The taxonomic status of the headshield slug genus Nakamigawaia Kuroda and Habe, 1961 (Gastropoda: Cephalaspidea: Aglajidae), with the description of a new species from the Western Pacific

Abstract

Nakamigawaia is a poorly understood genus of Aglajidae sea slugs with only two species formally ascribed. In this paper we explore new morpho-anatomical characters using stereo and scanning electron microscopy and employ different molecular approaches (a cytochrome c oxidase sub-unit I gene phylogeny, the Automatic Barcode Gap Discovery species delimitation method, and genetic distances) to compare specimens across the geographical span of the genus and from two distinct chromatic morphotypes occurring in the Western Pacific (blackish morph and white-dotted morph). Our results support the conspecificity of these two morphs and show they belong to an undescribed species here named Nakamigawaia nakanoae sp. nov. The species differs from the type species of the genus, N. spiralis, by the presence of a distinct open-dilated shell and differs from its Western Atlantic congener N. felis by subtle differences in the shell, male reproductive system and caudal lobes. Genetically (COI uncorrected p-distance) the two species (N. nakanoae and N. felis) are 18.8–20.1% distinct. The definition of the genus Nakamigawaia is discussed and the current assignment to the latter of lineages other than the type species is questioned.

Full text

The taxonomic status of the headshield slug genus
Nakamigawaia Kuroda and Habe, 1961 (Gastropoda:
Cephalaspidea: Aglajidae), with the description of a new
species from the Western Pacic
Emma Hellem
a
and Manuel António E. Malaquias
b
a
Department of Biological Sciences, Faculty of Mathematics and Natural Sciences, University of Bergen,
Bergen, Norway;
b
Department of Natural History, University Museum, University of Bergen, Bergen, Norway
ABSTRACT
Nakamigawaia is a poorly understood genus of Aglajidae sea slugs
with only two species formally ascribed. In this paper we explore
new morpho-anatomical characters using stereo and scanning elec-
tron microscopy and employ dierent molecular approaches (a
cytochrome c oxidase sub-unit I gene phylogeny, the Automatic
Barcode Gap Discovery species delimitation method, and genetic
distances) to compare specimens across the geographical span of
the genus and from two distinct chromatic morphotypes occurring
in the Western Pacic (blackish morph and white-dotted morph).
Our results support the conspecicity of these two morphs and
show they belong to an undescribed species here named
Nakamigawaia nakanoae sp. nov. The species diers from the
type species of the genus, N. spiralis, by the presence of a distinct
open-dilated shell and diers from its Western Atlantic congener
N. felis by subtle dierences in the shell, male reproductive system
and caudal lobes. Genetically (COI uncorrected p-distance) the two
species (N. nakanoae and N. felis) are 18.8–20.1% distinct. The
denition of the genus Nakamigawaia is discussed and the current
assignment to the latter of lineages other than the type species is
questioned.
ARTICLE HISTORY
Received 23 June 2021
Accepted 22 September 2021
KEYWORDS
biodiversity; DNA barcoding;
Mollusca; morphology;
phylogeny; Western Pacific
Introduction
The family Aglajidae is the second most diverse of the gastropod Heterobranchia order
Cephalaspidea, with an estimated 85 valid species worldwide divided into 15 genera
(Zamora-Silva and Malaquias 2018). These marine slugs inhabit tropical and temperate
waters predominantly at shallow depths where they are found crawling on sandy bot-
toms, coral rubble and algae (Burn and Thompson 1998; Gosliner et al. 2008; Camacho-
García et al. 2014; Zamora-Silva and Malaquias 2018), yet at least three species have been
reported from deeper bathymetries including abyssal depths (Zamora-Silva and
Malaquias 2018; Chaban et al. 2019). Aglajids are predators feeding mostly upon vagile
prey such as other sea slugs, nematodes, polychaetes and small sh (Zamora-Silva and
Malaquias 2016).
CONTACT Manuel António E. Malaquias Manuel.Malaquias@uib.no
JOURNAL OF NATURAL HISTORY
2021, VOL. 55, NOS. 35–36, 2231–2244
https://doi.org/10.1080/00222933.2021.1986165
© 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License
(http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any med-
ium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way.
Published online 11 Feb 2022

One of the genera of Aglajidae is the enigmatic Nakamigawaia Kuroda and Habe, 1961.
The genus was introduced for the species N. spiralis Kuroda and Habe, 1961 from Japan
(type locality: Kasajima beach, Sagami Bay) and originally included in the family
Chelidonuridae Kuroda and Habe 1961 (presently regarded as a synonym of Aglajidae;
MolluscaBase 2021). Kuroda and Habe (1961) provided only a brief description, in
Japanese, of the colouration of the animal and an illustration of the shell. The denition
of the genus and species remained largely elusive, and, for example, Rudman (1978) and
Gosliner (1980) did not consider this genus in their dedicated accounts on the systematics
of the aglajid slugs.
Baba (1985) was the rst author to provide a detailed description of the type species
N. spiralis, recognising at the same time similarities to representatives of the Aglajidae
genus Melanochlamys, which led the author to rmly suggest the inclusion of the genus
Nakamigawaia in the family. The validity of Nakamigawaia and its inclusion in the
Aglajidae have been corroborated by Camacho-García et al. (2014) and Zamora-Silva
and Malaquias (2018) based on molecular phylogenetics. In addition, Camacho-García
et al. (2014) rst suggested a close relationship between N. spiralis (based on specimens
from Papua New Guinea and the Philippines) and the morphologically similar tropical
western Atlantic species ‘Aglaja’ felis described by Marcus and Marcus (1970) from Puerto
Rico, which led the authors to reassign the latter species to the genus Nakamigawaia.
Despite the putative phylogenetic evidence for a common generic aliation of the
Atlantic and Pacic species, the occurrence of substantial dierences between their shells
(convex, dilated with an open whorl in ‘A’. felis, and spiralled in N. spiralis), led Ortea et al.
(2014) to argue these species warranted a dierent generic assignment, and introduced the
genus name Migaya for the Atlantic lineage. However, Zamora-Silva and Malaquias (2018),
based on extended genetic and taxon sampling, supported the validity of Nakamigawaia
for both Atlantic N. felis and Pacic N. spiralis and argued that dierent shell types could be
present in the same genus, and considered Migaya a junior synonym of Nakamigawaia.
Specimens from the western Atlantic have consistently been ascribed to the species
‘felis’, but interestingly those from the Pacic have been named erratically by authors and
attributed either to the genus Chelidonura or to Nakamigawaia, resulting in pervasive
taxonomic confusion and a still unsettled nomenclature, with the name ‘spiralis’, in fact,
being hardly used. For example, Susuki (2000, p. 16) and Ono (1999, p. 14, 15) identied
specimens from the Izu Peninsula and Kerama Islands (Japan) as Chelidonura sp.; and
Gosliner et al. (2008, p. 39) labelled specimens from the Philippines and Papua New
Guinea as Nakamigawaia felis, whereas the same authors later used the name
Nakamigawaia spiralis (Gosliner et al. 2015, p. 45) and Nakamigawaia sp. (Gosliner et al.
2018, p. 367). In 2018, Nakano (2018, p. 55; 2019, p. 55) identied specimens from Japan as
Nakamigawaia felis.
In addition, two distinct chromatic morphs occur in the western Pacic, namely one
that is consistent with the original description and having a uniform blackish pattern
(Kuroda and Habe 1961; Baba 1985), and the other characterised by a white-dotted
pattern on a brownish background (Ono 1999; Gosliner et al. 2008, 2015, 2018; Nakano
2018, 2019). In this work we use morpho-anatomical characters and a molecular phylo-
genetic framework to test the taxonomic aliation of these two morphs and of specimens
from the western Atlantic in order to shed light on the diversity and systematics of the
genus Nakamigawaia.
2232 E. HELLEM AND M. A. E. MALAQUIAS

Material and methods
Taxon sampling and literature review
Samples were obtained from the collections of the Department of Natural History,
University Museum, University of Bergen, Norway (ZMBN). Specimens of Nakamigawaia
from Taiwan (two black morph specimens and two white-dotted morph specimens),
Japan (black morph), Venezuela and the Bahamas were used for morphological studies,
for a total of 11 specimens (six of those sequenced for their DNA; Table 1). In addition, we
obtained from GenBank 16 sequences of the cytochrome c oxidase subunit I gene (COI),
representing the genera Chelidonura, Nakamigawaia, Philinopsis and Tubulophilinopsis.
The tree was rooted with the species Philine quadripartita (Table 1).
Table 1. List of specimens used for molecular analysis, including locality, voucher numbers and
GenBank accession numbers. *Listed as Aglaja felis in GeneBank; ** listed as Nakamigawaia spiralis
in GenBank. Novel sequences generated for this study are depicted in bold font.
Taxa
Sample
ID
Voucher
number Locality
COI gene GenBank
Accession number
*Nakamigawaia felis ZMBN84913a Venezuela Isla: Tortuga MF036535
*Nakamigawaia felis CASIZ175655 The Bahamas: Great Exuma, Stocking
Island
JN825179
*Nakamigawaia felis CASIZ175653 The Bahamas: Great Exuma, Stocking
Island
JN825178
Nakamigawaia felis EH05 ZMBN84913.1 Venezuela: Isla Tortuga OK094481
Nakamigawaia felis EH06 ZMBN84913.2 Venezuela Isla Tortuga OK094482
*Nakamigawaia felis CPIC-00684 The Bahamas: Great Exuma, Stocking
Island
JN825180
*Nakamigawaia felis CPIC-00685 The Bahamas: Great Exuma, Stocking
Island
JN825181
**Nakamigawaia
nakanoae sp. nov.
ZMBN95949 Australia: Lizard Island MF036536
*Nakamigawaia sp. CASIZ173495 The Philippines: Bohol Island, Panglao JN825177
Nakamigawaia
nakanoae sp. nov.
EH01 ZMBN116778.1 Taiwan: Shadao, Kenting National
Park, Pingtung County
OK094484
**Nakamigawaia
nakanoae sp. nov.
ZMBN95960 Australia: Lizard Island MF036537
Nakamigawaia
nakanoae sp. nov.
EH03 ZMBN116777.1 Taiwan: Shadao, Kenting National
Park, Pingtung Count
OK094486
Nakamigawaia
nakanoae sp. nov.
EH02 ZMBN116778.2 Taiwan: Shadao, Kenting National
Park, Pingtung County
OK094485
Nakamigawaia
nakanoae sp. nov.
EH04 ZMBN116777.2 Taiwan: Shadao, Kenting National
Park, Pingtung County
OK094483
Philinopsis speciosa ZMBN95993 Japan: Kyoda Beach, Okinawa MF036556
Philinopsis pusa ZMBN95958 The Bahamas: Great Exuma, Stocking
Island
MF036552
Chelidonura hirundinina ZMBN95971 Australia: Lizard Island MF036524
Chelidonura varians ZMBN95978 Australia: Lizard Island MF036530
Chelidonura alisonae ZMBN95991 Hawaii: Anini Beach, Kauai MF036523
Tubulophilinopsis
pilsbryi
ZMBN95948 Australia: Lizard Island MF036569
Tubulophilinopsis
lineolata
ZMBN95941 Australia: Lizard Island MF036565
Philine quadripartita - ?Mediterranean Sea KU557520
JOURNAL OF NATURAL HISTORY 2233

DNA extraction, amplication and sequencing
DNA was extracted from tissue clipped from the parapodial lobes of the animals following
the protocol of the Qiagen DNeasy Blood and Tissue Kit. Sequences of the gene COI were
amplied and sequenced using the universal primers by Folmer et al. (1994). Polymerase
chain reactions (PCRs) were performed in 25 µL Eppendorf tubes containing 1 μL of DNA,
2.5 μL Qiagen Buer, 2.5 μL dNTPs, 5 μL Qiagen Q-Solution, 2 μL forward and reverse
primers (1 μL for each primer direction), 0.15 μL of Qiagen HotStar TAQ DNA polymerase,
8.35 μL of Sigma water, and 3.5 μL of magnesium chloride. The PCR thermal cycle included
an initial denaturation at 95°C for 5 min, followed by 39 cycles of 45 s at 94°C, 45 s at 45°C
(annealing temperature), extension at 72°C for 2 min, and a nal extension at 72°C for
10 min.
PCRs that did not yield results were redone with the restriction enzyme Takara and the
primers GasF1_t1 (TGTAAAACGACGGCCAGTTTTCAACAAACCATAARGATATTGG) and
GasR1_t1 (CAGGAAACAGCTATGACACTTCWGGRTGHCCRAARAATCARAA) (Stein et al.
2013). In those cases the PCR was started with an initial denaturation step of 5 min at
95°C, followed by 5 cycles of 40 s at 94°C, 40 s at 45°C and 1 min at 72°C, then 35 cycles of
40 s at 94°C, 40 s at 51°C and 1 min at 72°C. This was followed by a nal extension step of
5 min at 72°C.
Gel electrophoresis was performed on a 1% agarose/buer to assess the quality and
quantity of the amplied DNA. The successful PCR products were puried with
Exonuclease I Shrimp Alkaline Phosphatase (ExoSAP). The total volume of each purica-
tion reaction was 10 μL, which consisted of 0.1 μL of Exo I (10 U/μL), 1 μL SAP (1 U/μL),
0.9 μL of Sigma water and 8 μL of PCR product. The mixtures were incubated at 37°C for
30 min, followed by 85°C for 15 min, and nally kept cool at 4°C.
Sanger sequencing reactions were prepared using primers diluted to 3.2 μM. Each
sequencing reaction contained 6.5 μL Sigma water, 1 μL buer, 1 μL BigDye, 1 μL primer
and 0.5 μL PCR product (or 1 μL PCR product for samples with a low amount of DNA, with
no changes made to the amount of Sigma water). Prior to sequencing the mixtures were
run in a thermal cycle including an initial step at 96°C for 5 min, followed by 25 cycles at
96°C for 10 sec, 5 sec at an annealing temperature of 50°C, and 60°C for 4 min, and nally
kept cool at 4°C. After the thermal cycle 10 μL Sigma water was added to each sample.
Samples were sequenced with an ABI 3730XL DNA Analyser (Applied Biosystems) at the
DNA Sequencing Facility, Department of Biological Sciences, University of Bergen,
Norway.
Phylogenetic and species delimitation analyses
Both forward and reverse DNA chromatograms were edited and assembled using
Geneious R11 (Biomatters, Auckland, New Zealand; Kearse et al. 2012). All sequences
were blasted using GenBank to test for contamination. Sequences were aligned with the
programme Muscle (Edgar 2004) implemented in Geneious using default parameters.
The alignment was trimmed at both ends where at least 50% of the individual
sequences had nucleotides and was translated into amino acids to test for the presence
of stop codons. The best-t model of evolution was estimated with JModelTest 2.1.10
(Darriba et al. 2012).
2234 E. HELLEM AND M. A. E. MALAQUIAS

Bayesian molecular phylogenetic inference was done in the program MrBayes 3.2.7
(Ronquist and Huelsenbeck 2003) through the platform CIPRES Science Gateway V.3.3
(Phylo.org) with three parallel runs of ve million generations with sampling every
100 generations. Node support was assessed using Bayesian posterior probabilities
(Felsenstein 1985). Convergence of runs was assessed using Tracer 1.7.1 (Rambaut
and Drummond 2007) with the burn-in set to 25%. The phylogenetic tree was
visualised in FigTree 1.4.4 (Rambaut and Drummond 2009) and edited in Adobe
Illustrator.
Uncorrected p-distances between and within species and colour morphs were
estimated with the programme MEGA X (Kumar et al. 2018). The Automatic
Barcode Gap Discovery method (ABGD; Puillandre et al. 2012), implementing the
Simple Distance algorithm and default settings, was used to aid in dening candidate
species.
Anatomical work and scanning electron microscopy
A total of 11 specimens (three representing the black morph and two the dotted-white
morph of N. ‘spiralis’, and six representing N. felis) were dissected for structures of taxonomic
relevance, namely the male reproductive system and the shell. Drawings of soft anatomical
structures were made under a stereomicroscope with the aid of a camera lucida. Shells were
cleansed with diluted commercial bleach, rinsed in distilled water and mounted on metallic
stubs covered with carbon sticky tabs, coated with gold–palladium and later studied and
imaged with a Fei Quanta 450 scanning electron microscope at the Electron Microscopy
Laboratory, Faculty of Mathematics and Natural Sciences, University of Bergen, Norway.
Results
Phylogenetic analyses
The monophyly of the genus Nakamigawia received marginal support in the COI phylo-
genetic analysis. Three sub-clades were rendered within Nakamigawaia: a clade with
Caribbean specimens compatible with the species N. felis, a clade with western Pacic
specimens (Taiwan and Lizard Island, Australia) with a mix of black and dotted-white
animals that we provisionally assigned to N. ‘spiralis’ (= N. nakanoae sp. nov.; name
adopted hereafter; see Taxonomic section), and a single specimen from the Philippines
(Nakamigawaia sp.; based on GenBank sequences) that split o alone and probably
corresponds to an unnamed species.
The COI genetic uncorrected p-distance between black and white-dotted morphs of
N. nakanoae sp. nov. was estimated at 0–0.4%, which – like the phylogenetic results –
suggests these two morphs are conspecic. The genetic distance within specimens of
N. felis was 0–0.7%, and within specimens of N. nakanoae sp. nov. it was 0–1.2%. The
genetic distance between N. felis and N. nakanoae sp. nov. was 18.8–20.1%. The ABGD
analysis rendered 11 candidate species including N. felis and N. nakanoae sp. nov. (see
Supplementary material).
JOURNAL OF NATURAL HISTORY 2235

Taxonomic section
Genus NAKAMIGAWAIA Kuroda and Habe, 1961
Nakamigawaia felis (Er. Marcus and Ev. Marcus, 1970)
(Figure 1(b), 3(e), (f), 4(b))
Diagnosis
External colouration plain black. Internal shell dilated with an open whorl extending half
a turn, protoconch dorsally covered by teleoconch, a plate-like structure can project
ventrally near protoconch. Caudal lobes of nearly equal length. Male reproductive system
with pyriform penial chamber and tubular, long, folded prostate. Prostate about 3 times
longer than penial chamber.
Figure 1. Live images of Nakamigawaia nakanoae sp. nov. (A, C, D) and Nakamigawaia felis (B). A,
black morph, Taiwan, ZMBN 116778, animal length (L) = 10 mm (paratype). B, the Bahamas, ZMBN
91108, L = 13 mm. C–D, white dotted morph, Taiwan, ZMBN 116777, L = 8 mm (paratype).
2236 E. HELLEM AND M. A. E. MALAQUIAS

Examined material
Venezuela, Isla Tortuga, Playa El Yaque’s Lagoon, 2 specimens dissected and sequenced,
ZMBN 84913, animal length (L) = 13 mm, coll. Manuel Malaquias, 16 March 2010. The
Bahamas, Eleuthera Island, Savannah Sound, 4 specimens dissected, ZMBN 91108, L = 10–
13 mm, coll. Manuel Malaquias, 14 April 2013.
External morphology (Figure 1(b)): Plain black including foot. Caudal lobes about the
same length.
Shell (Figure 3(e), (f)): Convex, dilated, with an open whorl extending half a turn.
Protoconch smooth, partly visible ventrally; dorsally fully covered by extension of teleo-
conch; a plate-like structure can project ventrally near protoconch (fragile, breaks o easily).
Male reproductive system (Figure 4(b)): Penial chamber pyriform, rounded distally and
funnel-like proximally. Length between genital opening and insertion of prostate around
1 mm. Prostate tubular, cylindrical, folded; length around 3 mm.
Ecology
Found crawling on sand between 0.2 and 1 m depth.
Distribution
Caribbean Sea, between the Bahamas in the north and Venezuela in the south (Malaquias
2014; Caballer et al. 2015).
Figure 2. Bayesian phylogeny of Aglajidae species with a focus on the genus Nakamigawaia, based on
the mitochondrial Cytochrome c oxidase subunit I (COI) gene. Numbers on branches are posterior
probabilities. Tree rooted with Philine quadripartita.
BM = black morph. DM = white dotted morph.
JOURNAL OF NATURAL HISTORY 2237

Figure 3. Shells of Nakamigawaia species. A, N. spiralis, holotype, NSMT-39805, ventral (upper) and
dorsal (lower) views (macrophotograph), Japan. B, N. nakanoae sp. nov., micrograph of dorsal view;
arrow pointing to protoconch, ZMBN 132073, animal length (L) = 7 mm (fixed length), Japan. C,
N. nakanoae sp. nov., micrograph of ventral view, ZMBN 132073, L = 7 mm (fixed length), Japan. D,
N. nakanoae sp. nov., micrograph of protoconch observed from ventral side of shell, ZMBN 116778,
L = 10 mm, Taiwan. E, N. felis, micrograph of dorsal view, ZMBN 91108, L = 13 mm, the Bahamas. F,
N. felis, micrograph of ventral view, ZMBN 91108, L = 12 mm, the Bahamas. Scale bars: a = 200 μm; b,
c, e, f = 500 μm; d = 100 μm.
2238 E. HELLEM AND M. A. E. MALAQUIAS

Nakamigawaia nakanoae Hellem and Malaquias sp. nov.
(Figures 1(a), (c), (d), 3(b)–(d), 4(a))
Zoobank: lsid:zoobank.org:act:EB686586-9EFD-448A-B743-15C0FDC583DF
Diagnosis
External colouration plain black or brownish dotted in white. Internal shell dilated with an
open whorl extending half a turn, protoconch dorsally visible near transition to teleo-
conch, otherwise covered by teleoconch layer; ventrally a funnel-like structure can be
present near protoconch. Right caudal lobe nearly half length of left lobe. Male repro-
ductive system with pyriform penial chamber and tubular, long, folded prostate. Prostate
about 5 times longer than penial chamber.
Type locality
Taiwan, Pingtung County, Kenting National Park, Shadao (21.912233N, 120.846
961E).
Figure 4. Male reproductive system of (A) Nakamigawaia nakanoae sp. nov. and (B) N. felis. A: Taiwan,
ZMBN 116778, animal length (L) = 10 mm. B: the Bahamas, ZMBN 91108, L = 13 mm. ga, genital
aperture; pa, penial atrium; pr, prostate. Scale bar: 1 mm.
JOURNAL OF NATURAL HISTORY 2239

Etymology
The species is named after Dr Rie Nakano, for her passion and contributions to the study
of the sea slug molluscs of Japan and for her continuous support throughout the years of
our research work.
Examined material
Taiwan, Pingtung County, Kenting National Park, Shadao, 1 specimen, ZMBN 130245
(black morph), L = 10 mm, holotype, coll. Manuel Malaquias, 7 May 2017. Taiwan,
Pingtung County, Kenting National Park, Shadao, 2 specimens, ZMBN 116814 (black
morph), L = 10 mm, paratypes, coll. Manuel Malaquias, 7 May 2017. Taiwan, Pingtung
County, Kenting National Park, Shadao, 2 specimens dissected and sequenced plus 46
observed, ZMBN 116778 (black morph), L = 8–10 mm, paratypes, coll. Manuel Malaquias,
7 May 2017. Taiwan, Pingtung County, Kenting National Park, Shadao, 2 specimens
dissected and sequenced plus 5 observed, ZMBN 116777 (white dotted morph),
L = 8 mm, paratypes, coll. Manuel Malaquias, 7 May 2017. Japan, Kagoshima, Amami-
Ohshima Island, Ayamaru, 1 specimen dissected, ZMBN 132073 (black morph), L = 7 mm
(xed length), coll. Rie Nakano, 19 May 2015.
External morphology (Figure 1(a), (c), (d)): Plain black including foot, or brownish back-
ground dotted in white, with rim of head-shield and parapodial lobes white or dashed-
white; foot sole brownish, white-dotted. Caudal lobes asymmetrical, with right lobe nearly
half length of left lobe.
Shell (Figure 3(b)–(d)): Convex, dilated, with an open whorl extending half a turn.
Protoconch smooth, entirely or partly visible ventrally; dorsally visible only near transition
to teleoconch; a funnel-like structure (fragile, breaks o easily) can be present ventrally
near protoconch.
Male reproductive system (Figure 4(a)): Penial chamber pyriform, rounded distally and
funnel-like proximally. Length between genital opening and insertion of prostate around
1 mm. Prostate tubular, cylindrical, folded; length around 5 mm. One specimen from
Japan (ZMBN 132073) has a 7.5 mm prostate.
Ecology
Specimens were found at depths between 1 and 6 m crawling on white sandy bottoms.
Distribution
Kenting in Taiwan (current study), Kerama Islands, Amami-Ohshima Island and Izu
Peninsula in Japan (Ono 1999; Susuki 2000), the Philippines and Papua New Guinea
(Gosliner et al. 2008), and in Lizard Island, eastern Australia (current study).
Discussion
There is widespread confusion and inconsistency about the naming of slugs with black-
coloured headshields occurring in the western Pacic (Kuroda and Habe 1961; Baba 1985;
Ono 1999; Gosliner et al. 2008, 2015, 2018; Nakano 2018, 2019) and controversy regarding
the denition of the genus Nakamigawaia (Ortea et al. 2014; Zamora-Silva and Malaquias
2018). The original description in Japanese of the type species N. spiralis is brief and
includes a single illustration of the shell (Figure 3A). The description refers to animals with
2240 E. HELLEM AND M. A. E. MALAQUIAS

black body colour and a shell coiling in three turns, opening downwards. Specimens were
collected by Mr Korokuro Nakamigawa at ‘Zushi beach’ [= Kasajima], Kanagawa
Prefecture, in a tide pool (Kuroda and Habe 1961; Baba 1985).
The rst comprehensive study of N. spiralis was performed by Baba (1985). The author
provided detailed data on the morphology and anatomy and sound evidence of its
taxonomic anity with the family Aglajidae. Striking features of the description by Baba
(1985) are the comparatively large size of the animals (ranging between 25 and 35 mm)
and the long prostate (about 20 mm for a specimen 25 mm in length overall; measure-
ments based on g. 4C in Baba 1985).
In our preliminary identication of black headshield slugs collected in southern Taiwan
and southern Japan we assigned these specimens to N. spiralis. However, detailed
comparative studies of the shell and reproductive system revealed substantial dierences
between what was observed and what is known about these structures in N. spiralis. The
shells in our specimens were dilated, with an open whorl extending half a turn (Figure 3),
and there were dierences in the soft anatomy: prostates were shorter, ranging between
3.5 and 6.5 mm in specimens that measured about 10 mm in total length when alive.
However, the dierence in the size of the reproductive system might be explained by the
unique length of the animals. Interestingly, such slugs with large black headshields have
not been observed or reported during at least the last decade (Rie Nakano, personal
communication). Therefore, based on dierences in the shell shape and apparent dissim-
ilarities in parts of the male reproductive system, we assign our specimens to a new
species, named Nakamigawaia nakanoae sp. nov. The known distribution of N. spiralis
from Sagami Bay to the Kyusyu region (Kuroda and Habe 1961; Baba 1985) suggests
a species of temperate anity, whereas N. nakanoae sp. nov. is conned to tropical
waters.
This study conrms the validity of the species N. felis and its tropical western Atlantic
geography, and suggests the occurrence of an additional species of Nakamigawaia
present in the Philippines at least. No phylogenetic or genetic dierentiation was
detected between the white-dotted and black morphs of N. nakanoae sp. nov. (COI
uncorrected p-distance ranged between 0% and 0.4%), whereas the large genetic dier-
ence between the latter species and N. felis (18.8–20.1%) suggests an old speciation event
or fast evolutionary rates in these species (Figure 2).
Externally, N. felis and the black morph of N. nakanoae sp. nov. are nearly indistinguish-
able, but there is one subtle dierence: namely, the right and left caudal lobes in the latter
species are of dierent lengths, whereas in N. felis they are of similar size (Figure 1). Also,
the internal shells of these two species are similar, but in N. felis the dorsal part of the
protoconch is covered by a teleoconch layer, whereas in N. nakanoae sp. nov. part of the
protoconch is dorsally visible (Figure 3). The male reproductive systems are also similar in
morphology, yet the ratio between the length of the prostate and the length of the penial
chamber (from the genital aperture to the insertion point of the prostate) is about 5× in
N. nakanoae sp. nov. and only 3× in N. felis (Figure 4).
The systematics of Nakamigawaia and its recognised lineages (N. spiralis, N. felis,
N. nakanoae sp. nov., Nakamigawaia sp. from the Philippines) admittedly remains elusive
after this work. Our COI gene tree barely recovers support for the monophyly of the genus,
but this is not surprising considering that it is a single-locus phylogeny. On the other hand,
the works of Camacho-García et al. (2014) and Zamora-Silva and Malaquias (2018), based on
JOURNAL OF NATURAL HISTORY 2241

a broader character set, strongly support the monophyly of Nakamigawaia. However, if the
genus Nakamigawaia is to be dened by the presence of a spiralled internal shell and a long
and convoluted prostate, then perhaps N. felis, N. nakanoae sp. nov. and eventually
Nakamigawaia sp. will have to be assigned to a distinct genus. In this case, the junior
synonym name Migaya Ortea, Caballer and Espinosa, 2014 is available. Nevertheless,
answering this question warrants the study of new specimens of the true N. spiralis with
spiralled shells and its comparison in a phylogenetic framework with other aglajids includ-
ing the species currently assigned to Nakamigawaia. Until additional evidence is available,
we believe that the use of the generic name Nakamigawaia for these lineages better
promotes taxonomic stability.
Acknowledgements
We are very thankful to Rie Nakano in Japan and Kasunori Hasegawa at the National Museum
of Nature and Science, Tokyo, for sharing material with us, valuable discussions about type
material and type localities, and helping translating Japanese texts; to Chung-Chi Hwan
(National University of Kaohsiung, Taiwan) for help organising a eldtrip to Taiwan and for
his friendship; to Trond Oskars (Statsforvalteren at Møre og Romsdal, Norway) for help
sampling in Taiwan and companionship; and to the authorities of the Kenting National Park
in Taiwan for granting collecting permits and access to laboratory and accommodation
facilities. At the University of Bergen, we are indebted to Irene Hegstad (Electron Microscopy
Lab, Faculty of Mathematics and Natural Sciences) for help with electron scanning microscopy,
and to Louise Lindblom and Justine Siegwald for help with DNA bench work. In addition, we
are grateful to the two anonymous referees whose comments and suggestions contributed to
a better version of this work.
Disclosure statement
No potential conict of interest was reported by the authors.
Funding
This work was funded by the Department of Natural History, University Museum of Bergen,
University of Bergen, Norway.
References
Baba K. 1985. Anatomical review of a Cephalaspidean mollusc, Nakamigawaia spiralis Kuroda &
Habe in Habe 1961, (Aglajidae), from Japan. Spec Publ Mukaishima Mar Biol Stat. 231:1–5.
Burn R, Thompson TE. 1998. Order Cephalaspidea. In: Beesley GJ, Ross B, Wells AA, editors. Fauna of
Australia, Mollusca: the Southern Synthesis. Melbourne: CSIRO Press; p. 943–959.
Caballer M, Ortea J, Rivero N, Tucker GC, Malaquias MAE, Narciso S. 2015. The opisthobranch
gastropods (Mollusca: Heterobranchia) from Venezuela: an annotated and illustrated inventory
of species. Zootaxa. 4034:201–256. doi:10.11646/zootaxa.4034.2.1.
Camacho-García YE, Ornelas-Gatdula E, Gosliner TM, Valdés Á. 2014. Phylogeny of the family
Aglajidae (Pilsbry, 1895) (Heterobranchia: Cephalaspidea) inferred from mtDNA and nDNA. Mol
Phylogenet Evol. 71:113–126. doi:10.1016/j.ympev.2013.11.010.
2242 E. HELLEM AND M. A. E. MALAQUIAS

Chaban EM, Ekimova IA, Schepetov DM, Kohnert PC, Schrödl M, Chernyshev AV. 2019.
Euopisthobranch mollusks of the order Cephalaspidea (Gastropoda: Heterobranchia) of the
Kuril-Kamchatka Trench and the adjacent Pacic abyssal plain with descriptions of three new
species of the genus Spiraphiline (Philinidae). Prog Oceanogr. 178:102185. doi:10.1016/j.
pocean.2019.102185.
Darriba D, Taboada GL, Doallo R, Posada D. 2012. JModeltest 2: more models, new heuristics and
parallel computing. Nat Methods. 9:772. doi:10.1038/nmeth.2109.
Edgar RC. 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput.
Nucleic Acids Res. 32:1792–1797. doi:10.1093/nar/gkh340.
Felsenstein J. 1985. Condence limits on phylogenies: an approach using the bootstrap. Evol.
39:783–791. doi:10.1111/j.1558-5646.1985.tb00420.x.
Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R. 1994. DNA primers for amplication of mitochon-
drial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol
Biotechnol. 3:294–299.
Gosliner TM. 1980. Systematics and phylogeny of the Aglajidae (Opisthobranchia: Mollusca). Zool
J Lin Soc. 68:325–360. doi:10.1111/j.1096-3642.1980.tb01925.x.
Gosliner TM, Behrens DW, Valdés Á. 2008. Indo-Pacic nudibranchs and sea slugs: a eld guide to
the world’s most diverse fauna. Washington: Sea Challengers Natural History Books & California
Academy of Sciences.
Gosliner TM, Behrens DW, Valdés Á. 2015. Nudibranch & Sea Slug identication - Indo-Pacic.
Second ed. Jacksonville (Florida, USA): New World Publications Inc.
Gosliner TM, Behrens DW, Valdés Á. 2018. Nudibranch & Sea Slug identication - Indo-Pacic.
Second ed. Jacksonville (Florida, USA): New World Publications Inc.
Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S,
Duran C, et al. 2012. Geneious Basic: an integrated and extendable desktop software platform for
the organization and analysis of sequence data. Bioinformatics. 28:1647–1649. doi:10.1093/
bioinformatics/bts199.
Kumar S, Stecher G, Li M, Knyaz C, Tamura K, Battistuzzi FU. 2018. MEGA X: molecular evolutionary
genetics analysis across computing platforms. Mol Biol Evol. 35:1547–1549. doi:10.1093/molbev/
msy096.
Kuroda T, Habe T. 1961. Nakamigawaia spiralis. In: Habe T, editor. Coloured illustrations of the shells
of Japan (II). Vol. 183: p. 66. pls., 42 1-Chome Uehonmachi Higashi-Ku, Osaka, Japan. (In Japanese).
Malaquias MAE. 2014. New data on the heterobranch gastropods (‘opisthobranchs’) for the Bahamas
(tropical western Atlantic Ocean). Mar Biodivers Rec. 7:e27. doi:10.1017/S175526721400030X.
Marcus E, Marcus E. 1970. Opisthobranchs from Curaçao and faunistically related regions. Stud
Fauna Curaçao Other Caribb Isl. 122:1–129.
MolluscaBase. eds. (2021). Chelidonuridae Habe. MolluscaBase. 1961. http://marinespecies.org/
aphia.php?p=taxdetails&id=22981
Nakano R. 2018. Field guide to sea slugs and Nudibranches of Japan. Tokyo: Bun-ichi Co., Ltd.
Nakano R. 2019. Field guide to sea slugs and Nudibranches of Japan. Second ed. Tokyo: Bun-ichi Co.,
Ltd.
Ono A. 1999. Opisthobranchs of Kerama Islands. Japan: TBS-Britannica & Co., Ltd.
Ortea J, Caballer M, Moro L, Espinosa J. 2014. What the shell tells in Aglajidae: a new genus for Aglaja
felis (Opisthobranchia: Cephalaspidea). Rev Acad Canar Cienc. 26:83–119.
Puillandre N, Lambert A, Brouillet S, Achaz G. 2012. ABGD, Automatic Barcode Gap Discovery for
primary species delimitation. Mol Ecol. 21:1864–1877. doi:10.1111/j.1365-294X.2011.05239.x.
Rambaut A, Drummond A, 2009. FigTree v1.4.4. http://tree.bio.ed.ac.uk/software/gtree/
Rambaut A, Drummond AJ 2007. Tracer v1.7.1. http://tree.bio.ed.ac.uk/software/tracer/
Ronquist F, Huelsenbeck JP. 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models.
Bioinformatics. 19:1572–1574. doi:10.1093/bioinformatics/btg180.
Rudman WB. 1978. A new species and genus of the Aglajidae and the evolution of the
Philinacean opisthobranch molluscs. Zool J Lin Soc. 62:89–107. doi:10.1111/j.1096-3642.1978.
tb00524.x.
JOURNAL OF NATURAL HISTORY 2243

Stein ED, White BP, Mazor RD, Miller PE, Pilgrim EM, Steinke D. 2013. Evaluating ethanol-based
sample preservation to facilitate use of DNA barcoding in routine freshwater biomonitoring
programs using benthic macroinvertebrates. PLoS One. 8:1–7. doi:10.1371/journal.pone.0051273.
Susuki K. 2000. Opisthobranchs of Izu Peninsula. Tokyo (Japan): TBS-Brittanica Co., Ltd.
Zamora-Silva A, Malaquias MAE. 2016. Diet preferences of the Aglajidae: a family of cephalaspidean
gastropod predators on tropical and temperate shores. J Mar Biolog Assoc UK. 96:1101–1112.
doi:10.1017/S0025315415000739.
Zamora-Silva A, Malaquias MAE. 2018. Molecular phylogeny of the Aglajidae head-shield sea slugs
(Heterobranchia: Cephalaspidea): new evolutionary lineages revealed and proposal of a new
classication. Zool J Lin Soc. 183:1–51. doi:10.1093/zoolinnean/zlx064.
2244 E. HELLEM AND M. A. E. MALAQUIAS