y-larva with lecithotrophic nauplii for

which both nauplii and the cyprid are known. All

cuticular surface structures (pores and setae) for

both the last-stage nauplius (LSN) (63 structures

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40 J. OLESEN ET AL.

over the entire body) and the cyprid (120 structures)

are fully mapped. This is done for the first time for

y-larvae to provide a baseline for exploring the

importance of pore/setae patterns in classifying

species of this group.

• Hansenocaris demodex is the first formally described

lecithotrophic y-larva for which more than one stage

in the naupliar development has been studied. The

naupliar phase consists of at least five to six instars,

approaching the seven instars currently inferred

for the best-studied planktotrophic species, H. itoi.

• The largest molecular diversity dataset for

Facetotecta compiled so far is presented here, with

22 individual y-larvae sequenced anew, representing

four different types (including H. demodex).

• Our preliminary phylogenetic tree, based on partial

18S rDNA sequences, shows significant congruence

with larval morphology, supporting the utility of

hypervariable regions of this marker as a barcoding

tool for y-larvae.

• Based on 18S rDNA sequences of specimens

identified from photographs, four out of six unnamed

‘species’ uploaded to GenBank in a previous work

(Pérez-Losada et al., 2009) could be identified as

belonging to either Itô’s (1986a) ‘Pacific type I’ or to

a form nicknamed ‘Big brown’.

ACKNOWLEDGEMENTS

Fieldwork at the Sesoko Station in 2018 and 2019

could not have been carried out without the helpful

support of Yoshikatsu Nakano and other members

of the staff at the station, who kindly provided

excellent lab facilities. We are also grateful for the

support from Ming-Jay Ho at the Marine Science

Thematic Center, Academia Sinica at Green Island,

Taiwan. Special thanks go to Vanessa Pei-Chen Tsai

for tireless support of all aspects of the work at both

Green Island and Academia Sinica in Taipei. This

work has been generously supported by a VILLUM

Experiment grant (project no. 17467) to JO from the

Velux Foundations. ND was jointly supported by a

double-degree graduate grant from the Biodiversity

Research Center, Academia Sinica, and the Natural

History Museum of Denmark. MJG’s work was

enabled by support to the Center of Excellence for

the Oceans at National Taiwan Ocean University

from the Featured Areas Research Center Program

of the Taiwan Ministry of Education’s Higher

Education Sprout Project and by a grant from

Taiwan’s Ministry of Science and Technology (MOST

108-2611-M-019-002-). FP acknowledges the projects

‘CIDEGENT/2019/028 – BIOdiversity PAtterns

of Crustacea from Karstic Systems (BIOPACKS):

molecular, morphological, and functional adaptations’

funded by the Conselleria d’Innovació, Universitats,

Ciència i Societat Digital and ‘PRO2020-S02-PALERO

– Fauna aquàtica en coves anquihalines del País

Valencià: un mon encara per descriure’ funded by the

Institut d’Estudis Catalans. Joachim Haug (Munich)

and Rony Huys (London) are thanked for valuable

comments on a late version of the manuscript. JO and

ND dedicate this paper to mentor, former advisor and

friend Jens Thorvald Høeg for his great contributions

to barnacle larval studies and for providing invaluable

inspiration and support at the onset of the study.

AUTHOR CONTRIBUTIONS

JO, ND and MJG conceived the project and designed

the experimental approach. JO, ND, FP, DEJ, YF and

MJG performed experiments and carried out fieldwork.

JO took photographs/videos and prepared figures. ND

carried out molecular lab work and sequence analyses.

FP designed oligonucleotide primers and supervised

molecular work. BKKC contributed reagents,

molecular lab facilities, and supervised molecular

work. JO secured funding for fieldwork. BKKC and JO

secured funding for molecular work. JO wrote the first

draft. ND, FP, DEJ, BKKC and MJG edited the first

and subsequent drafts. All authors read and approved

the final version of the manuscript.

CONFLICT OF INTEREST

All authors declare that they have no conflicts of

interest.

DATA AVAILABILITY

Most morphological data supporting the taxa

described here are available directly as figures in

the paper. A short video showing live larvae from

the paper can be seen here: https://youtu.be/seo-

63AK10E. More images/videos are available from

the corresponding author, JO, upon reasonable

request. Sequences used to estimate phylogenies

are deposited in GenBank under accession numbers

OM135272-OM135293. The code, alignment

and IQ-TREE log files are stored at https://doi.

org/10.6084/m9.figshare.17803628.v1.

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Abundance survey of crustacean "y-larvae" (Thecostraca: Facetotecta) over a fringing reef in Okinawa, Japan, with special reference to the two dominant planktotrophic and lecithotrophic naupliar types

Article

Full-text available

Jan 2025

Facetotecta, or "y-larvae", are mysterious planktonic crustaceans that are known only from their larval instars, but which are often assumed to be endoparasitic as adults. Dozens of mostly undescribed forms occur in the shallow-water plankton over a fringing reef at Sesoko Island, Okinawa, Japan. Recently, it has become possible to discriminate the different forms of their nauplius-stage larvae ("y-nauplii") in a replicable way. A large year-to-year overlap in morphospecies recovered during fieldwork at Sesoko Island in 2018, 2019, and 2023 suggests that a full inventory is close at hand. To date, 49 morphospecies of y-nauplii (8 planktotrophic, 41 lecithotrophic) have been recognized in the area, among which three have been formally described. A detailed analysis of the temporal fluctuations in abundance during October 2023 showed that most morphospecies were rare, but two were particularly common: Type A*, a planktotroph with a long (> 3 weeks) period of naupliar development allowing for long-distance dispersal, and Type AG*, a lecithotroph with a short (3 days) period of development that implies rapid local recolonization. During the survey, both types showed distinct, largely non-overlapping peaks in abundance, related perhaps to their different dispersal/feeding strategies. An examination of the morphology of the swimming/feeding appendages in y-nauplii of Types A* and AG*, together with a mapping of feeding versus non-feeding nauplii on a recent comprehensive phylogeny of Facetotecta, suggests that broader taxonomic coverage of naupliar feeding structures in this group may provide useful information regarding the evolutionary direction of planktotrophy versus lecithotrophy in marine larvae.

Two New Species of Lecithotrophic Nauplius y with Remarkable Labra from Okinawa, Japan, and a Family-Group Name for y-Larvae (Crustacea: Thecostraca: Facetotecta: Hansenocarididae fam. nov.)

Article

Full-text available

Oct 2022

Two large (ca. 0.5 mm long), rare, and probably closely related species of Facetotecta (y-larvae), Hansenocaris crista-labri sp. nov. and Hansenocaris aquila sp. nov., are described on the basis of last-stage lecithotrophic nauplii reared from plankton at Sesoko Island, Okinawa, Japan. The two species resemble each other in having a labrum with a row of spines and a long, attenuate trunk region that terminates in a long, heavily spinose dorsocaudal spine. The labrum of H. cristalabri sp. nov. has an enormous, cockscomb-like ventral process that bears a row of distally directed, dagger-like spines along its anterior side, while the spine-bearing keel of the labrum of H. aquila sp. nov. extends posteriorly into a robust, eagle-like beak. The labral "crest" of H. cristalabri sp. nov. has no equivalent in any other described y-larva, nor in any other crustacean nauplius; its possible functions are discussed. Another diagnostic feature of H. cristalabri sp. nov., absent in H. aquila sp. nov., is a pair of shallow, rounded notches bounded by sharp spinules on the far posteriolateral margins of the cephalic shield. Both new species have longitudinal spine rows on the trunk dorsum, two rows in H. cristalabri sp. nov. and four in H. aquila sp. nov., something not previously documented for y-nauplii. The plate arrangement of the cephalic shield in H. cristalabri sp. nov. is described in detail, with an attempt to homologize the pattern with that of other y-nauplii (especially Hansenocaris furcifera Itô, 1989). The body surface of H. cristalabri sp. nov. has fewer setae and pores than any other late-or last-stage facetotectan nauplius described to date, suggesting paedomorphic development. A formal diagnosis is presented for the family-group taxon Hansenocarididae fam. nov.; this name, while already in use, has until now been nomenclaturally unavailable.

Phylogenomics of Enigmatic Crustacean Y-Larvae Reveals Multiple Origins of Parasitism in Barnacles

Preprint

Jan 2025

Taxonomic diversity of marine planktonic ‘y-larvae’ (Crustacea: Facetotecta) from a coral reef hotspot locality (Japan, Okinawa), with a key to y-nauplii

Article

Full-text available

Mar 2024

The enigmatic ‘y-larvae’ (Thecostraca: Facetotecta) are microscopic marine planktonic crustaceans that were discovered more than a century ago, yet to this day their adults remain unknown. Despite occurring locally in large diversities, and therefore presumably being of ecological importance, only 17 species have been described globally, rendering it practically impossible to identify any y-larval specimen from any locality. The fact that species have been based on different life stages (nauplii and/or cyprids) further hampers identification. Y-larvae include many forms with planktotrophic (feeding) nauplii and even more with lecithotrophic (non-feeding) nauplii. At one coral-reef locality on the shore of Sesoko Island (Okinawa, Japan), extensive fieldwork in 2018 and 2019 confirmed an enormous taxonomic diversity of y-larvae there. Here, we present morphological diagnoses and an identification key for 34 lecithotrophic y-naupliar types (or morphospecies), which will correspond minimally to the same number of species when described. As a temporary measure, all are referred to by alphabetical parataxonomic designations, except for three that have been formally described already within the genus Hansenocaris Itô, 1985. To this should be added an additional 7–9 planktotrophic y-naupliar morphospecies, which are only treated briefly. Most often, y-larval taxonomy has been based on the cyprid stage, but the large morphological diversity of y-nauplii suggests that nauplii are at least as important for taxonomy. Lecithotrophic y-nauplii display a multitude of body shapes, the form-evolution of which is discussed here with reference to a recent molecular phylogeny of Facetotecta partly based on material from the same site. An indirect estimate of the relative abundances of all 34 lecithotrophic y-naupliar morphospecies is presented, based on laboratory-reared final-instar specimens. This treatment is intended as a step towards a proper taxonomy and a revised classification of Facetotecta, which will involve detailed descriptions of both nauplii and cyprids. Until such work progresses, the present overview of the y-naupliar fauna of a single Okinawan locality known to be a hotspot of y-larval diversity is offered as a baseline for further surveys of Facetotecta elsewhere in the Indo-West Pacific and beyond.

Autofluorescence imaging of exuviae as a tool for studying slide preparations of micro-arthropods, exemplified by a museum collection of the enigmatic crustacean “y-larvae” (Pancrustacea: Facetotecta)

Article

Full-text available

Feb 2024
ZOOMORPHOLOGY

In recent years, fluorescence microscopy has revitalized the study of invertebrate comparative morphology. Here we explore the usefulness of combining confocal laser scanning microscopy (CLSM) and cuticular autofluorescence to examine the taxonomically challenging marine planktonic “y-larvae” (Pancrustacea: Facetotecta). To gauge the effectiveness of CLSM with autofluorescence in producing taxonomically useful images, we applied it to seven distinct y-naupliar species or morphospecies that had previously undergone scrutiny by other techniques. The specimens were part of a museum collection of glycerin-jelly slides of exuviae of last-stage y-nauplii, a key instar for studying the taxonomy of y-larvae. For Hansenocaris demodex, the level of detail obtained from a single specimen was comparable to that previously obtained by scanning electron microscopy (SEM). For Hansenocaris aquila, revisiting the original holotype specimen resulted in a dramatic increase in our understanding of the species’ morphology, including taxonomically pivotal information about its spinose labrum and a digitally rotated lateral view. CLSM analyses of the other five specimens, which represented a broad spectrum of y-naupliar morphology, efficiently generated more such information. Novel observations were made concerning putative external rudiments of both the first and second maxillae in late nauplii as well as the extreme dorso-ventral flattening of some naupliar types. CLSM observation of museum slides of naupliar exuviae using cuticular autofluorescence thus shows great promise of becoming an excellent tool for studying the morphology and taxonomy of y-larvae, and we suggest that this technique might also profitably be applied to other forms of larval exuviae.

”Y-larvae” – a 100-year-old mystery in marine biology [Translated from Danish]

Article

Full-text available

Dec 2023

Jørgen Olesen

Are there still unsolved mysteries in marine biology? The answer is "yes"! 100 years of searching has still not provided the answer to what the adult version of the mysterious, microscopic (<0.5 mm) " y-larvae" looks like. The larvae, i.e. the early stages of development, on the other hand, have been found free- swimming everywhere in the world's oceans, also in Denmark, more precisely in Øresund, but usually only a few individuals at a time. Recently, it has been shown that y-larvae are very common in coastal areas in Japan, e.g. around Okinawa, where there are at least 40 different species. Only a few are described and named. Three seasons of fieldwork on Okinawa in 2018, 2019 and 2023 have resulted in the collection of over 15,000 individuals now stored at the Natural History Museum of Denmark. The work to describe all the species has now started, but the mystery remains, as the adult "y-larvae" are still unknown, and their full life cycle therefore not yet understood. We suppose that the adult stage of "y-larvae" is endoparasitic, perhaps in corals, and that it is probably quite small, which could explain why it has not yet been found. Although the full life cycle of y-larvae is not known, it is important to describe their great species richness in the plankton of the world's oceans

Novel molecular resources for single-specimen barcoding of enigmatic crustacean y-larvae

Article

Full-text available

Mar 2024

Despite discovery more than 100 years ago and documented global occurrence from shallow waters to the deep sea, the life cycle of the enigmatic crustacean y-larvae isincompletely understood and adult forms remain unknown. To date, only 2 of the 17 formally described species, all based on larval stages, have been investigated using an integrative taxonomic approach. This approach provided descriptions of the morphology of the naupliar and cyprid stages, and made use of exuvial voucher material and DNA barcodes. To improve our knowledge about the evolutionary history and ecological importance of y-larvae, we developed a novel protocol that maximises the amount of morpho-ecological and molecular data that can be harvested from single larval specimens. This includes single-specimen DNA barcoding and daily imaging of y-nauplii reared in culture dishes, mounting of the last naupliar exuviae on a slide as a reference voucher, live imaging of the y-cyprid instar that follows, and fixation, DNA extraction, amplification and sequencing of the y-cyprid specimen. Through development and testing of a suite of new primers for both nuclear and mitochondrial protein-coding and ribosomal genes, we showcase how new sequence data can be used to estimate the phylogeny of Facetotecta. We expect that our novel procedure will help to unravel the complex systematics of y-larvae and show how these fascinating larval forms have evolved. Moreover, we posit that our protocols should work on larval specimens from a diverse array of moulting marine invertebrate taxa.

The Biology and Life Cycle of Enigmatic Crustacean Y-Larvae: A Review

Chapter

Full-text available

Aug 2023

Resolution of recalcitrant nodes in the Tree of Life has been substantially eased recently by increased worldwide sampling and advancements in sequencing technology. It has become routine to use molecular data to characterise and taxonomically allocate tens to hundreds of taxa based on the DNA occurring in a few drops of water. Despite this, the adult stages of one invertebrate taxon, the enigmatic crustacean ‘y-larvae’ (Thecostraca: Facetotecta), have never been found, and the true diversity of this group has long remained unknown. Here, we review the current state of our knowledge concerning these mysterious larval forms and provide a significant body of new morphological and ecological data to make y-larvae accessible to a wider community of biologists. After summarising the history of y-larva studies, we review the current state of facetotectan systematics and outline in detail the structural fea- tures of each known phase in the life history, from y-nauplius through y-cypris to ypsigon. In particular, we document a suite of new ultrastructural details of the ypsigon, the putatively invasive phase if, as suspected, the adults are parasitic. Scanning and transmission electron microscopy are used to show that after a moult the ypsigon inherits numerous structures directly from the preceding y-cypris larva, including parts of the nervous system, sensory organs, pores, and antennal musculature. A comparison of the ypsigon to the very similar, yet surely independently evolved and thus merely analogous invasive stages of parasitic barnacles (Rhizocephala) provides a broader phylogenetic and functional context for these findings. These structural traits agree with molecular phylogenetic data placing Facetotecta as an early- branching sister clade to the cirripede barnacles, including Rhizocephala. We then review the ecology and biogeography of y-larvae in the global plankton and offer a comprehensive map and list of recorded localities. Finally, we statistically test the abundance of the different life-history stages of y-larvae in the surface plankton at one of our primary study sites (Sesoko Island, Okinawa, Japan) in relation to various environmental factors that may drive their occurrence there. We found evidence of crepuscular emer- gence around dawn (7 am–9 am) and dusk (5 pm–7 pm). This review is the most comprehensive synthesis of information on Facetotecta to date. Despite our continued ignorance of the adults, we hope it will serve both as a starting point for future scientists embarking on studies of this challenging group of crustaceans and as an inspiration for those working on other kinds of planktonic larvae.

Single-specimen systematics resolves the phylogeny and diversity conundrum of enigmatic crustacean y-larvae

Article

Apr 2023
MOL PHYLOGENET EVOL

Resolving the evolutionary history of organisms is a major goal in biology. Yet for some taxa the diversity, phylogeny, and even adult stages remain unknown. The enigmatic crustacean "y-larvae" (Facetotecta) is one particularly striking example. Here we use extensive video-imaging and single-specimen molecular sequencing of >200 y-larval specimens to comprehensively explore for the first time their evolutionary history and diversity. This integrative approach revealed five major clades of Facetotecta, four of which encompass a considerable larval diversity. Whereas morphological analyses recognized 35 y-naupliar "morphospecies", molecular species delimitation analyses suggested the existence of between 88 and 127 species. The phenotypic and genetic diversity between the morphospecies suggests that a more elaborate classification than the current one-genus approach is needed. Morphology and molecular data were highly congruent at shallower phylogenetic levels, but no morphological synapomorphies could be unambiguously identified for major clades, which mostly comprise both planktotrophic and lecithotrophic y-nauplii. We argue that lecithotrophy arose several times independently whereas planktotrophic y-nauplii, which are structurally more similar across clades, most likely display the ancestral feeding mode of Facetotecta. We document a remarkably complex and highly diverse phylogenetic backbone for a taxon of marine crustaceans, the full life cycle of which remains a mystery.

Novel molecular resources for single-larva barcoding of enigmatic crustacean y-larvae

Preprint

Full-text available

Dec 2022

The enigmatic “y-larvae” (Pancrustacea: Facetotecta) still have an incompletely understood lifecycle, and their adult forms remain unknown despite their discovery more than 100 years ago and their documented global occurrence from shallow waters to the deep-sea. Only two of the 17 formally described species, all based on larval stages, have been investigated using an integrative taxonomic approach that, besides providing descriptions of the morphology of the naupliar and cyprid stages, also made use of exuvial voucher material and DNA barcodes. To improve our knowledge about the systematics and phylogenetics of y-larvae, we developed a novel protocol that maximizes the amount of morphological, ecological, and molecular data that can be harvested from single individuals of these tiny larvae. This revolves around single larva barcoding, and includes daily imaging of y-nauplii reared in culture dishes, mounting of their last naupliar exuviae on a slide as a reference voucher, live imaging of the y-cyprid instar that follows, and fixation, DNA extraction, amplification, and sequencing of the y-cyprid specimen. By developing and testing a suite of new primers for both nuclear and mitochondrial protein-coding and ribosomal genes, we estimated the most comprehensive phylogeny of Facetotecta to date. We expect that our novel procedure will help to unravel the complex systematics of y-larvae and show how these fascinating larval forms have evolved. Moreover, we posit that our protocols should work on larval specimens of a diverse array of molting marine invertebrate taxa.

The evolutionary diversity of barnacles, with an updated classification of fossil and living forms

Article

Full-text available

Feb 2021

We present a comprehensive revision and synthesis of the higher-level classification of the barnacles (Crustacea: Thecostraca) to the genus level and including both extant and fossils forms. We provide estimates of the number of species in each group. Our classification scheme has been updated based on insights from recent phylogenetic studies and attempts to adjust the higher-level classifications to represent evolutionary lineages better, while documenting the evolutionary diversity of the barnacles. Except where specifically noted, recognized taxa down to family are argued to be monophyletic from molecular analysis and/or morphological data. Our resulting classification divides the Thecostraca into the subclasses Facetotecta, Ascothoracida and Cirripedia. The whole class now contains 14 orders, 65 families and 367 genera. We estimate that barnacles consist of 2116 species. The taxonomy is accompanied by a discussion of major morphological events in barnacle evolution and justifications for the various rearrangements we propose.

Naupliar development of Facetotecta (Crustacea: Thecostraca) and the nature of the first nauplius instar in the Crustacea - pro et contra the Hexanauplia concept

Article

Full-text available

Feb 2021

From a number of partial moult series obtained by laboratory culture of plankton-caught specimens, we described all naupliar instars of the facetotectan species, Hansenocaris itoi. It reveals seven naupliar instars, instead of the five that were previously supposed for the Facetotecta. This number of naupliar instars is unique not only for Facetotecta, but also for Thecostraca and Hexanauplia as well. We studied the external morphology of each naupliar instar in detail with light microscopy and SEM. Nauplius 1 is entirely non-feeding and differs from subsequent instars in having a smooth, unsculptured cuticle and undeveloped armament of the labrum and the limbs. The subsequent naupliar development is characterized by an increase in size and in the number of cuticular plates and by the appearance of different armament of the limbs. We compare nauplius 1 across all Thecostraca and discuss its nature in relation to the larval development of Crustacea. We also discuss the presence of seven naupliar instars in Facetotecta and the concept of Hexanauplia.

A new species of Sessilogoga Grygier, 1990 parasitic in an antipatharian from Green Island, Taiwan, with notes on its nauplius larvae and the synapomorphies and apparent gonochorism of the genus (Crustacea: Thecostraca: Ascothoracida)

Article

Full-text available

Jun 2020

More than 40 specimens of a new endoparasitic ascothoracidan species, described herein as Sessilogoga captiva Kolbasov & Grygier sp. nov. in the family Synagogidae Gruvel, 1905, were found at 35 m depth near Green Island off southeastern Taiwan infecting a colony of the antipatharian Antipathes sp. aff. A. atlantica Gray, 1857. Its sole known congener is S. elongata Grygier, 1990a, an endoparasite of an unidentified antipatharian from Guam. We photographed some specimens in life and used scanning electron microscopy (SEM) and light microscopy to extensively document the fine-scale external morphology of both sexes, including carapace ornamentation (with special attention to lattice organs), the frontal filament complex, the antennular armature, and details of the mouthparts. We also examined the first two naupliar larval stages by SEM. Among the three most plesiomorphic genera of ascothoracidans, the two species of Sessilogoga Grygier, 1990a exhibit several synapomorphies, probably connected with their endoparasitic way of life, that distinguish them from the vagile, possibly micro-predatory species of Synagoga Norman, 1888 and the ectoparasitic, possibly vagile species of Waginella Grygier, 1983a: (i) a reduced frontal filament complex, (ii) absence of epaulets on the sixth thoracomere, (iii) reduction of the setation of the proximal antennular segments, and (iv) more seminal receptacles in any given pair of thoracopods. The new species appears to be gonochoric with females generally larger than males; our find of a juvenile female smaller than any adult male tends to refute the likelihood of protandry.

Revision of the West African species of Scyllarus Fabricius, 1775 (Decapoda: Achelata: Scyllaridae), with the description of three phyllosoma stages of S. caparti Holthuis, 1952 and an updated identification key

Article

Full-text available

May 2020
J CRUSTACEAN BIOL

West African species of Scyllarus Fabricius, 1775 (Achelata, Scyllaridae) are poorly known, mostly due to the difficulties of sampling Eastern Atlantic tropical waters. Recent expeditions carried out by the Universidad de Cádiz and the Instituto Español de Oceanografía collected phyllosoma larvae from Cape Verde Islands (CVI) and fresh Scyllarus adults from continental West Africa. Larval stages VII, IX, and X (final stage) of S. caparti Holthuis, 1952 are analyzed using DNA barcoding methods and described for the first time. A comprehensive identification key is provided, summarizing our current knowledge on the phyllosomas of Scyllarus. Together with a revision of museum collections, the new molecular and morphological data obtained here supports the polyphyletic origin of Acantharctus Holthuis, 2002. The West African A. posteli (Forest, 1963) is found to belong to Scyllarus and it is closest to another species from Atlantic shallow waters (i.e. S. paradoxus Miers 1881), whereas the Pacific Ocean A. delfini (Bouvier, 1909) would belong to Crenarctus Holthuis, 2002.

Diversity of Parasitic Peltogastrid Barnacles (Crustacea: Cirripedia: Rhizocephala) on Hermit Crabs in Korea

Article

Full-text available

Nov 2019

We performed a diversity study on parasitic barnacles (Crustacea: Cirripedia: Rhizocephala: Peltogastridae) that parasitize hermit crabs in Korea. Their morphological, ecological, molecular (cytochrome c oxidase subunit I, 16S rRNA), and biogeographical characteristics were examined. Three species were identified based on GenBank sequences and the external morphology of the externa. In addition, this study proposes four new candidate species. This is the first report on the family Peltogastridae from Korea. Six hermit crab species were found to be new hosts to peltogastrids. Korean peltogastrids are less prevalent on their host hermit crabs than those from Japan are, especially in the west coast of Korea. Peltogasterella gracilis is widely distributed throughout Korea, Peltogaster lineata is located on the east coast, and Peltogaster postica is only located on Jeju Island.

Identification of Y-Nauplii (Facetotecta) in Andaman Sea, India

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Full-text available

Jan 2019

The Upper Cambrian Rehbachiella and the phylogeny of Branchiopoda and Crustacea

Chapter

Jul 1993

Dieter Waloszek

More than 130 specimens representing various growth stages of Rehbachiella kinnekullensis Müller, 1983, have permitted a detailed description of its ontogeny. It begins with a nauplius already able to swim and feed actively. The 30th stage is about 1.7 mm long, but still immature. Because the type specimens belong to earlier instars, the original diagnosis of Muller (1983) is emended. Details of the limb apparatus of late instars suggest that the animals were able to filter-feed by this stage, possibly while swimming close to the bottom. Two larval series are distinguished by size and morphology in their early stages, but their structural differences become almost balanced subsequently. This is interpreted as intraspecific differentiation rather than as existence of two species. The entire postnaupliar feeding apparatus of Branchiopoda, which is basically adapted to filtration, is recongnized here as an apomorphic character of this group. Branchiopoda comprise the two monophyletic units Anostraca and Phyllopoda (Calmanostraca, with Notostraca and Kazacharthra, and Onychura). Rehbachiella shares all major aspects of the branchiopod apparatus, which led to identify it as an ancestral marine branchiopod. Moreover, there are indications that Rehbachiella is a representative of the anostracan lineage, i.e. a representative of the stem-group of Sarsostraca, which include the Devonian Lipostraca and the extant Euanostraca. The long larval sequence of Rehbachiella and selective external features, including the locomotory and feediang apparatus, are evaluated for their bearing upon the phylogeny of Branchiopoda and Curstacea in general. This study on Rehbachiella supports the monophyly of the crown-group Crustacea (sensu Walossek & Muller 1990). It also has revealed that only the first maxilla was morphologically and functionally included into the crustacean head, while subsequent limbs were addted to the head in a stepwise manner and became modified separately within the different crustacean lineages, which is of great relevance when evaluating the relationships between these.

A new species of the Y-larva genus Hansenocaris Itô, 1985 (Crustacea: Thecostraca: Facetotecta) from the Azores, with notes on its morphology and biogeography

Article

Sep 2021

A new facetotectan species, described herein as Hansenocaris spiridonovi Kolbasov, Savchenko et Høeg sp.n. and based on its cypridiform stage, was found in the plankton off Azores Islands. We employed scanning electron microscopy to document the fine-scale external morphology of this species. We discuss the findings of facetotectan larvae showing cosmopolitan distribution of these crustaceans. The presence of Y-cyprid exuviae in plankton, findings of y-larvae in the deep sea plankton, occurrence in the oceanic plankton, as well as their permanent presence in the tropical waters may evidence on a pelagic host.

Secrets from the deep: Pseudotanaidae (Crustacea: Tanaidacea) diversity from the Kuril–Kamchatka Trench

Article

Apr 2020
PROG OCEANOGR

The Biomass Composition of the Oceans: A Blueprint of Our Blue Planet

Article

Dec 2019

Obtaining a quantitative global picture of life in the great expanses of the oceans is a challenging task. By integrating data from across existing literature, we provide a comprehensive view of the distribution of marine biomass between taxonomic groups, modes of life, and habitats.

Integrative taxonomy of crustacean y-larvae (Thecostraca: Facetotecta) using laboratory-rearing and molecular analyses of single specimens, with the description of a new vermiform species

Abstract

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