Molecular phylogeny of the Aglajidae head-shield sea slugs (Heterobranchia: Cephalaspidea): new evolutionary lineages revealed and proposal of a new classification

Abstract

Aglajidae is a family of predominantly colourful shallow water marine slugs widely distributed in coral, rocky and sandy habitats of tropical and temperate waters worldwide. The monophyly of the group is supported by morphological traits, but recent molecular phylogenetic studies questioned not only the monophyletic status of the family but also the definition of its traditional genera and relationships. Therefore, in order to test the monophyly of the family and of its genera and to elucidate internal sister relationships, we here present a new multilocus phylogenetic study based on Bayesian inference and maximum likelihood analyses of the largest ever assembled taxon and gene data sets of Aglajidae including 412 specimens from 63 species (74% of the global diversity) with representatives of type species of all genera and 835 DNA sequences of the gene markers COI, 16S rRNA, 28S rRNA and Histone-3. The genera Aglaja, Chelidonura, Odontoglaja and Philinopsis were confirmed as not monophyletic. Fifteen evolutionary lineages of generic status were recovered and seven are here described as new, namely Biuve, Camachoaglaja, Mannesia, Mariaglaja, Niparaya, Spinophallus and Tubulophilinopsis. The valid taxonomic status of Spinoaglaja is confirmed and Migaya is considered to be a junior synonym of Nakamigawaia. Our results support the hypothesis that events of radula loss or gain have happened independently at least twice in the evolutionary history of Aglajidae, but lack of node support in the deeper parts of our tree obscured inference on the sister relationships of genera. A new classification for Aglajidae is proposed and diagnostic features for all 15 genera are given. © 2017 The Linnean Society of London, Zoological Journal of the Linnean Society.

Full text

© 2017 The Linnean Society of London, Zoological Journal of the Linnean Society, 2017, XX, 1–51 1
Zoological Journal of the Linnean Society, 2017, XX, 1–51. With 6 figures.
Molecular phylogeny of the Aglajidae head-shield sea
slugs (Heterobranchia: Cephalaspidea): new evolutionary
lineages revealed and proposal of a new classification
ANDREA ZAMORA-SILVA* and MANUEL ANTÓNIO E. MALAQUIAS
Phylogenetic Systematics and Evolution Research Group, Section of Taxonomy and Evolution,
Department of Natural History, University Museum of Bergen, University of Bergen, PB 7800,
5020-Bergen, Norway
Received 29 August 2016; revised 26 July 2017; accepted for publication 12 August 2017
Aglajidae is a family of predominantly colourful shallow water marine slugs widely distributed in coral, rocky and
sandy habitats of tropical and temperate waters worldwide. The monophyly of the group is supported by morphologi-
cal traits, but recent molecular phylogenetic studies questioned not only the monophyletic status of the family but
also the definition of its traditional genera and relationships. Therefore, in order to test the monophyly of the family
and of its genera and to elucidate internal sister relationships, we here present a new multilocus phylogenetic study
based on Bayesian inference and maximum likelihood analyses of the largest ever assembled taxon and gene data
sets of Aglajidae including 412 specimens from 63 species (74% of the global diversity) with representatives of type
species of all genera and 835 DNA sequences of the gene markers COI, 16S rRNA, 28S rRNA and Histone-3. The gen-
era Aglaja, Chelidonura, Odontoglaja and Philinopsis were confirmed as not monophyletic. Fifteen evolutionary line-
ages of generic status were recovered and seven are here described as new, namely Biuve, Camachoaglaja, Mannesia,
Mariaglaja, Niparaya, Spinophallus and Tubulophilinopsis. The valid taxonomic status of Spinoaglaja is confirmed
and Migaya is considered to be a junior synonym of Nakamigawaia. Our results support the hypothesis that events
of radula loss or gain have happened independently at least twice in the evolutionary history of Aglajidae, but lack
of node support in the deeper parts of our tree obscured inference on the sister relationships of genera. A new clas-
sification for Aglajidae is proposed and diagnostic features for all 15 genera are given.
ADDITIONAL KEYWORDS: Aglajids – biodiversity – morphology – systematics.
INTRODUCTION
Head-shield marine slugs or aglajids (family Aglajidae)
occur worldwide across tropical and temperate lati-
tudes predominantly in shallow waters inhabiting
coral reefs, sandy bottoms and rocky environments
with algae (Burn & Thompson, 1998; Gosliner, Behrens
& Valdés, 2008; Camacho-García et al., 2014). The
recent discovery of a specimen of Aglaja sp. in Japan
at 235-m depth (ZMBN 95935) confirms the occur-
rence of representatives of the group at greater depths
(Bouchet et al., 2008). These slugs are active predators
(Turner, 1978) and fast hunters feeding almost exclu-
sively upon vagile preys such as other shelled and non-
shelled sea slugs, nematodes, polychaetes and small
fish (Zamora-Silva & Malaquias, 2016).
The family Aglajidae has been an important model
for comparative studies on the evolution of reproduc-
tive, colour and life-history strategies in sea slug gas-
tropods (Anthes & Michiels, 2007; Anthes, Schulenburg
& Michiels, 2008; Turner & Wilson, 2011) and is
the second most diverse family of the heterobranch
Order Cephalaspidea, with an estimated 85 valid spe-
cies worldwide divided among nine accepted genera:
Aglaja Renier, 1807; Chelidonura A. Adams, 1850;
Melanochlamys Cheeseman, 1881; Migaya Ortea,
Caballer & Espinosa, 2014; Nakamigawaia Kuroda
& Habe, 1961; Navanax Pilsbry, 1895; Noalda Iredale,
1936; Odontoglaja Rudman, 1978; and Philinopsis
*Corresponding author. E-mail: andrea.zamora@uib.no
[Version of Record, published online 1 November
2017; http://zoobank.org/urn:lsid:zoobank.
org:pub:C7375EA1-7EE1-46A8-ADCF-8FC6AA1CE065]
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2 A. ZAMORA-SILVA AND M. A. E. MALAQUIAS
© 2017 The Linnean Society of London, Zoological Journal of the Linnean Society, 2017, XX, 1–20
Pease, 1860 (Bouchet, 2015). Animals in this family
are characterized by the presence of sensory bristles
surrounding the mouth, a yellow gland in the man-
tle cavity and a muscular buccal bulb devoid of both
radula and jaws except in the genus Odontoglaja
(Rudman, 1978; Gosliner, 1980). The body is cylindri-
cal and is divided into the cephalic (or anterior) shield
and posterior shield (or visceral hump) containing
the shell and ending in two caudal lobes. The shell of
aglajids is internal and reduced in all linages but one
(Noalda has a partially external shell). The shape of
the shell varies from the typical cephalaspidean bub-
ble form as in Noalda (Burn & Thompson, 1998) to a
plate-like kind with the apex curled and the posterior
part of the outer lip developed into a wing-like expan-
sion (see Ortea et al., 2012 for a review). The penis of
aglajids bears, in most cases, a conical penial papilla
and one or two prostate glands (Rudman, 1972a, b, c;
Rudman, 1974; Gosliner, 1980).
Much debate surrounds the origin and sister
relationships of Aglajidae. Fischer (1887) intro-
duced the name Cephalaspidea for a group of nine
families joined by the presence of a large cephalic
shield with burrowing function, in which Aglajidae
+ Gastropteridae + Philinidae formed a group.
Guiart (1901) suggested that Philine gave rise
to the Aglajidae without intermediate ancestors,
while Ghiselin (1966) considered aglajids a group
derived from primitive scaphandrids. Rudman
(1978) described the first radula-bearing genus
(Odontoglaja) in the family, discussed the morpho-
anatomical similarities between Aglajidae and
Philinidae and suggested that both groups evolved
independently from a Cylichna-like ancestor. Later,
Gosliner (1980) re-evaluated several morpho-
anatomical and ecological characters of the agla-
jids and pointed out that the similarities between
Aglajidae and Philinidae are too great to exclude a
direct ancestor/descendent relationship, a hypoth-
esis that is strongly supported by contemporary
molecular phylogenies (e.g. Oskars et al., 2015).
Aglajidae has been suggested to be paraphyletic by
cladistic analysis (Dayrat & Tillier, 2001), but subse-
quent molecular phylogenies supported the mono-
phyly of the family and its sister relationship with
Philinidae (Grande et al., 2004; Vonnemann et al.,
2005; Malaquias et al., 2009; Göbbeler & Klussmann-
Kolb, 2011; Ornelas-Gatdula et al., 2012; Oskars et al.,
2015). The 16S rRNA-based phylogeny of Gonzales &
Gosliner (2014) hypothesized that philinid species with
pigmented bodies and plate-less muscular gizzards
could be more closely related to aglajid slugs than to
philinid species; however, their results were not sta-
tistically supported and this hypothesis remains to be
thoroughly tested.
Anthes & Michiels (2007) published the first molec-
ular phylogeny with a relatively good coverage of
Aglajidae diversity including six genera and 31 spe-
cies. Nevertheless, the number of species per genus
was rather low with the exception of Chelidonura
and Philinopsis represented by 12 and ten species,
respectively. Their results rendered Chelidonura
and Melanochlamys paraphyletic and pointed to the
potential paraphyly of Philinopsis. The genera Aglaja,
Navanax and Odontoglaja were represented by sin-
gle species, and the latter was found nested within a
cluster of species of Chelidonura [Anthes & Michiels
(2007: 905)]. The results obtained by Malaquias et al.
(2009) and Oskars et al. (2015) have also suggested
the paraphyletic status of the genus Chelidonura and
a putative close affiliation between the radula-bear-
ing Odontoglaja and species attributed to the former
genus.
The latest molecular phylogeny of Aglajidae
(Camacho-García et al., 2014) included 243 new
sequences and 60 sequences from GenBank, repre-
senting 54 species and eight out of the nine accepted
genera (Noalda was not included, and Migaya was
represented by its type species M. felis, but attributed
to the genus Nakamigawaia). The authors confirmed
the monophyly of the family and postulated the
monophyletic status of the genera Melanochlamys,
Nakamigawaia and Odontoglaja. Also, they retrieved
a ‘superclade’ containing a paraphyletic Chelidonura
(split in three sub-clades; two with Indo-West Pacific
(IWP) species and one with Atlantic species), a mono-
phyletic Navanax, a clade with all species of Aglaja
but one (A. regiscorona), and one species of Philinopsis
(P. falciphallus). All remaining species of Philinopsis
clustered together outside this ‘superclade’.
Odontoglaja was found sister to all other Aglajidae
taxa, which led Camacho-García et al. (2014) to sug-
gest a single event of radula loss during the evolution
of Aglajidae. Additionally, the authors proposed the
synonymization of the genus Spinoaglaja Ortea, Moro
& Espinosa, 2007 with Philinopsis and stressed the
need for further phylogenetic analyses with extended
gene coverage to re-evaluate the whole systematics of
the family Aglajidae.
Based on shell differences between the Caribbean
Nakamigawaia felis and N. spiralis (the latter is the
Japanese type species of the genus) and on phyloge-
netic arguments derived from the hypothesis proposed
by Camacho-García et al. (2014), Ortea et al. (2014)
introduced the genus Migaya for the western Atlantic
(WA) species (see Discussion for a critical appraisal
on the validity of the genus Migaya). Some interesting
questions about the phylogeny of Aglajidae remain
open. For instance, the taxonomic and systematic sta-
tus of the paraphyletic Chelidonura, the definition
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MOLECULAR PHYLOGENY OF THE AGLAJIDAE SEA SLUGS 3
© 2017 The Linnean Society of London, Zoological Journal of the Linnean Society, 2017, XX, 1–20
and relationship of the poorly known genera Migaya,
Nakamigawaia, Noalda and Spinoaglaja. There is
also a need to re-evaluate the taxonomic affiliation
of some lineages of Aglaja and Philinopsis and to find
new synapomorphies and names to define the gen-
era and the new clades of the Aglajidae revealed by
phylogenetic analyses. Therefore, we here present a
new phylogenetic study of the entire family Aglajidae,
with analyses of four molecular markers and a com-
prehensive taxon sampling, which includes repre-
sentatives from across the geographical distribution
range of all genera but Noalda. The aims of this
study are (1) to test the monophyly of all traditional
genera and of those recently proposed (Migaya) or
synonymized (Spinoaglaja); (2) to define sister rela-
tionships between genera and (3) to propose a new
generic classification for the family and establish
synapomorphies for all new clades.
MATERIAL AND METHODS
Taxon sampling and ouTgroup selecTion
We first performed an exhaustive literature review
to gain a sound overview of the number and diver-
sity of Aglajidae species and to establish the type
species and type localities for each genus. Specimens
were obtained through fieldwork in Bermuda,
El Salvador, Japan, México (Atlantic and Pacific
coasts), Red Sea (Egypt), Tenerife (Canary Islands,
Spain) and Venezuela; from collections of worldwide
natural history museums and research institutions;
and from a network of international colleagues
(see Acknowledgements for full list). In total, 412
Aglajidae specimens representing all genera were
gathered and identified by the authors with assis-
tance of expert taxonomists (Table 1).
This study includes the largest data set of Aglajidae
ever analysed, which consists of 835 DNA sequences
(345 newly generated and 490 mined from GenBank) of
the mitochondrial genes cytochrome c oxidase I (COI)
and 16S rRNA (16S) and nuclear genes 28S rRNA (28S)
and Histone-3 (H3). A total of 63 valid species includ-
ing the type species of nine genera, namely Aglaja
tricolorata, Chelidonura hirundinina, Melanochlamys
cylindrica, Migaya felis (as N. felis), Nakamigawaia
spiralis, Navanax inermis, Odontoglaja guamensis,
Philinopsis speciosa and Spinoaglaja petra, were
included for analyses. This represents ~74% of the
global diversity of Aglajidae. Eight species from the
families Gastropteridae, Haminoeidae and Philinidae
were included as outgroup taxa, and the phylogenetic
trees were rooted with a representative from the basal
cephalaspidean family Diaphanidae (Oskars et al.,
2015) (Table 1).
dna exTracTion, amplificaTion and sequencing
DNA was extracted from tissue obtained from the
parapodial lobes or from whole specimens using the
Qiagen DNeasy Blood and Tissue Kit following the
protocol recommended by the manufacturer. Partial
sequences of ~579 bp of the COI, 403 bp of the 16S,
1467 bp of the 28S and 269 bp of the H3 genes were
amplified and sequenced using the primers listed in
Table 2. PCRs were preformed in 50 μL reactions con-
taining 1 μL of DNA, 5 μL of Qiagen Buffer 10× (15
mM MgCl2), 5 μL of dNTPs (2.5 mM), 10 μL of Qiagen
Q-Solution, 4 μL of forward and reverse primers (2 μL
each primer direction) and 0.5 μL of Qiagen Taq DNA
polymerase (5U/μL; QUIAGEN 203601) plus specific
amounts of Sigma water and magnesium chloride
(COI and 16S: 17.5 μL of Sigma water and 7 μL of
MgCl2; 28S: 22.5 μL of Sigma water and 2 μL of MgCl2;
H3: 20.5 μL of Sigma water and 7 μL of MgCl2). PCR
thermal cycles were as follows: an initial denatura-
tion at 95 °C for 3 min, followed by 39 cycles of 45 s at
94 °C, 45 s at a gene-specific annealing temperature
(45 °C for 16S, COI, H3; and 52 °C for 28S), extension
at 72 °C for 2 min, and a final extension at 72 °C for
10 min. Successful PCR products were purified with
Exonuclease I Shrimp Alkaline Phosphatase (ExoSAP)
protocol. The total volume of each purification reaction
was 16 μL, which consisted of 0.4 μL of Exo I (10 U/μL),
4 μL SAP (1 U/μL), 3.6 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 finally
kept cool at 4 °C. Purified samples were sequenced
with an Automatic Sequencer 3730XL.
phylogeneTic analyses
The forward and reverse DNA chromatograms were
edited and assembled using the software Sequencher
(v. 4.10.1, Gene Codes Corp). All sequences were
blasted in GenBank to check for contamination. Single
gene sequences were aligned with 15 iterations using
‘Muscle Alignment’ (Edgar, 2004) and default param-
eters executed for this aligning method in Geneious
6.0.6 (Biomatters Ltd.). Alignments were trimmed
at each end to a position where at least 50% of the
sequences had nucleotides and missing positions at
the ends were coded as missing data (N). Saturation
in the protein-coding genes COI and H3 was tested for
third codon positions with the programme MEGA 5.1
(Tamura et al., 2011) by plotting general time-reversi-
ble distances (GTR) against total substitutions (transi-
tions + transversions). Saturation was detected in third
codon positions, and therefore, two data sets with and
without third codons were assembled in SeaView 4.5.0
(Gouy, Guindon & Gascuel, 2010). Gap-rich regions
were identified in the alignments of the ribosomal
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4 A. ZAMORA-SILVA AND M. A. E. MALAQUIAS
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Table 1. List of specimens used in this study, with collection sites, voucher numbers, GenBank accession numbers and revised identification after our phyloge-
netic results
Taxa Revised identification Voucher number Collection site GenBank accession numbers
16S rRNA COI 28S rRNA H3
Genus Aglaja Renier, 1807
Aglaja sp. Melanochlamys mader-
ensis (Watson, 1897)
ZMBN95936 El Palmar, Tenerife, Spain MF036365 – MF036466 MF036625
Aglaja sp. (deep-sea
Aglajid)
Tubulophilinopsis sp. 1 ZMBN95935 Nagasaki Bay, Japan – – MF036518 MF036678
A. felis Er. Marcus &
Ev. Marcus, 1970
Nakamigawaia felis (Er.
Marcus & Ev. Marcus,
1970)
ZMBN84913 Isla Tortuga, Venezuela MF036366 MF036535 MF036467 MF036627
A. hummelincki
Er. Marcus & Ev.
Marcus, 1970
Camachoaglaja berolina
(Er. Marcus & Ev.
Marcus, 1970)*
LACM176425 The Bahamas – HQ011869 – HQ011883
A. hummelincki
Er. Marcus & Ev.
Marcus, 1970
Camachoaglaja berolina
(Er. Marcus & Ev.
Marcus, 1970)*
LACM176427 The Bahamas HQ011852 HQ011866 – HQ011886
A. hummelincki
Er. Marcus & Ev.
Marcus, 1970
Camachoaglaja mari-
agordae (Ortea,
Espinosa & Moro,
2004)*
LACM3127 The Bahamas HQ011859 – – HQ011902
A. hummelincki
Er. Marcus & Ev.
Marcus, 1970
Camachoaglaja berolina
(Er. Marcus & Ev.
Marcus, 1970)*
LACM3126 The Bahamas – HQ011876 – HQ011901
A. ocelligera (Bergh,
1893)
Aglaja ocelligera (Bergh,
1893)
CPIC00679 San Diego, California, USA – JN825205 – JN824999
A. ocelligera (Bergh,
1893)
Aglaja ocelligera (Bergh,
1893)
– San Diego, California, USA JN825080 – – –
A. regiscorona
Bertsch, 1972
Niparaya regiscorona
(Bertsch, 1972)
E632 Kwajalein Atoll, Marshall
Islands
MF036393 – MF036488 MF036648
A. regiscorona
Bertsch, 1972
Niparaya regiscorona
(Bertsch, 1972)
ZMBN95965 Honokowai Park, Hawaii MF036389 MF036548 MF036485 MF036644
A. regiscorona
Bertsch, 1972
Niparaya regiscorona
(Bertsch, 1972)*
CASIZ24042 Bahía de las Cruces, BC,
México
– – – JN825003
A. tricolorata Renier,
1807
Aglaja tricolorata Renier,
1807
ZMBN95937 Spain – – MF036425 MF036575
A. tricolorata Renier,
1807
Aglaja tricolorata Renier,
1807
Isolate E19 Giglio, Italy AM421854 AM421902 – –
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MOLECULAR PHYLOGENY OF THE AGLAJIDAE SEA SLUGS 5
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Taxa Revised identification Voucher number Collection site GenBank accession numbers
16S rRNA COI 28S rRNA H3
A. tricolorata Renier,
1807
Aglaja tricolorata Renier,
1807
CPIC00680 Cadiz, Spain – JN825149 – JN825004
A. tricolorata Renier,
1807
Aglaja tricolorata Renier,
1807
BMNH20060327 Algarve, Portugal – – DQ927215 –
Genus Chelidonura A. Adams, 1850
Chelidonura sp. (as
Navanax sp. in
Gosliner et al. 2008)
Mannesia sp.* CASIZ166759 Malico Bay, Maui, Hawaii – – – JN825013
Chelidonura sp.
M-11-001
C. alisonae Gosliner,
2011
ZMBN95966 Heikili Point, Maui, Hawaii – KU183993 MF036432 –
Chelidonura sp.
M-10-014
C. alisonae Gosliner,
2011
ZMBN96006 Mala Wharf, Maui, Hawaii MF036329 – – MF036589
Chelidonura sp. Mariaglaja sandrana
(Rudman, 1973)
ZMBN95974 Lizard Island, NE Australia MF036360 MF036534 MF036461 MF036621
Chelidonura sp. Tubulophilinopsis sp. 2923/SUL.11 Selat Lembeh, Indonesia – – MF036517 MF036677
C. africana Pruvot-
Fol, 1953
Camachoaglaja africana
(Pruvot-Fol, 1953)
ZMBN96017 Playa Hombre, Gran
Canaria, Spain
MF036322 MF036521 – MF036581
C. africana Pruvot-
Fol, 1953
Camachoaglaja africana
(Pruvot-Fol, 1953)
SAB2011.D/10.B2 Playa Hombre, Gran
Canaria, Spain
MF036321 – MF036428 MF036579
C. africana Pruvot-
Fol, 1953
Camachoaglaja africana
(Pruvot-Fol, 1953)*
MNCN150544368 Madeira, Portugal HQ011851 HQ011863 – HQ011878
C. africana Pruvot-
Fol, 1953
Camachoaglaja africana
(Pruvot-Fol, 1953)*
– Algarve, Portugal – DQ983184 – –
C. africana Pruvot-
Fol, 1953
Camachoaglaja africana
(Pruvot-Fol, 1953)*
BMNH20030343 Algarve, Portugal – DQ974654 DQ927216 –
C. africana Pruvot-
Fol, 1953
Camachoaglaja africana
(Pruvot-Fol, 1953)*
MNCN150546493 Algarve, Portugal HQ011850 – – –
C. africana Pruvot-
Fol, 1953
Camachoaglaja africana
(Pruvot-Fol, 1953)*
MNCN150546488 Algarve, Portugal HQ011849 – – –
C. alexisi Gosliner,
2015
Mariaglaja alexisi
(Gosliner, 2015)*
CASIZ121878 Mainit Bubbles, Batangas,
The Philippines
JN825093 JN825153 – JN825014
C. alisonae Gosliner,
2011
Chelidonura alisonae
Gosliner, 2011
ZMBN95991 Anini Beach, Kauai, Hawaii MF036328 MF036523 MF036433 MF036588
C. alisonae Gosliner,
2011
Chelidonura alisonae
Gosliner, 2011
CASIZ165872 Heikili Point, Maui, Hawaii JN825091 JN825151 – JN825012
C. amoena Bergh,
1905
Chelidonura amoena
Bergh, 1905
ZMBN95996 Manza Beach, Okinawa,
Japan
MF036330 – MF036435 MF036612
Table 1. Continued
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6 A. ZAMORA-SILVA AND M. A. E. MALAQUIAS
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Taxa Revised identification Voucher number Collection site GenBank accession numbers
16S rRNA COI 28S rRNA H3
C. amoena Bergh,
1905
Chelidonura amoena
Bergh, 1905
– Lizard Island, NE Australia – AM421901 – –
C. amoena Bergh,
1905
Chelidonura amoena
Bergh, 1905
Isolate E2 Selat Lembeh, Indonesia – AM421900 AM421963 –
C. amoena Bergh,
1905
Chelidonura amoena
Bergh, 1905
– Albrolhosld Islands,
NW-Australia
– – AM421962 –
C. amoena Bergh,
1905
Chelidonura amoena
Bergh, 1905
– Lizard Island, NE Australia AM421841 – – –
C. amoena Bergh,
1905
Chelidonura amoena
Bergh, 1905
CASIZ070125 Okinawa, Japan JN825083 – – JN825005
C. amoena Bergh,
1905
Chelidonura amoena
Bergh, 1905
CASIZ173355 Bohol Island, Panglao, The
Philippines
JN825084 JN825150 – JN825006
C. berolina Er. Marcus
& Ev. Marcus, 1970
Camachoaglaja berolina
(Er. Marcus & Ev.
Marcus, 1970)*
ZMBN83014 Shelly Bay, Bermuda MF036324 MF036522 – MF036582
C. berolina Er. Marcus
& Ev. Marcus, 1970
Camachoaglaja berolina
(Er. Marcus & Ev.
Marcus, 1970)*
LACM176427 The Bahamas HQ011852 HQ011866 – HQ011886
C. berolina Er. Marcus
& Ev. Marcus, 1970
Camachoaglaja berolina
(Er. Marcus & Ev.
Marcus, 1970)*
LACM176435 The Bahamas HQ011853 HQ011867 – HQ011899
C. berolina Er. Marcus
& Ev. Marcus, 1970
Camachoaglaja berolina
(Er. Marcus & Ev.
Marcus, 1970)*
LACM176434 Bermuda HQ011858 HQ011872 – HQ011898
C. berolina Er. Marcus
& Ev. Marcus, 1970
Camachoaglaja berolina
(Er. Marcus & Ev.
Marcus, 1970)*
LACM176430 The Bahamas HQ011855 HQ011870 – HQ011893
C. berolina Er. Marcus
& Ev. Marcus, 1970
Camachoaglaja berolina
(Er. Marcus & Ev.
Marcus, 1970)*
LACM176429 The Bahamas HQ011857 HQ011868 – HQ011892
C. berolina Er. Marcus
& Ev. Marcus, 1970
Camachoaglaja berolina
(Er. Marcus & Ev.
Marcus, 1970)*
Isolate 66 Yucatán, México HQ011854 – – HQ011879
C. berolina Er. Marcus
& Ev. Marcus, 1970
Camachoaglaja berolina
(Er. Marcus & Ev.
Marcus, 1970)*
LACM176436 Florida HQ011856 – – –
C. cf. amoena Bergh,
1905
Chelidonura sp. RMNH.MOL.41697 Selat Lembeh, Indonesia – – – MF036604
Table 1. Continued
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MOLECULAR PHYLOGENY OF THE AGLAJIDAE SEA SLUGS 7
© 2017 The Linnean Society of London, Zoological Journal of the Linnean Society, 2017, XX, 1–20
Taxa Revised identification Voucher number Collection site GenBank accession numbers
16S rRNA COI 28S rRNA H3
C. cf. cubana Ortea &
Martínez, 1997
Chelidonura cubana
(Ortea & Martínez,
1997)
ZMBN95986 Discovery Bay, Jamaica – – – MF036592
C. cf. cubana Ortea &
Martínez, 1997
Chelidonura cubana
(Ortea & Martínez,
1997)
ZMBN95985 Discovery Bay, Jamaica MF036332 – – MF036591
C. cubana Ortea &
Martínez, 1997
Chelidonura cubana
(Ortea & Martínez,
1997)
CNMO2979 Arrecife Madagascar,
Campeche, México
MF036331 – – MF036590
C. cubana Ortea &
Martínez, 1997
Chelidonura cubana
(Ortea & Martínez,
1997)
CPIC00681 Yucatán, México JN825087 – – JN825008
C. cubana Ortea &
Martínez, 1997
Chelidonura cubana
(Ortea & Martínez,
1997)
CPIC00682 Yucatán, México JN825088 – – JN825009
C. electra Rudman,
1970
Chelidonura electra
Rudman, 1970
Isolate 149 Lizard Island, NE Australia AM421843 AM421899 AM421964 –
C. electra Rudman,
1970
Chelidonura electra
Rudman, 1970
CASIZ175762 Mooloolaba, Queensland,
Australia
JN825089 – – JN825010
C. electra Rudman,
1970
Chelidonura electra
Rudman, 1970
CASIZ179921 Espiritu Santo Island,
Vanuatu
JN825090 – – JN825011
C. electra Rudman,
1970
Chelidonura electra
Rudman, 1970
CASCephas5 Madagascar – – DQ927217 –
C. flavolobata Heller
& Thompson, 1983
Chelidonura flavolobata
Heller & Thompson,
1983
Isolate RM17 Mangrove Bay, Egypt, Red
Sea
AM421845 AM421897 AM421967 –
C. flavolobata Heller
& Thompson, 1983
Chelidonura flavolobata
Heller & Thompson,
1983
Isolate RM08 Mangrove Bay, Egypt, Red
Sea
AM421844 – AM421968 –
C. fulvipunctata Baba,
1938
Biuve fulvipunctata
(Baba, 1938)*
E508b Kwajalein Atoll, Marshall
Islands
MF036319 KU183996 – –
C. fulvipunctata Baba,
1938
Biuve fulvipunctata
(Baba, 1938)*
E528a Kwajalein Atoll, Marshall
Islands
– KU183999 MF036426 –
C. fulvipunctata Baba,
1938
Biuve fulvipunctata
(Baba, 1938)*
E528b Kwajalein Atoll, Marshall
Islands
MF036320 MF036520 MF036427 MF036578
C. fulvipunctata Baba,
1938
Biuve fulvipunctata
(Baba, 1938)*
Isolate 120 Lizard Island, NE Australia AM421849 AM421896 AM421971 –
Table 1. Continued
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8 A. ZAMORA-SILVA AND M. A. E. MALAQUIAS
© 2017 The Linnean Society of London, Zoological Journal of the Linnean Society, 2017, XX, 1–20
Taxa Revised identification Voucher number Collection site GenBank accession numbers
16S rRNA COI 28S rRNA H3
C. hirundinina (Quoy
& Gaimard, 1833)
Chelidonura hirundin-
ina (Quoy & Gaimard,
1833)
ZMBN95971 Lizard Island, NE Australia – MF036524 MF036438 MF036594
C. hirundinina (Quoy
& Gaimard, 1833)
Chelidonura sp. CNMO3034 Arrecife Madagascar,
Campeche, México
MF036339 – MF036443 MF036601
C. hirundinina (Quoy
& Gaimard, 1833)
Chelidonura hirundin-
ina (Quoy & Gaimard,
1833)
E115 Kwajalein Atoll, Marshall
Islands
MF036334 – MF036436 MF036595
C. hirundinina (Quoy
& Gaimard, 1833)
Chelidonura sp. ZMBN95979 Discovery Bay, Jamaica MF036344 MF036528 MF036446 MF036605
C. hirundinina (Quoy
& Gaimard, 1833)
Chelidonura hirundin-
ina (Quoy & Gaimard,
1833)
ZMBN95959 Heikili Point, Maui, Hawaii – – MF036437 MF036593
C. hirundinina (Quoy
& Gaimard, 1833)
Camachoaglaja mari-
agordae (Ortea,
Espinosa & Moro,
2004)
ZMBN96016 Maria la Gorda,
Guanahacabibes, Cuba
MF036327 – MF036431 MF036586
C. hirundinina (Quoy
& Gaimard, 1833)
Chelidonura sp. ZMBN96012 Maria la Gorda,
Guanahacabibes, Cuba
MF036342 MF036526 MF036444 MF036602
C. hirundinina (Quoy
& Gaimard, 1833)
Camachoaglaja mari-
agordae (Ortea,
Espinosa & Moro,
2004)
ZMBN96014 Maria la Gorda,
Guanahacabibes, Cuba
MF036325 – MF036430 MF036585
C. hirundinina (Quoy
& Gaimard, 1833)
Chelidonura hirundin-
ina (Quoy & Gaimard,
1833)
ZMBN95994 Manza Beach, Okinawa,
Japan
MF036335 – MF036441 MF036597
C. hirundinina (Quoy
& Gaimard, 1833)
Chelidonura hirundin-
ina (Quoy & Gaimard,
1833)
ZMBN95992 Manza Beach, Okinawa,
Japan
– – MF036439 MF036596
C. hirundinina (Quoy
& Gaimard, 1833)
Chelidonura hirundin-
ina (Quoy & Gaimard,
1833)
LACM176438 The Bahamas HQ011862 HQ011877 – HQ011905
C. hirundinina (Quoy
& Gaimard, 1833)
Chelidonura hirundin-
ina (Quoy & Gaimard,
1833)
Isolate 54 Lizard Island, NE Australia AM421846 AM421882 AM421970 –
C. hirundinina (Quoy
& Gaimard, 1833)
Chelidonura hirundin-
ina (Quoy & Gaimard,
1833)
CASIZ171259 Palmyra Atoll, Sand Island,
USA
JN825096 JN825156 – JN825017
Table 1. Continued
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MOLECULAR PHYLOGENY OF THE AGLAJIDAE SEA SLUGS 9
© 2017 The Linnean Society of London, Zoological Journal of the Linnean Society, 2017, XX, 1–20
Taxa Revised identification Voucher number Collection site GenBank accession numbers
16S rRNA COI 28S rRNA H3
C. hirundinina (Quoy
& Gaimard, 1833)
Chelidonura hirundin-
ina (Quoy & Gaimard,
1833)
CASIZ175674 Great Exuma, Stocking
Island, The Bahamas
JN825097 JN825157 – JN825018
C. hirundinina (Quoy
& Gaimard, 1833)
Chelidonura hirundin-
ina (Quoy & Gaimard,
1833)
CASIZ175675 Great Exuma, Stocking
Island, The Bahamas
JN825098 JN825158 – JN825019
C. hirundinina (Quoy
& Gaimard, 1833)
Mariaglaja sp.* CASIZ177071 Espiritu Santo Island,
Vanuatu
– JN825159 – JN825020
C. hirundinina (Quoy
& Gaimard, 1833)
Chelidonura hirundin-
ina (Quoy & Gaimard,
1833)
CASIZ177328 Luzon Island, Batangas,
The Philippines
JN825099 JN825160 – JN825021
C. hirundinina (Quoy
& Gaimard, 1833)
Chelidonura hirundin-
ina (Quoy & Gaimard,
1833)
CASIZ185070 West Papua, Raja Ampat,
Indonesia
JN825100 JN825161 – JN825022
C. hirundinina (Quoy
& Gaimard, 1833)
Chelidonura hirundin-
ina (Quoy & Gaimard,
1833)
CASIZ185125 Iles Radama, Nosy
Kalakajoro, Madagascar
JN825101 – – JN825023
C. hirundinina (Quoy
& Gaimard, 1833)
Chelidonura hirundin-
ina (Quoy & Gaimard,
1833)
Isolate 180 Lizard Island, NE Australia – AM421881 AM421969 –
C. hirundinina (Quoy
& Gaimard, 1833)
Chelidonura sp. ZMBN95998 Discovery Bay, Jamaica MF036340 – – –
C. inornata Baba,
1949
Chelidonura sp. EO41/10.10.11 Kwajalein Atoll, Marshall
Islands
MF036348 – MF036450 MF036609
C. inornata Baba,
1949
Chelidonura sp. EO41/4.25.11 Kwajalein Atoll, Marshall
Islands
MF036349 – – –
C. inornata Baba,
1949
Mariaglaja inornata
(Baba, 1949)
EO41/1.1.11 Kwajalein Atoll, Marshall
Islands
MF036354 MF036533 MF036455 MF036615
C. inornata Baba,
1949
Chelidonura sp. ZMBN95989 Lizard Island, NE Australia MF036347 – MF036449 MF036608
C. inornata Baba,
1949
Chelidonura sp. ZMBN95984 Lizard Island, NE Australia MF036346 – – –
C. inornata Baba,
1949
Chelidonura sp. ZMBN95983 Lizard Island, NE Australia – MF036529 MF036448 MF036607
C. inornata Baba,
1949
Chelidonura sp. ZMBN96001 Merizo, Guam, USA – MF036527 MF036445 MF036603
C. inornata Baba,
1949
Mariaglaja inornata
(Baba, 1949)
ZMBM94027 Manza Beach, Okinawa,
Japan
MF036355 – MF036456 MF036616
Table 1. Continued
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10 A. ZAMORA-SILVA AND M. A. E. MALAQUIAS
© 2017 The Linnean Society of London, Zoological Journal of the Linnean Society, 2017, XX, 1–20
Taxa Revised identification Voucher number Collection site GenBank accession numbers
16S rRNA COI 28S rRNA H3
C. inornata Baba,
1949
Chelidonura livida
Yonow, 1994*
Isolate 45 Lizard Island, NE Australia AM421842 AM421898 AM421965 –
C. inornata Baba,
1949
Chelidonura sp.* – Lizard Island, NE Australia – – AY427473 –
C. inornata Baba,
1949
Mariaglaja inornata
(Baba, 1949)*
CPIC00683 Merizo, Guam, USA JN825105 JN825163 – JN825026
C. inornata Baba,
1949
Chelidonura sp.* CASIZ173698 Palmyra Atoll, Sand Island,
USA
JN825103 – – JN825024
C. inornata Baba,
1949
Mariaglaja inornata
(Baba, 1949)*
CASIZ182855 Luzon Island, Batangas,
The Philippines
JN825104 JN825162 – JN825025
C. juancarlosi Ortea &
Espinosa, 1998
Camachoaglaja berolina
(Er. Marcus & Ev.
Marcus, 1970)*
LACM173215 The Bahamas – – – HQ011880
C. leopoldoi Ortea,
Moro & Espinosa,
1997
Camachoaglaja africana
(Pruvot-Fol, 1953)
ZMBN95955 Tenerife, Canary Islands,
Spain
MF036323 – – MF036580
C. livida Yonow, 1994 Chelidonura livida
Yonow, 1994
ZMBN96002 Mangrove Bay, Egypt MF036336 MF036525 – MF036598
C. livida Yonow, 1994 Chelidonura livida
Yonow, 1994
Isolate RM40 Mangrove Bay, Egypt – AM421895 AM421972 –
C. livida Yonow, 1994 Chelidonura livida
Yonow, 1994
CASIZ173455 Iles Radama, Nosy
Kalakajoro, Madagascar
– JN825164 – JN825027
C. livida Yonow, 1994 Chelidonura livida
Yonow, 1994
– Mangrove Bay, Egypt AM421848 – – –
C. mandroroa
Gosliner, 2011
Mariaglaja mandroroa
(Gosliner, 2011)*
CASIZ182002 Iles Radama, Nosy
Kalakajoro, Madagascar
JN825094 JN825154 – JN825015
C. mariagordae Ortea,
Espinosa & Moro,
2004
Camachoaglaja mari-
agordae (Ortea,
Espinosa & Moro,
2004)
ZMBN96010 Maria la Gorda,
Guanahacabibes, Cuba
MF036326 – – MF036584
C. mariagordae Ortea,
Espinosa & Moro,
2004
Camachoaglaja berolina
(Er. Marcus & Ev.
Marcus, 1970)*
LACM176436 Key Largo, Florida – – – HQ011900
C. mariagordae Ortea,
Espinosa & Moro,
2004
Camachoaglaja berolina
(Er. Marcus & Ev.
Marcus, 1970)*
LACM176431 The Bahamas – – – HQ011895
Table 1. Continued
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MOLECULAR PHYLOGENY OF THE AGLAJIDAE SEA SLUGS 11
© 2017 The Linnean Society of London, Zoological Journal of the Linnean Society, 2017, XX, 1–20
Taxa Revised identification Voucher number Collection site GenBank accession numbers
16S rRNA COI 28S rRNA H3
C. normani Ornelas-
Gatdula, Dupont &
Valdés, 2011
Camachoaglaja mari-
agordae (Ortea,
Espinosa & Moro,
2004)*
LACM3128 Great Exuma, Stocking
Island, The Bahamas
HQ011861 – – HQ011904
C. pallida A. Adams,
1850
Chelidonura pallida
A. Adams, 1850
ZMBN95981 Lizard Island, NE Australia MF036337 – MF036442 MF036599
C. pallida A. Adams,
1850
Chelidonura pallida
A. Adams, 1850
ZMBN95980 Lizard Island, NE Australia MF036338 – MF036517 MF036600
C. pallida A. Adams,
1850
Mariaglaja sandrana
(Rudman, 1973)*
Isolate 243 Lizard Island, NE Australia AM421853 AM421876 AM421946 –
C. punctata Eliot,
1903
Chelidonura punctata
Eliot, 1903
CASIZ173409 Iles Radama, Nosy
Kalakajoro, Madagascar
JN825106 – – JN825028
C. sandrana ‘black’
Rudman, 1973
Mariaglaja sandrana
(Rudman, 1973)
ZMBN95982 Lizard Island, NE Australia MF036362 – MF036463 MF036622
C. sandrana ‘black’
Rudman, 1973
Mariaglaja sandrana
(Rudman, 1973)
ZMBN95969 Selat Lembeh, Indonesia MF036358 – MF036459 MF036617
C. sandrana ‘black’
Rudman, 1973
Mariaglaja sandrana
(Rudman, 1973)
ZMBN95950 Lizard Island, NE Australia MF036357 – MF036458 –
C. sandrana ‘black’
Rudman, 1973
Mariaglaja sandrana
(Rudman, 1973)*
CASIZ185065 West Papua, Raja Ampat,
Indonesia
JN825111 JN825170 – JN825034
C. sandrana ‘pale’
Rudman, 1973
Chelidonura sp. ZMBN95976 Lizard Island, NE Australia MF036345 – MF036447 MF036606
C. sandrana Rudman,
1973
Mariaglaja sandrana
(Rudman, 1973)
ZMBN95977 Lizard Island, NE Australia MF036361 – MF036462 –
C. sandrana Rudman,
1973
Mariaglaja sandrana
(Rudman, 1973)
ZMBM94028 Lizard Island, NE Australia MF036356 – MF036457 MF036619
C. sandrana Rudman,
1973
Mariaglaja sandrana
(Rudman, 1973)
ZMBN95970 Lizard Island, NE Australia MF036359 – MF036460 MF036620
C. sandrana Rudman,
1973
Mariaglaja sandrana
(Rudman, 1973)*
– Lizard Island, NE Australia – AM421875 – –
C. sandrana Rudman,
1973
Mariaglaja sandrana
(Rudman, 1973)*
Isolate 387 Lizard Island, NE Australia AM421852 – – –
C. sandrana Rudman,
1973
Mariaglaja sandrana
(Rudman, 1973)*
Isolate 380 Mangrove Bay, Egypt, Red
Sea
– AM421873 AM421943 –
C. sandrana Rudman,
1973
Mariaglaja sandrana
(Rudman, 1973)*
– Mangrove Bay, Egypt, Red
Sea
– AM421872 – –
C. sandrana Rudman,
1973
Mariaglaja sandrana
(Rudman, 1973)*
Isolate RM38 Mangrove Bay, Egypt, Red
Sea
– AM421871 AM421942 –
Table 1. Continued
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12 A. ZAMORA-SILVA AND M. A. E. MALAQUIAS
© 2017 The Linnean Society of London, Zoological Journal of the Linnean Society, 2017, XX, 1–20
Taxa Revised identification Voucher number Collection site GenBank accession numbers
16S rRNA COI 28S rRNA H3
C. sandrana Rudman,
1973
Mariaglaja sandrana
(Rudman, 1973)*
– Lizard Island, NE Australia – – AM421944 –
C. sandrana Rudman,
1973
Mariaglaja sandrana
(Rudman, 1973)*
– Lizard Island, NE Australia – – AM421945 –
C. sandrana Rudman,
1973
Mariaglaja sandrana
(Rudman, 1973)*
– Lizard Island, NE Australia AM421851 – – –
C. sandrana Rudman,
1973
Mariaglaja sandrana
(Rudman, 1973)*
– Lizard Island, NE Australia AM421850 – – –
C. sandrana Rudman,
1973
Mariaglaja sandrana
(Rudman, 1973)*
CASIZ171450 Panglao, The Philippines JN825108 JN825166 – JN825030
C. sandrana Rudman,
1973
Mariaglaja sandrana
(Rudman, 1973)*
CASIZ173311 Panglao, The Philippines – JN825167 – JN825031
C. sandrana Rudman,
1973
Mariaglaja sandrana
(Rudman, 1973)*
CASIZ167962 Nabwageta Island, Papua
New Guinea
JN825107 JN825165 – JN825029
C. sandrana Rudman,
1973
Mariaglaja sandrana
(Rudman, 1973)*
CASIZ175649 Ajotau, Papua New Guinea JN825109 JN825168 – JN825032
C. sandrana Rudman,
1973
Mariaglaja sandrana
(Rudman, 1973)*
CASIZ185131 Espiritu Santo Island,
Vanuatu
JN825110 JN825169 – JN825033
C. sandrana Rudman,
1973
Mariaglaja sandrana
(Rudman, 1973)*
CASIZ185126 Espiritu Santo Island,
Vanuatu
JN825112 JN825171 – JN825035
C. tsurugensis Baba &
Abe, 1964
Mariaglaja sandrana
(Rudman, 1973)*
CASIZ079256 Seragaki, Okinawa, Japan JN825113 – – JN825036
C. tsurugensis Baba &
Abe, 1964
Mariaglaja sandrana
(Rudman, 1973)*
CASIZ115743 Seragaki, Okinawa, Japan JN825114 – – –
C. tsurugensis Baba &
Abe, 1964
Mariaglaja sandrana
(Rudman, 1973)*
CASIZ185130 Espiritu Santo Island,
Vanuatu
JN825115 JN825172 – JN825037
C. varians Eliot, 1903 Chelidonura varians
Eliot, 1903
ZMBN95978 Lizard Island, NE Australia MF036351 MF036530 MF036452 –
C. varians Eliot, 1903 Chelidonura varians
Eliot, 1903
– Lizard Island, NE Australia – AM421894 – –
C. varians Eliot, 1903 Chelidonura varians
Eliot, 1903
Isolate 55 Lizard Island, NE Australia AM421847 AM421893 AM421973 –
C. varians Eliot, 1903 Chelidonura varians
Eliot, 1903
CPIC1009 Uama Island, Papua New
Guinea
JN825117 JN825175 – JN825040
C. varians Eliot, 1903 Chelidonura varians
Eliot, 1903
CASIZ173326 Bohol Island, Panglao, The
Philippines
JN825116 JN825173 – JN825038
Table 1. Continued
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MOLECULAR PHYLOGENY OF THE AGLAJIDAE SEA SLUGS 13
© 2017 The Linnean Society of London, Zoological Journal of the Linnean Society, 2017, XX, 1–20
Taxa Revised identification Voucher number Collection site GenBank accession numbers
16S rRNA COI 28S rRNA H3
C. varians Eliot, 1903 Chelidonura varians
Eliot, 1903
CASIZ175334 Bohol Island, Panglao, The
Philippines
– JN825174 – JN825039
C. varians Eliot, 1903 Chelidonura varians
Eliot, 1903
RMNH.MOL.41702 Selat Lembeh, Indonesia MF036350 – MF036450 MF036610
Genus Melanochlamys Cheeseman, 1881
Melanochlamys sp. Melanochlamys sp. 06AUS1 Lizard Island, NE Australia EU604715 EU604697 – –
Melanochlamys sp. Melanochlamys sp. 06AUS3 Lizard Island, NE Australia EU604716 EU604698 – –
Melanochlamys sp. Melanochlamys sp. Isolate 169 Lizard Island, NE Australia AM421824 AM421868 – –
Melanochlamys sp. Melanochlamys sp. Isolate 175 Lizard Island, NE Australia AM421823 AM421867 – –
M. cylindrica
Cheeseman, 1881
Melanochlamys cylin-
drica Cheeseman,
1881
Isolate 06NZ1 Northland, New Zealand EU604717 EU604699 EU604736 –
M. cylindrica
Cheeseman, 1881
Melanochlamys cylin-
drica Cheeseman,
1881
Isolate 06NZ2 Northland, New Zealand EU604718 EU604700 EU604735 –
M. cylindrica
Cheeseman, 1881
Melanochlamys cylin-
drica Cheeseman,
1881
Isolate 06NZ3 Northland, New Zealand EU604719 EU604701 – –
M. cylindrica
Cheeseman, 1881
Melanochlamys cylin-
drica Cheeseman,
1881
Isolate 06NZ4 Northland, New Zealand EU604720 EU604702 – –
M. cylindrica
Cheeseman, 1881
Melanochlamys cylin-
drica Cheeseman,
1881
Isolate 06NZ5 Northland, New Zealand EU604721 EU604703 – –
M. cylindrica
Cheeseman, 1881
Melanochlamys cylin-
drica Cheeseman,
1881
Isolate 06NZ7 Northland, New Zealand EU604722 EU604704 – –
M. cylindrica
Cheeseman, 1881
Melanochlamys cylin-
drica Cheeseman,
1881
Isolate 06NZ8 Northland, New Zealand EU604723 EU604705 – –
M. cylindrica
Cheeseman, 1881
Melanochlamys cylin-
drica Cheeseman,
1881
Isolate 06NZ9 Northland, New Zealand EU604724 EU604706 – –
M. diomedea (Bergh,
1893)
Melanochlamys diome-
dea (Bergh, 1893)
Isolate E14 Friday Harbor, USA AM421825 AM421866 AM421935 –
M. diomedea (Bergh,
1893)
Melanochlamys diome-
dea (Bergh, 1893)
06SJI2 San Juan Island, USA EU604730 EU604712 – –
Table 1. Continued
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14 A. ZAMORA-SILVA AND M. A. E. MALAQUIAS
© 2017 The Linnean Society of London, Zoological Journal of the Linnean Society, 2017, XX, 1–20
Taxa Revised identification Voucher number Collection site GenBank accession numbers
16S rRNA COI 28S rRNA H3
M. diomedea (Bergh,
1893)
Melanochlamys diome-
dea (Bergh, 1893)
06SJI3 San Juan Island, USA EU604731 EU604713 EU604734 –
M. diomedea (Bergh,
1893)
Melanochlamys diome-
dea (Bergh, 1893)
06SJI4 San Juan Island, USA EU604732 EU604714 – –
M. diomedea (Bergh,
1893)
Melanochlamys diome-
dea (Bergh, 1893)
06SJI1 San Juan Island, USA EU604729 EU604711 EU604733 –
M. diomedea (Bergh,
1893)
Melanochlamys diome-
dea (Bergh, 1893)
LACM7320 Cohen Island, Alaska, USA JN825118 – – JN825042
M. diomedea (Bergh,
1893)
Melanochlamys ezoensis
(Baba, 1957)
ZMBN95934 Peter the Great Bay, Russia MF036363 – MF036464 MF036623
M. diomedea (Bergh,
1893)
Melanochlamys ezoensis
(Baba, 1957)
ZMBN95968 Peter the Great Bay, Russia MF036364 – MF036465 MF036624
M. fukudai (Cooke et
al. 2014)
Melanochlamys fukudai
(Cooke et al. 2014)
CPIC00905 Hokkaido, Japan KJ704889 – KJ704930 KJ704974
M. fukudai (Cooke et
al. 2014)
Melanochlamys fukudai
(Cooke et al. 2014)
CPIC00777.D312 Souma, Fukushima, Japan KJ704868 KJ704906 – KJ704953
M. fukudai (Cooke et
al. 2014)
Melanochlamys fukudai
(Cooke et al. 2014)
CPIC00860.SC132 Miura, Kanagawa, Japan KJ704871 KJ704911 – KJ704955
M. fukudai (Cooke et
al. 2014)
Melanochlamys fukudai
(Cooke et al. 2014)
CPIC00859.SC145 Kisarazu, Japan KJ704883 KJ704924 – KJ704968
M. kohi (Cooke et al.
2014)
Melanochlamys kohi
(Cooke et al. 2014)
LACM3266 Wando Island, South Korea KJ704890 KJ704931 – KJ704975
M. kohi (Cooke et al.
2014)
Melanochlamys kohi
(Cooke et al. 2014)
CPIC00792.D285 Shiohama, Ichikawa, Japan KJ704892 KJ704933 – KJ704977
M. ezoensis (Bergh,
1893)
Melanochlamys ezoensis
(Baba, 1957)
CASIZ158983 San Francisco, California,
USA
JN825148 – – JN825041
M. ezoensis (Bergh,
1893)
Melanochlamys ezoensis
(Baba, 1957)
CPIC00771 San Francisco, California,
USA
KJ704858 – – KJ704942
M. ezoensis (Bergh,
1893)
Melanochlamys ezoensis
(Baba, 1957)
CPIC0097.SC164 Hokkaido, Japan KJ704859 KJ704899 – KJ704943
M. ezoensis (Bergh,
1893)
Melanochlamys ezoensis
(Baba, 1957)
CPIC 00906.SC163 Peter the Great Bay, Russia KJ704867 – – KJ704951
M. ezoensis (Bergh,
1893)
Melanochlamys ezoensis
(Baba, 1957)
CMNH-ZM04529 Katsuura, Japan – – – KJ704952
M. lorrainae
(Rudman, 1968)
Melanochlamys lorrainae
(Rudman, 1968)
O6NZ1 Northland, New Zealand – EU604707 – –
Table 1. Continued
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MOLECULAR PHYLOGENY OF THE AGLAJIDAE SEA SLUGS 15
© 2017 The Linnean Society of London, Zoological Journal of the Linnean Society, 2017, XX, 1–20
Taxa Revised identification Voucher number Collection site GenBank accession numbers
16S rRNA COI 28S rRNA H3
M. lorrainae
(Rudman, 1968)
Melanochlamys lorrainae
(Rudman, 1968)
O6NZ2 Northland, New Zealand – EU604708 – –
M. lorrainae
(Rudman, 1968)
Melanochlamys lorrainae
(Rudman, 1968)
O6NZ3 Northland, New Zealand EU604727 EU604709 – –
M. lorrainae
(Rudman, 1968)
Melanochlamys lorrainae
(Rudman, 1968)
O6NZ4 Northland, New Zealand EU604728 EU604710 EU604737 –
Genus Nakamigawaia Kuroda & Habe, 1961
Nakamigawaia sp. Nakamigawaia spiralis
Kuroda & Habe, 1961
ZMBM94029 Lizard Island, NE Australia MF036369 – MF036469 MF036628
Nakamigawaia sp. Nakamigawaia spiralis
Kuroda & Habe, 1961
ZMBN95960 Lizard Island, NE Australia MF036371 MF036537 MF036471 –
N. felis (Er. Marcus &
Ev. Marcus, 1970)
Nakamigawaia spiralis
Kuroda & Habe, 1961*
CASIZ167971 Duperre Islets, Papua New
Guinea
– JN825176 – JN825044
N. felis (Er. Marcus &
Ev. Marcus, 1970)
Nakamigawaia sp.* CASIZ173495 Bohol Island, Panglao, The
Philippines
JN825119 JN825177 – JN825045
N. felis (Er. Marcus &
Ev. Marcus, 1970)
Nakamigawaia felis (Er.
Marcus & Ev. Marcus,
1970)
CASIZ175653 Great Exuma, Stocking
Island, The Bahamas
JN825120 JN825178 – JN825046
N. felis (Er. Marcus &
Ev. Marcus, 1970)
Nakamigawaia felis (Er.
Marcus & Ev. Marcus,
1970)
CASIZ175655 Great Exuma, Stocking
Island, The Bahamas
JN825121 JN825179 – JN825047
N. spiralis Kuroda &
Habe, 1961
Nakamigawaia spiralis
Kuroda & Habe, 1961
ZMBN95949 Lizard Island, NE Australia MF036370 MF036536 MF036470 MF036626
Nakamigawaia cf.
spiralis Kuroda &
Habe, 1961
Nakamigawaia spiralis
Kuroda & Habe, 1961
ZMBN95972 Lizard Island, NE Australia MF036372 – MF036472 MF036629
Genus Navanax Pilsbry, 1895
N. aenigmaticus
(Bergh, 1893)
Navanax aenigmaticus
(Bergh, 1893)
ZMBN95938 El Salvador MF036374 MF036538 MF036474 MF036631
N. aenigmaticus
(Bergh, 1893)
Navanax gemmatum
(Mörch, 1863)
ZMBN96009 Cuba MF036383 MF036542 MF036482 MF036638
N. aenigmaticus
(Bergh, 1893)
Navanax gemmatum
(Mörch, 1863)
ZMBN96008 Cuba MF036382 MF036541 MF036481 MF036637
N. aenigmaticus
(Bergh, 1893)
Navanax aenigmaticus
(Bergh, 1893)
CNMO3221 El Faro, Michoacán, México MF036379 – MF036479 MF036635
N. aenigmaticus
(Bergh, 1893)
Navanax aenigmaticus
(Bergh, 1893)
CNMO3474 Manzanillo, Colima, México MF036377 MF036539 MF036477 MF036633
Table 1. Continued
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16 A. ZAMORA-SILVA AND M. A. E. MALAQUIAS
© 2017 The Linnean Society of London, Zoological Journal of the Linnean Society, 2017, XX, 1–20
Taxa Revised identification Voucher number Collection site GenBank accession numbers
16S rRNA COI 28S rRNA H3
N. aenigmaticus
(Bergh, 1893)
Navanax gemmatum
(Mörch, 1863)
CNMO2962 Veracruz, México MF036385 MF036545 MF036483 MF036641
N. aenigmaticus
(Bergh, 1893)
Navanax aenigmaticus
(Bergh, 1893)
ZSMMol20110048 Laguna Grande, Perú JN402135 JN402041 – JN402071
N. aenigmaticus
(Bergh, 1893)
Navanax aenigmaticus
(Bergh, 1893)
ZSMMol20100036 Juan López, Chile JN402132 JN402039 – JN402073
N. aenigmaticus
(Bergh, 1893)
Navanax aenigmaticus
(Bergh, 1893)
ZSMMol20100037 Juan López, Chile JN402122 JN402034 – JN402079
N. aenigmaticus
(Bergh, 1893)
Navanax aenigmaticus
(Bergh, 1893)
ZSMMol20110046 Mancora, Perú JN402129 JN402032 – JN402080
N. aenigmaticus
(Bergh, 1893)
Navanax aenigmaticus
(Bergh, 1893)
ZSMMol20110051 Juan López, Chile JN402127 JN402031 – JN402082
N. aenigmaticus
(Bergh, 1893)
Navanax aenigmaticus
(Bergh, 1893)
ZSMMol20110019 Laguna Grande, Perú JN402126 JN402029 – JN402084
N. aenigmaticus
(Bergh, 1893)
Navanax aenigmaticus
(Bergh, 1893)
ZSMMol20100753 Laguna Grande, Perú JN402133 JN402027 – JN402087
N. aenigmaticus
(Bergh, 1893)
Navanax aenigmaticus
(Bergh, 1893)
LACM19726844 Puntarenas, Costa Rica – JN402065 – JN402088
N. aenigmaticus
(Bergh, 1893)
Navanax aenigmaticus
(Bergh, 1893)
LACM153304 Canal de Jicarón, Panama JN402139 JN402064 – JN402092
N. aenigmaticus
(Bergh, 1893)
Navanax aenigmaticus
(Bergh, 1893)
LACM176390 Baja California Sur, México JN402142 JN402061 – JN402093
N. aenigmaticus
(Bergh, 1893)
Navanax aenigmaticus
(Bergh, 1893)
LACM176393 Islas Marietas, Jalisco,
México
JN402146 JN402058 – JN402099
N. aenigmaticus
(Bergh, 1893)
Navanax aenigmaticus
(Bergh, 1893)
LACM3418910 Galapagos Islands, Ecuador – – – JN402101
N. aenigmaticus
(Bergh, 1893)
Navanax aenigmaticus
(Bergh, 1893)
CASIZ073370 Isla Cocos, Costa Rica – – – JN402103
N. aenigmaticus
(Bergh, 1893)
Navanax aenigmaticus
(Bergh, 1893)
CASIZ066862 San Juan del Sur,
Nicaragua
– – – JN402104
N. aenigmaticus
(Bergh, 1893)
Navanax aenigmaticus
(Bergh, 1893)
CASIZ175758 Isla Uva, Panama JN402152 JN402050 – JN402111
N. aenigmaticus
(Bergh, 1893)
Navanax aenigmaticus
(Bergh, 1893)
MZUCR6923 Playa Sámara, Costa Rica JN402148 JN402053 – JN402114
N. aenigmaticus
(Bergh, 1893)
Navanax aenigmaticus
(Bergh, 1893)
MZUCR6927 Playa Ballena, Costa Rica JN402150 JN402054 – JN402115
Table 1. Continued
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MOLECULAR PHYLOGENY OF THE AGLAJIDAE SEA SLUGS 17
© 2017 The Linnean Society of London, Zoological Journal of the Linnean Society, 2017, XX, 1–20
Taxa Revised identification Voucher number Collection site GenBank accession numbers
16S rRNA COI 28S rRNA H3
N. aenigmaticus
(Bergh, 1893)
Navanax aenigmaticus
(Bergh, 1893)
LACM176391 Manzanillo, Colima, México JN402143 JN402060 – JN402116
N. aenigmaticus
(Bergh, 1893)
Navanax aenigmaticus
(Bergh, 1893)
LACM176392 Manzanillo, Colima, México JN402144 JN402059 – JN402117
N. aenigmaticus
(Bergh, 1893)
Navanax aenigmaticus
(Bergh, 1893)
MZUCR6213 Isla Uva, Panama JN402149 JN402051 – –
N. gemmatum (Mörch,
1863)
Navanax gemmatum
(Mörch, 1863)
ZSMMol20100505 São Paulo, Brazil JN402124 JN402042 – JN402070
N. gemmatum (Mörch,
1863)
Navanax gemmatum
(Mörch, 1863)
ZSMMol20100506 São Paulo, Brazil JN402130 JN402043 – JN402069
N. gemmatum (Mörch,
1863)
Navanax gemmatum
(Mörch, 1863)
ZSMMol20090614 Pernambuco, Brazil JN402125 JN402026 – JN402086
N. gemmatum (Mörch,
1863)
Navanax gemmatum
(Mörch, 1863)
LACM173263 St. Anne´s Bay, Jamaica JN402136 JN402067 – JN402089
N. gemmatum (Mörch,
1863)
Navanax gemmatum
(Mörch, 1863)
LACM2004948 Cucaçao, Netherdlands
Antilles
– – – JN402096
N. gemmatum (Mörch,
1863)
Navanax gemmatum
(Mörch, 1863)
CASIZ087317 The Bahamas – – – JN402102
N. gemmatum (Mörch,
1863)
Navanax gemmatum
(Mörch, 1863)
LACM176389 Peanut Island, Florida,
USA
JN402153 JN402056 – JN402105
N. gemmatum (Mörch,
1863)
Navanax gemmatum
(Mörch, 1863)
LACM173262 St. Anne’s Bay, Jamaica JN402137 JN402055 – JN402106
N. gemmatum (Mörch,
1863)
Navanax gemmatum
(Mörch, 1863)
CASIZ175767 Punta Cahuita, Costa Rica JN402151 JN402046 – JN402107
N. gemmatum (Mörch,
1863)
Navanax gemmatum
(Mörch, 1863)
Isolate Naen390 Punta Cahuita, Costa Rica JN402156 JN402047 – JN402108
N. inermis (J.
G. Cooper, 1862)
Navanax inermis (J.
G. Cooper, 1862)
ZMBN95952 Manzanillo, Colima, México MF036388 MF036546 MF036484 MF036643
N. inermis (J.
G. Cooper, 1862)
Navanax aenigmaticus
(Bergh, 1893)
CNMO3461 Manzanillo, Colima, México MF036376 – MF036476 MF036632
N. inermis (J.
G. Cooper, 1862)
Navanax inermis (J.
G. Cooper, 1862)
LACM176388 Long Beach, California,
USA
JN402154 JN402045 – JN402119
N. inermis (J.
G. Cooper, 1862)
Navanax inermis (J.
G. Cooper, 1862)
Isolate E21 La Paz, Baja California,
México
AM421855 AM421877 – –
N. inermis (J.
G. Cooper, 1862)
Navanax inermis (J.
G. Cooper, 1862)
– Gulf of California, México – – AY427472 –
Table 1. Continued
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18 A. ZAMORA-SILVA AND M. A. E. MALAQUIAS
© 2017 The Linnean Society of London, Zoological Journal of the Linnean Society, 2017, XX, 1–20
Taxa Revised identification Voucher number Collection site GenBank accession numbers
16S rRNA COI 28S rRNA H3
N. nyalnya (C. nyan-
yana Edmunds,
1968)
Navanax orbignyanus
(Rochebrune, 1881)
ZSMMol20070253 Miamia, Ghana – JN402038 – JN402074
N. nyalnya (C. nyan-
yana Edmunds,
1968)
Navanax orbignyanus
(Rochebrune, 1881)
ZSMMol20070241 Miamia, Ghana JN402121 JN402040 – –
N. nyalnya (C. nyan-
yana Edmunds,
1968)
Navanax orbignyanus
(Rochebrune, 1881)
LACM153125 Cape Verde JN402138 JN402066 – JN402090
N. polyalphos
(Gosliner &
Williams, 1972)
Navanax polyalphos
(Gosliner & Williams,
1972)
CPIC00686 Long Beach, California,
USA
JN825079 JN825182 – JN825050
N. polyalphos
(Gosliner &
Williams, 1972)
Navanax polyalphos
(Gosliner & Williams,
1972)
LACMA8477 Bahía de San Carlos, Sea of
Cortes, México
– – – JN825051
Genus Odontoglaja Rudman, 1978
Odontoglaja sp. Odontoglaja guamensis
Rudman, 1978*
43 MAEM-2006 Kalakajoro, Madagascar – DQ974655 DQ927218 –
O. guamensis
Rudman, 1978
Odontoglaja guamensis
Rudman, 1978
ZMBN95963 Kosrae Island, Micronesia MF036395 – MF036491 MF036652
O. guamensis
Rudman, 1978
Odontoglaja guamensis
Rudman, 1978
ZMBN95961 Bile Bay, Guam MF036394 – MF036490 MF036650
O. guamensis
Rudman, 1978
Odontoglaja guamensis
Rudman, 1978
Isolate E23 Orpheus Island, NE
Australia
AM421830 AM421869 AY427471 –
O. guamensis
Rudman, 1978
Odontoglaja guamensis
Rudman, 1978
CASIZ175764 Luzon Island, Batangas,
The Philippines
JN825124 JN825183 – JN825053
O. guamensis
Rudman, 1978
Odontoglaja guamensis
Rudman, 1978
CASIZ175944 Pineapple Point, Malaysia JN825125 – – JN825054
O. guamensis
Rudman, 1978
Odontoglaja guamensis
Rudman, 1978
CPIC00688 Merizo, Guam, USA JN825127 JN825185 – JN825056
O. guamensis
Rudman, 1978
Odontoglaja guamensis
Rudman, 1978
CPIC00687 Merizo, Guam, USA JN825126 JN825184 – JN825055
O. mosaica Gosliner,
2011
Odontoglaja mosaica
Gosliner, 2011
CASIZ175943 Iles Redama, Nosy
Kalakajoro, Madagascar
– – – JN825052
O. sabadiega (Ortea,
Moro & Espinosa,
1997)
Mannesia sabadiega
(Ortea, Moro &
Espinosa, 1997)
ZMBM94030 Las Cruces, Tenerife, Spain – MF036532 – MF036614
Table 1. Continued
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MOLECULAR PHYLOGENY OF THE AGLAJIDAE SEA SLUGS 19
© 2017 The Linnean Society of London, Zoological Journal of the Linnean Society, 2017, XX, 1–20
Taxa Revised identification Voucher number Collection site GenBank accession numbers
16S rRNA COI 28S rRNA H3
O. sabadiega (Ortea,
Moro & Espinosa,
1997)
Mannesia sabadiega
(Ortea, Moro &
Espinosa, 1997)
ZMBN96000 Madeira, Portugal MF036353 MF036531 MF036452 MF036613
O. sabadiega (Ortea,
Moro & Espinosa,
1997)
Mannesia sabadiega
(Ortea, Moro &
Espinosa, 1997)
ZMBN81686 Falial, Portugal MF036352 – – –
Genus Philinopsis Pease, 1860
Philinopsis sp.
(as P. anneae in
Ornelas-
Gadtula & Valdés,
2012)
Spinoaglaja petra (Ev.
Marcus, 1976)*
LACM3226 Sand Dollar Beach,
Stocking I.
JX188006 JX188012 – JX188015
Philinopsis sp.
(as P. anneae in
Ornelas-
Gadtula & Valdés,
2012)
Spinoaglaja petra (Ev.
Marcus, 1976)*
LACM3225 Sand Dollar Beach,
Stocking I.
– JX188011 – –
P. coronata Gosliner,
2011
Spinophallus coronata
(Gosliner, 2011)*
CASIZ182887 Luzon, Batangas, The
Philippines
JN825130 JN825188 – JN825058
P. cf. ctenophoraphaga
Gosliner, 2011
Philinopsis cte-
nophoraphaga
Gosliner, 2011
CASIZ177788 Ambil Island, The
Philippines
– JN825204 – –
P. cyanea (Martens,
1879)
Philinopsis speciosa
Pease, 1860
ZMBN95951 Lizard Island, NE Australia MF036401 – MF036497 MF036658
P. cyanea (Martens,
1879)
Philinopsis speciosa
Pease, 1860
ZMBN95997 Kyoda Beach, Okinawa,
Japan
MF036404 MF036558 – MF036661
P. cyanea (Martens,
1879)
Philinopsis speciosa
Pease, 1860*
Isolate 71 Lizard Island, NE Australia AM421833 AM421891 AM421953 –
P. cyanea (Martens,
1879)
Philinopsis speciosa
Pease, 1860*
Isolate 239 Lizard Island, NE Australia AM421832 AM421890 AM421951 –
P. cyanea (Martens,
1879)
Philinopsis speciosa
Pease, 1860*
CASIZ097543 Isabela Island, Tagus,
Galapagos, Ecuador
JN825131 JN825189 – JN825059
P. cyanea (Martens,
1879)
Philinopsis speciosa
Pease, 1860*
Isolate E25 Palau AM421834 AM421889 AM421952 –
P. depicta (Renier,
1807)
Philinopsis depicta
(Renier, 1807)
ZMBM94031 Palmachim, Israel MF036396 – MF036492 MF036653
P. depicta (Renier,
1807)
Philinopsis depicta
(Renier, 1807)
Isolate E17 Mediterranean, Spain AM421831 AM421892 AM421954 –
Table 1. Continued
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20 A. ZAMORA-SILVA AND M. A. E. MALAQUIAS
© 2017 The Linnean Society of London, Zoological Journal of the Linnean Society, 2017, XX, 1–20
Taxa Revised identification Voucher number Collection site GenBank accession numbers
16S rRNA COI 28S rRNA H3
P. depicta (Renier,
1807)
Philinopsis depicta
(Renier, 1807)
MNCN1505/17675 Spain JN825132 – – –
P. falciphallus
Gosliner, 2011
Spinophallus falciphal-
lus (Gosliner, 2011)
E518 Kwajalein Atoll, Marshal
Islands
MF036410 MF036562 MF036501 MF036665
P. falciphallus
Gosliner, 2011
Spinophallus falciphal-
lus (Gosliner, 2011)*
CASIZ177678 Luzon, Batangas, The
Philippines
JN825133 – – JN825060
P. falciphallus
Gosliner, 2011
Spinophallus falciphal-
lus (Gosliner, 2011)*
CASIZ177758 Luzon, Batangas, The
Philippines
JN825134 – – –
P. gardineri (Eliot,
1903)
Tubulophilinopsis gar-
dineri (Eliot, 1903)
ZMBN95944 Lizard Island, NE Australia MF036412 MF036564 MF036503 MF036667
P. gardineri (Eliot,
1903)
Tubulophilinopsis gar-
dineri (Eliot, 1903)
ZMBN95943 Lizard Island, NE Australia – – MF036502 MF036666
P. gardineri (Eliot,
1903)
Tubulophilinopsis gar-
dineri (Eliot, 1903)
EO95 Kwajalein Atoll, Mashall
Islands
MF036413 MF036563 MF036505 –
P. gardineri (Eliot,
1903)
Tubulophilinopsis gar-
dineri (Eliot, 1903)*
Isolate 168 Lizard Island, NE Australia AM421837 AM421887 AM421957 –
P. gardineri (Eliot,
1903)
Tubulophilinopsis gar-
dineri (Eliot, 1903)*
CASIZ167960 Yasawa Group, Sawa-
I-Lau, Fiji Islands
JN825135 JN825190 – JN825061
P. gardineri (Eliot,
1903)
Tubulophilinopsis gar-
dineri (Eliot, 1903)*
CPIC00689 Merizo, Guam, USA JN825136 JN825191 – JN825063
P. gardineri (Eliot,
1903)
Philinopsis speciosa
Pease, 1860
ZMBN95993 Kyoda Beach, Okinawa,
Japan
MF036402 – – MF036659
P. gigliolii
(Taparrone–
Canefri, 1874)
Philinopsis speciosa
Pease, 1860
ZMBN95995 Kyoda Beach, Okinawa,
Japan
MF036403 MF036557 MF036434 MF036660
P. lineolata (H. Adams
& A. Adams, 1854)
Tubulophilinopsis pils-
bry (Eliot, 1900)
ZMBN95946 Lizard Island, NE Australia – – MF036508 MF036669
P. lineolata (H. Adams
& A. Adams, 1854)
Tubulophilinopsis
lineolata (H. Adams &
A. Adams, 1854)
ZMBN95941 Lizard Island, NE Australia MF036415 MF036565 MF036507 –
P. lineolata (H. Adams
& A. Adams, 1854)
Tubulophilinopsis
lineolata (H. Adams &
A. Adams, 1854)
ZMBN95940 Lizard Island, NE Australia MF036414 – MF036506 MF036668
P. lineolata (H. Adams
& A. Adams, 1854)
Tubulophilinopsis
lineolata (H. Adams &
A. Adams, 1854)*
Isolate 165 Lizard Island, NE Australia AM421839 AM421884 AM421958 –
Table 1. Continued
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MOLECULAR PHYLOGENY OF THE AGLAJIDAE SEA SLUGS 21
© 2017 The Linnean Society of London, Zoological Journal of the Linnean Society, 2017, XX, 1–20
Taxa Revised identification Voucher number Collection site GenBank accession numbers
16S rRNA COI 28S rRNA H3
P. lineolata (H. Adams
& A. Adams, 1854)
Tubulophilinopsis
lineolata (H. Adams &
A. Adams, 1854)*
Isolate 123 Lizard Island, NE Australia AM421838 AM421883 – –
P. miqueli Pelorce,
Horst & Hoarau,
2013
Melanochlamys miqueli
(Pelorce, Horst &
Hoarau, 2013)*
CPIC00903 La Ciotat, Calanque du
Mugel, France
KJ704851 KJ704896 KJ704937
P. pilsbryi (Eliot,
1900)
Tubulophilinopsis pils-
bryi (Eliot, 1900)
ZMBN95948 Lizard Island, NE Australia MF036416 MF036569 MF036509 MF036670
P. pilsbryi (Eliot,
1900)
Tubulophilinopsis pils-
bryi (Eliot, 1900)
E455a Kwajalein Atoll, Marshall
Islands
MF036418 MF036567 MF036511 MF036671
P. pilsbryi (Eliot,
1900)
Tubulophilinopsis pils-
bryi (Eliot, 1900)
E182 Kwajalein Atoll, Marshall
Islands
MF036417 MF036566 MF036510 MF036672
P. pilsbryi (Eliot,
1900)
Tubulophilinopsis pils-
bryi (Eliot, 1900)
E455b Kwajalein Atoll, Marshall
Islands
MF036419 MF036568 MF036512 –
P. pilsbryi (Eliot,
1900)
Tubulophilinopsis pils-
bryi (Eliot, 1900)*
CASIZ157033 Balayan Bay, Batangas, The
Philippines
JN825140 JN825194 – JN825067
P. pilsbryi (Eliot,
1900)
Tubulophilinopsis pils-
bryi (Eliot, 1900)*
CASIZ178483 Espiritu Santo Island,
Vanuatu
JN825142 JN825196 – JN825069
P. pilsbryi (Eliot,
1900)
Tubulophilinopsis pils-
bryi (Eliot, 1900)*
Isolate 167 Lizard Island, NE Australia AM421840 – AM421956 –
P. pilsbryi (Eliot,
1900)
Tubulophilinopsis pils-
bryi (Eliot, 1900)*
259620 The Philippines – – AY427474 –
P. pilsbryi (Eliot,
1900)
Tubulophilinopsis pils-
bryi (Eliot, 1900)*
Isolate E8 Lizard Island, NE Australia – – AM421955 –
P. pusa (Ev. Marcus &
Er. Marcus, 1966)
Philinopsis pusa (Ev.
Marcus & Er. Marcus,
1966)
ZMBN95958 Great Exuma, Stocking
Island, The Bahamas
– MF036552 MF036493 MF036654
P. pusa (Ev. Marcus &
Er. Marcus, 1966)
Philinopsis pusa (Ev.
Marcus & Er. Marcus,
1966)
LACM173220 Great Exuma, Stocking
Island, The Bahamas
JN825144 JN825199 – JN825072
P. pusa (Ev. Marcus &
Er. Marcus, 1966)
Philinopsis pusa (Ev.
Marcus & Er. Marcus,
1966)
LACM173221 Great Exuma, Stocking
Island, The Bahamas
JN825145 JN825200 – JN825073
P. reticulata (Eliot,
1903)
Tubulophilinopsis reticu-
lata (Eliot, 1903)*
Isolate 166 Lizard Island, NE Australia AM421836 AM421888 AM421960 –
P. reticulata (Eliot,
1903)
Tubulophilinopsis reticu-
lata (Eliot, 1903)
ZMBN95947 Lizard Island, NE Australia MF036420 – MF036514 MF036674
Table 1. Continued
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22 A. ZAMORA-SILVA AND M. A. E. MALAQUIAS
© 2017 The Linnean Society of London, Zoological Journal of the Linnean Society, 2017, XX, 1–20
Taxa Revised identification Voucher number Collection site GenBank accession numbers
16S rRNA COI 28S rRNA H3
P. reticulata (Eliot,
1903)
Tubulophilinopsis reticu-
lata (Eliot, 1903)
E454a Kwajalein Atoll, Marshall
Islands
MF036421 MF036571 MF036516 MF036675
P. reticulata (Eliot,
1903)
Tubulophilinopsis reticu-
lata (Eliot, 1903)
E454c Kwajalein Atoll, Marshall
Islands
MF036423 MF036573 MF036515 MF036676
P. reticulata (Eliot,
1903)
Tubulophilinopsis reticu-
lata (Eliot, 1903)
E454b Kwajalein Atoll, Marshall
Islands
MF036422 MF036572 – –
P. reticulata (Eliot,
1903)
Tubulophilinopsis reticu-
lata (Eliot, 1903)*
Isolate 128 Lizard Island, NE Australia AM421835 AM421885 – –
P. reticulata (Eliot,
1903)
Tubulophilinopsis reticu-
lata (Eliot, 1903)*
AM421886 Lizard Island, NE Australia – AM421886 – –
P. speciosa Pease,
1860
Philinopsis speciosa
Pease, 1860
ZMBN95967 Black Rock, Maui, Hawaii MF036400 MF036555 MF036496 MF036657
P. speciosa Pease,
1860
Philinopsis speciosa
Pease, 1860
ZMBM81665 Bile Bay, Guam, USA MF036399 MF036554 MF036495 MF036656
P. taronga (Allan,
1933)
Philinopsis taronga
(Allan, 1933)
K02 Blairgowrie Marina,
Victoria, Australia
MF036405 – – –
Genus Spinoaglaja Ortea, Moro & Espinosa, 2007
S. petra (Ev. Marcus,
1976)
Spinoaglaja petra (Ev.
Marcus, 1976)
ZMBM83019 Little Head Park, St. Davis,
Bermuda
MF036408 MF036560 MF036499 –
S. petra (Ev. Marcus,
1976)
Spinoaglaja petra (Ev.
Marcus, 1976)*
LACM172268 Great Exuma, Stocking
Island, Bahamas
JN825137 – – JN825064
S. petra (Ev. Marcus,
1976)
Spinoaglaja petra (Ev.
Marcus, 1976)*
LACM172269 Great Exuma, Stocking
Island, Bahamas
JN825138 JN825192 – JN825065
S. petra (Ev. Marcus,
1976)
Spinoaglaja petra (Ev.
Marcus, 1976)
ZMBM83022 Ferry Reach, Bermuda MF036409 MF036561 MF036500 MF036664
S. orientalis (Baba,
1949)
Spinoaglaja orientalis
(Baba, 1949)*
CASIZ121169 Kwajalein Atoll, Marshall
Islands
JN825081 – – –
S. orientalis (Baba,
1949)
Spinoaglaja orientalis
(Baba, 1949)*
CASIZ177703 Maricaban Island,
Batangas, The
Philippines
JN825082 – – JN825002
S. orientalis Baba,
1949
Spinoaglaja orientalis
(Baba, 1949)
ZMBN95999 Manza Beach, Okinawa,
Japan
MF036407 MF036559 – MF036663
OUTGROUPS
Genus Diaphana T. Brow, 1827
D. globosa (Lovén,
1846)
ZMBN88018 Hauglandsosen, Norway KJ022791 KF992162 KJ023056 KJ022930
Table 1. Continued
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MOLECULAR PHYLOGENY OF THE AGLAJIDAE SEA SLUGS 23
© 2017 The Linnean Society of London, Zoological Journal of the Linnean Society, 2017, XX, 1–20
Taxa Revised identification Voucher number Collection site GenBank accession numbers
16S rRNA COI 28S rRNA H3
Genus Haminoea Turton & Kingston in Carrington, 1830
H. elegans (Gray,
1825)
BMNH20070180 Florida, USA – KF615829 KF615797 –
H. navicula (da Costa,
1778)
BMNH20070020 Aveiro, Portugal EU314806 KF615837 KF615805 KF615803
Genus Phanerophtalmus A. Adams, 1850
Phanerophtalmus sp. BMNH20050661 Sulawesi, Indonesia KJ022784 KF992160 DQ927241 KJ022938
P. cylindricus (Pease,
1861)
ZMBN81693 Maui, Hawaii KJ022819 KF992189 KJ023030 KJJ022900
Genus Sagaminopteron Tokioka & Baba, 1964
S. psychedelicum
Carlson & Hoff,
1974
CAS-Cephas3 Kalakajoro, Madagascar KJ022787 DQ974667 DQ927225 KJ022934
Genus Siphopteron Gosliner, 1989
S. tigrinum Gosliner,
1989
CAS-Cephas4 Kalakajoro, Madagascar KJ022788 DQ974668 DQ927226 KJ022933
Genus Philine Ascanius, 1772
P. babai Valdés, 2008 IM–2009–4352 Bohol Sea, The Philippines KJ022854 KF877702 KJ022989 KJ022968
P. quadrata (S. Wood,
1839)
ZMBN88012 Off Lofoten, Norway KJ022793 JX944809 KJ023010 KJ022952
Taxonomic names of GenBank sequences that were modified are marked with an asterisk.
Table 1. Continued
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24 A. ZAMORA-SILVA AND M. A. E. MALAQUIAS
© 2017 The Linnean Society of London, Zoological Journal of the Linnean Society, 2017, XX, 1–20
16S and 28S genes and excluded using Gblocks 0.91b
(Castresana, 2000) for relaxed and stringent settings
under default parameters (Talavera & Castresana,
2007; Kück et al., 2010).
In total, 11 data sets were assembled for phylo-
genetic analyses: individual-gene COI and H3 with
and without third codon positions, individual-gene
16S and 28S initial alignments, 16S and 28S under
relaxed and stringent Gblocks settings, and an all-
genes concatenated data set based on the individual-
gene alignments that yielded the best resolved/higher
posterior probability (PP) value trees. Concatenated
alignments consisted only of samples represented by
the four genes. The best-fit model of evolution for each
of the 11 alignments was chosen using the Akaike
information criterion (Akaike, 1974) implemented in
JModelTest 2.1.4 (Posada & Crandall, 1998; Darriba
et al., 2012; see Table 3). Bayesian inference (BI) anal-
yses were performed in MrBayes 3.1.2b (Ronquist &
Huelsenbeck, 2003), with three parallel runs of 15
million generations for each individual-gene data
set and 20 million generations for the all-genes
Table 3. Summary of the best-fit evolutionary models and parameters estimated in JModeltest
Parameters 16S rRNA COI 28S rRNA Histone-3 All-genes
combined
Data set Relaxed
outcome
3rd codon position
included
All positions
included
3rd codon position
included
16S + COI + 28S
+ H3
Alignment length 403 bp 579 bp 1467 bp 269 bp 2718 bp
Number of sequences 254 195 132 226 331
Best-fit model TVM + I + G TVM + I + G GTR + I + G TrN + G –
Frequency A 0.34 0.47 0.1713 0.23 0.3267
Frequency C 0.11 0.12 0.2895 0.36 0.2064
Frequency G 0.17 0.13 0.3611 0.23 0.2275
Frequency T 0.38 0.28 0.1781 0.19 0.2394
Gamma shape parameter
(G)
0.45 0.48 0.5190 0.25 0.5730
Proportion of invariant
sites (I)
0.21 0.48 0.5040 – 0.5910
R-matrix (A–C) 1.14 9.09 0.8045 1.77 1.0666
R-matrix (A–G) 6.45 122.73 1.9349 2.79 7.4741
R-matrix (A–T) 2.17 7.51 1.5421 1.23 3.1684
R-matrix (C–G) 1.18 10.82 0.6187 1.29 1.5107
R-matrix (C–T) 6.45 122.73 7.2434 4.97 10.1621
R-matrix (G–T) 1.00 1.00 1.0000 1.00 1.0000
Table 2. Forward (F) and reverse (R) primers used in this study
Gene Primer name Sequences 5′ 3′Source
16S rRNA 16S ar-L CGCCTGTTTATCAAAAACAT Palumbi et al. (1991)
16S br-R CCGGTCTGAACTCAGATCACGT Palumbi et al. (1991)
Cytochrome
c oxidase
subunit I
(COI)
COI LCO (F) GGTCAACAAATCATAAAGATATTGG Folmer et al. (1994)
HCO 2198 (R) TAAACTTCAGGGTGACCAAAAATCA Folmer et al. (1994)
28S rRNA 28S LSU5 (F) TAGGTCGACCCGCTGAAYTTAAGCA Littlewood, Curini-Galletti &
Herniou (2000)
28S 900 (F) CCGTCTTGAAACACGGACCAAG Olson et al. (2003)
28S LSU1600
(R)
AGCGCCATCCATTTTCAGG Williams, Reid & Littlewood (2003)
28S ECD2S (R) CTTGGTCCGTGTTTCAAGACGG Littlewood et al. (2000)
Histone-3 H3AD5′3′ (F) ATG-GCT-CGT-ACC-AAG-CAG-ACV-GC Colgan et al. (1998)
H3BD5′3′ (R) ATA-TCC-TTR-GGC-ATR-ATR-GTG-AC Colgan et al. (1998)
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MOLECULAR PHYLOGENY OF THE AGLAJIDAE SEA SLUGS 25
© 2017 The Linnean Society of London, Zoological Journal of the Linnean Society, 2017, XX, 1–20
concatenated data set with sampling every 100 gen-
erations. Robustness of each node was assessed using
Bayesian PPs in MrBayes and only those with PP
≥ 0.95 were considered supported (Camacho-García
et al., 2014). The ten individual-gene Bayesian trees
were compared by gene and those that produced bet-
ter-resolved topologies with higher PP and maximum
likelihood (ML) values were selected for concatena-
tion. The concatenated data set was analysed parti-
tioned by gene and each partition was run under the
best-fit model of evolution selected by JModelTest.
Convergence of runs was inspected in Tracer v1.5
(Rambaut & Drummond, 2007) with a burn-in set to
25%. Additionally, an ML analysis was performed on
the all-genes combined data set using RAxML v.7.2.6
(Stamatakis, 2006). The ML analysis was run for 20
replicates with a random seed using the GTR + G +
I model with partitions by gene and 1000 bootstrap
replicates. Only clades with bootstrap values (BS) ≥
75 were considered significant (Felsenstein, 1985).
Phylograms were annotated and converted to graph-
ics in FigTree v1.3.1 (Rambaut & Drummond, 2009)
and labelled in Adobe Illustrator.
morphological and scanning elecTron
microscopy
Specimens from various generic lineages recognized
by the molecular analyses in this work were dissected
for structures of systematic relevance discussed along
this study (digestive and male reproductive systems
and shells). Drawings of soft structures were pro-
duced with the aid of a camera lucida, whereas the
shells were mounted on scanning electron microscopy
(SEM) metallic stubs and coated with gold–palladium
for SEM photography. Macrophotography was used for
shells larger than 1 mm.
RESULTS
sequence analyses
The four gene fragments were successfully ampli-
fied for most of the specimens with the primers
listed in Table 2. Unfortunately, no sequences could
be obtained for the only available and formalin-fixed
specimen of the rare genus Noalda. The length of the
final alignments, number of sequences per alignment
and best-fit evolutionary models are summarized in
Table 3.
The data sets selected for concatenation were the
16S-relaxed, 28S-relaxed, and COI and H3 with third
protein codon positions included. All individual-gene
analyses were congruent regarding supported nodes
(PP > 0.95), but in general, the individual-gene data
sets produced poorly resolved trees (Supporting
Information, Figs S1–S4) when compared to the con-
catenated analyses (Figs 1, 2A, B).
monophyly of The family aglajidae and
phylogeneTic relaTionships aT genus level
The monophyly of the family Aglajidae received max-
imum support in both BI and ML analyses (Fig. 2A,
B). Our phylogenetic analyses retrieved eight clades
consistent with the previously established genera
Aglaja, Chelidonura, Melanochlamys, Nakamigawaia,
Navanax, Odontoglaja, Philinopsis and Spinoaglaja;
all including representatives of the type species.
Seven additional clades to which no name has been
proposed before were recognized and are here for-
mally named and described (Biuve, Camachoaglaja,
Mannesia, Mariaglaja, Niparaya, Spinophallus and
Tubulophilinopsis). These 15 clades of generic taxo-
nomic status were supported by both ML and BI
molecular phylogenetics and are in addition character-
ized by unique morpho-anatomical characters (Figs 1,
2A, B; see Systematic descriptions). Only Chelidonura
received a bootstrap lower than the cut-off value here
assumed as statistically significant (BS = 71), but
BI rendered nearly maximum support for the clade
(PP = 0.97) (Fig. 2A, B; Supporting Information, Figs
S1–S4; see Systematic descriptions). Inference of sis-
ter relationships between genera of the Aglajidae was
hampered by a general lack of basal node support
(Fig. 2A, B).
The monophyly of Melanochlamys (including the
type species M. cylindrica) was supported in both BI
and ML analyses (PP/BS = 0.98/76), together with that
of Navanax (PP/BS = 1/100; including the type spe-
cies N. inermis) and Nakamigawaia (PP/BS = 1/100;
including the type species N. spiralis) (Figs 1, 2).
Four genera were rendered not monophyletic (Aglaja,
Chelidonura, Odontoglaja and Philinopsis). Taxa tra-
ditionally assigned to Aglaja and Odontoglaja were
split in two clades each with maximum support, while
species of Chelidonura and Philinopsis were separated
in four distinct clades (Fig. 2A, B).
The true-Chelidonura (including the type species
C. hirundinina from Mauritius) was supported by
BI analysis (PP = 0.97) and nearly supported by ML
analysis (BS = 71). This clade is largely dominated by
IWP species, but includes two species from the WA
(C. cubana and the Caribbean C. ‘hirundinina’). A sec-
ond clade with only IWP (PP/BS = 1/97) is here ascribed
to the new genus Mariaglaja (see Systematic descrip-
tions). A third clade with only eastern Atlantic (EA)
and WA species was retrieved (PP/BS = 1/100) and is
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26 A. ZAMORA-SILVA AND M. A. E. MALAQUIAS
© 2017 The Linnean Society of London, Zoological Journal of the Linnean Society, 2017, XX, 1–20
here ascribed to the new genus Camachoaglaja (see
Systematic descriptions). A fourth smaller clade with
apparently two species, namely C. fulvipunctata and a
sibling restricted to the Hawaiian Is (PP/BS = 1/100), is
here attributed to the new genus Biuve (see Systematic
descriptions) (Fig. 2A, B; Tables 4 and 5).
The radula-bearing genus Odontoglaja was not
monophyletic, with species recovered in two well-sup-
ported separated clades: the true Odontoglaja with
IWP species including the type species O. guamensis
from Guam (PP/BS = 1/100) and the EA species here
ascribed to the new monotypic genus Mannesia (see
Systematic descriptions). The latter was sister to the
new genus Biuve (PP/BS = 0.95/75; Fig. 2A, B).
The genus Aglaja with A. ocelligera and the type spe-
cies A. tricolorata from the Israelian Mediterranean
Sea was monophyletic with maximum support (PP/
BS = 1/100). However, ‘Aglaja’ regiscorona has
branched off alone and is here ascribed to the new
genus Niparaya (see Systematic descriptions). Most
likely, our samples identified as N. regiscorona belong
to two distinct cryptic species (one from Hawaii and
the other from the western Pacific) (Fig. 2A, B).
Species traditionally ascribed to the genus Philinopsis
split in four clades (Fig. 2A, B). The first, Philinopsis-
proper (PP/BS = 1/97) with the type species P. speciosa
contains species from the IWP and WA. A second and
diverse clade contains IWP species only and is here
named Tubolophilinopsis (PP/BS = 1/97). A small clade
with two western Pacific species is here attributed to
the new genus Spinophallus (PP/BS = 0.97/98). The
fourth clade corresponds to the genus Spinoaglaja,
Figure 1. New classification of Aglajidae. Hypothesis based on Bayesian and maximum likelihood analyses of the com-
bined genes cytochrome c oxidase subunit I (COI), 16S rRNA, 28S rRNA and Histone-3. Figures on branches are posterior
probabilities and bootstrap values (PP/BS). Outgroup taxa removed for clarity (see Fig. 2A, B).
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MOLECULAR PHYLOGENY OF THE AGLAJIDAE SEA SLUGS 27
© 2017 The Linnean Society of London, Zoological Journal of the Linnean Society, 2017, XX, 1–20
Figure 2. Continued
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28 A. ZAMORA-SILVA AND M. A. E. MALAQUIAS
© 2017 The Linnean Society of London, Zoological Journal of the Linnean Society, 2017, XX, 1–20
Figure 2. Molecular phylogeny of the family Aglajidae obtained by Bayesian and maximum likelihood analyses based on
the combined genes cytochrome c oxidase subunit I (COI), 16S rRNA, 28S rRNA and Histone-3. Geographical localities
of samples are shown in parentheses after voucher numbers. Sequences of Navanax and Mariaglaja sandrana from the
same locality were removed because of size restrictions. Figures on branches are posterior probabilities and bootstrap
values (PP/BS).
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MOLECULAR PHYLOGENY OF THE AGLAJIDAE SEA SLUGS 29
© 2017 The Linnean Society of London, Zoological Journal of the Linnean Society, 2017, XX, 1–20
Table 4. New systematic classification of the family Aglajidae including all valid genera and species with synonyms
Genus Type species/locality Species
Aglaja Renier, 1807 A. tricolorata Renier, 1807 (*) A. berrieri (Dieuzeide, 1935)
Synonyms: Doridium
Meckel, 1809
Type locality: Near Caesarea,
Mediterranean coast of Israel
Syn: Doridium berrieri Dieuzeide, 1935
Acera Cuvier, 1810 A. ceylonica Bergh, 1900
Bullidium Leue, 1813 Synonyms: A. laurentiana Watson, 1897
Bullula Agassiz, 1846 Doridium tricoloratum Renier, 1807 A. minuta Pruvot-Fol, 1953
Doridium membranaceum Meckel, 1809 A. ocelligera (Bergh, 1893) (*)
Doridium meckelii Delle Chiaje, 1824 Syn: Doridium adellae (Dall, 1894)
Acera meckelii (Delle Chiaje, 1824) Syn: Doridium ocelligerum Bergh, 1893
Acera marmorata Cantraine, 1841 Syn: Chelidonura phocae Marcus, 1961
Doridium tuberculatum Delle Chiaje,
1841
A. unsa Marcus & Er. Marcus, 1967
Aglaja taila Marcus Ev. & Er. Marcus,
1966
Biuve gen. nov. B. fulvipunctata (Baba, 1938) (*)
Type locality: Kii, Japan
Synonyms:
Chelidonura conformata Burn, 1966
Chelidonura mediterranea Swennen,
1961
Chelidonura pervarva (Risbec, 1928)
Camachoaglaja
gen. nov.
C. africana (Pruvot-Fol, 1953) (*) C. berolina Er. (Marcus & Ev. Marcus, 1970) (*)
Type locality: Playa del Hombre, Taliarte,
Tenerife, Canary Islands
Syn: Aglaja hummelincki Er. Marcus & Ev.
Marcus, 1970
Syn: Chelidonura juancarlosi Ortea & Espinosa,
1998 new synonym
Synonyms: C. larramendii (Ortea, Espinosa & Moro, 2009)
Chelidonura italica Sordi, 1980 C. mariagordae (Ortea, Espinosa & Moro,
2004) (*)
Chelidonura leopoldoi Ortea, Moro &
Espinosa, 1997 new synonym
Syn: Chelidonura normani Ornelas-Gadtula,
Dupont & Valdés, 2011
C. pusilla (Ortea, Moro & Espinosa, 2014)
C. quadrata (Ortea, Caballer & Espinosa, 2014)
C. sabina (Er. Marcus & Ev. Marcus, 1970)
Chelidonura
A. Adams, 1850
C. hirundinina (Quoy & Gaimard,
1833) (*)
C. alisonae Gosliner, 2011 (*)
Synonyms:
Hirundinella Gray,
1850
Type locality. Fouquets, Mauritius C. amoena Bergh, 1905 (*)
C. castanea Yonow, 1994
Synonyms: C. cubana Ortea & Martínez, 1997 (*)
Bulla hirundinina Quoy & Gaimard,
1833
C. electra Rudman, 1970 (*)
Hirundinella hirundinina Gray, 1847 C. elegans Bergh, 1900
Chelidonura adamsi Angas, 1867 C. flavolobata Heller & Thompson, 1983 (*)
Chelidonura philinopsis Eliot, 1903 C. livida Yonow, 1994 (*)
C. obscura Bergh, 1901
C. orchidaea Perrone, 1990
C. pallida Risbec, 1951 (*)
C. perparva (Risbec, 1851)
C. plebeia Bergh, 1900
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30 A. ZAMORA-SILVA AND M. A. E. MALAQUIAS
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Table 4. Continued
Genus Type species/locality Species
C. punctata Eliot, 1903 (*)
Syn: Chelidonura hirundinina var. punctata Eliot,
1903
C. sanguinea (Allan, 1933)
C. varians Eliot, 1903 (*)
Syn: Chelidonura velutina (Bergh, 1905)
Syn: Aglaja velutina Bergh, 1908
Mannesia gen. nov. M. sabadiega (Ortea, Moro & Espinosa,
1996) (*)
Type locality: Mar de las Calmas, Isla del
Hierro, Canary Islands
Synonyms:
Chelidonura sabadiega Ortea, Moro &
Espinosa, 1996
Odontoglaja sabadiega (Ortea, Moro &
Espinosa, 2002)
Mariaglaja
gen. nov.
M. alexisi (Gosliner, 2015) (*) M. inornata (Baba, 1949) (*)
Type locality: Mainit Bubbles, Mabini,
Batangas, the Philippines
M. mandroroa (Gosliner, 2011) (*)
M. sandrana (Rudman, 1973) (*)
Syn: Chelidonura babai Gosliner, 1988
Syn: Chelidonura tsurugensis (Baba & Habe,
1959) new synonym
Melanochlamys
Cheeseman, 1881
M. cylindrica Cheeseman, 1881 (*) M. algirae (A. Adams in G.B. Sowerby II, 1850)
Synonyms: Nona
H. Adams &
A. Adams, 1854
Type locality: Tamaki Heads, Auckland
Harbor, New Zealand
Syn: Bulla algirae A. Adams in G. B. Sowerby II,
1850
Syn: Smaragdinella algirae (A. Adams in G.B
Sowerby II, 1850)
Synonyms: Syn: Doridium seurati Vayssière, 1926
Aglaja cylindrica Cheeseman, 1881 Syn: Melanochlamys seurati Vayssière, 1926
M. barryi Gosliner, 1990
M. chabanae Breslau, Valdés &
Chichvarkhin, 2016
M. diomedea (Bergh, 1894) (*)
Syn: Aglaja diomedea Bergh, 1894
Syn: Melanochlamys nana (Steinberg & Jones,
1960)
Syn: Aglaja nana Steinberg & Jones, 1960
M. ezoensis (Baba, 1957) (*)
Syn: Aglaja ezoensis Baba, 1957
M. fukudai (*)
M. handrecki Burn, 2010
M. kohi (*)
M. lorrainae (Rudman, 1968) (*)
M. maderensis (Watson, 1897) (*)
Syn: Doridium maderense Watson, 1897
Syn: Aglaja maderensis (Watson, 1897)
M. miqueli (Pelorce, Horst & Hoarau,
2013) (*) new combination
Syn: Philinopsis miqueli (Pelorce, Horst & Hoarau,
2013) new synonym
M. papillata Gosliner, 1990
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MOLECULAR PHYLOGENY OF THE AGLAJIDAE SEA SLUGS 31
© 2017 The Linnean Society of London, Zoological Journal of the Linnean Society, 2017, XX, 1–20
Genus Type species/locality Species
M. queritor (Burn, 1857)
Syn: Aglaja queritor Burn, 1957
Syn: Aglaja henri Burn, 1969
M. wildpretii Ortea, Bacallado & Moro, 2003
Nakamigawaia
Kuroda & Habe,
1961
N. spiralis Kuroda & Habe, 1961 (*) N. felis (Er. Marcus & Ev. Marcus, 1970) (*)
Type locality: Mukaishima, Japan
Navanax Pilsbry,
1895
Navanax inermis (J. G. Cooper,
1863) (*)
N. aenigmaticus (Bergh, 1893) (*)
Synonyms: Strategus
J. G. Cooper, 1862
Type locality: Cabo San Lucas, Baja
California
Syn: Navarchus aenigmaticus Bergh, 1893
Navarchus J. G.
Cooper, 1863
N. gemmatus (Mörch, 1863) (*)
Posterobranchaea d’
Orbigny, 1835
Synonyms: Syn: Doridium gemmatum Mörch, 1863
Posterobranchia d’
Orbigny, 1835
Posterobranchaea maculata d’ Orbigny,
1835
Syn: Aglaja gemmata (Mörch, 1863)
Posterobranchus d’
Orbigny, 1835
Aglaja maculata (d’Orbigny, 1835) Syn: Aglaja punctiluscens (Bergh, 1893)
Strategus inermis (Cooper, 1863) Syn: Posterobranchus punctilucens Bergh, 1893
Navarchus inermis (Cooper, 1862) Syn: Doridium punctilucens Bergh, 1893
Chelidonura inermis (Cooper, 1862) Syn: Chelidonura evelinae Marcus, 1955
Aglaja inermis (Cooper, 1862) Syn: Chelidonura evelinae dica Marcus & Marcus,
1970
Doridium purpureum (Bergh, 1894) Syn: Aglaja evelinae evelinae (Marcus & Marcus,
1970)
Aglaja bakeri MacFarland, 1924 N. orbignyanus (Rochebrune, 1881) (*)
Syn: Aglaja orbignyana (Rochebrune, 1881)
Syn: Posterobranchus orbygnianus Rochebrune,
1881
Syn: Chelidonura nyanyana Edmunds, 1968
N. polyalphos (Gosliner & Williams, 1972) (*)
Syn: Chelidonura polyalphos Gosliner & Williams,
1972
Niparaya gen. nov. N. regiscorona (Bertsch, 1972) (*)
Type locality: Bahía de las Cruces, Baja
California
Synonyms:
Aglaja regiscorona Bertsch, 1972
Odontoglaja
Rudman, 1978
O. guamensis Rudman, 1978 (*) O. mosaica Gosliner, 2011 (*)
Type locality: Guam
Philinopsis Pease,
1860
P. speciosa Pease, 1860 (*) P. aliciae Gosliner, 2015
Synonyms: Eidothea
Risso, 1826
Type locality: Hawaiian Archipelago? P. bagaensis Ortea, Moro & Espinosa, 2007
P. buntot Gosliner, 2015
Synonyms: P. batabanoensis Ortea, Moro & Espinosa, 2007
Philinopsis gigliolii (Tapparrone –
Canefri, 1874) new synonym
P. ctenophoraphaga Gosliner, 2011 (*)
Aglaja gigliolii Tapparrone – Canefri,
1874 new synonym
P. depicta (Renier, 1807) (*)
Doridium guttatum Martens, 1879 Syn: Aglaja depicta Renier, 1807
Doridium cyaneum Martens, 1879 Syn: Doridium depictum Renier, 1807
Table 4. Continued
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32 A. ZAMORA-SILVA AND M. A. E. MALAQUIAS
© 2017 The Linnean Society of London, Zoological Journal of the Linnean Society, 2017, XX, 1–20
Genus Type species/locality Species
Doridium cyaneum var. vittata Martens,
1879
Syn: Melanochlamys depicta (Renier, 1807)
Philinopsis cyanea (Martens, 1879) Syn: Doridium coriaceum Meckel, 1809
Aglaja cyanea (Martens, 1879) Syn: Acera carnosa Cuvier, 1810
Doridium marmoratum Smith, 1884 Syn: Bulla carnosa Cuvier, 1810
Doridium capensis Bergh, 1907 Syn: Doridium carnosum (Cuvier, 1810)
Philinopsis capensis Bergh, 1907 Syn: Doridium aplysiaeforme Delle Chiaje, 1825
Aglaja iwasai Hirase, 1936 Syn: Eidothea marmorata Risso, 1826
Syn: Aglaja pelsunca Ev. Marcus & Er. Marcus,
1966
P. dubia (O’Donoghue, 1929)
Syn: Aglaja dubia O’Donoghue, 1929
Syn: Melanochlamys dubia (O’Donoghue, 1929)
P. nutalli Pilsbry, 1896
P. minor (Tchang-Si, 1934)
Syn: Aglaja depicta var. minor Tchang-Si, 1934
P. pusa (Ev. Marcus & Er. Marcus, 1966) (*)
Syn: Aglaja pusa Ev. Marcus & Er. Marcus, 1966
P. quinza (Ev. Marcus, 1979)
Syn: Aglaja quinza Ev. Marcus, 1979
P. taronga (Allan, 1933) (*)
Syn: Aglaja taronga Allan, 1933
Syn: Chelidonura aureopunctata Rudman, 1968
P. troubridgensis (Verco, 1909)
Syn: Aglaja troubridgensis Verco, 1909
P. virgo (Rudman, 1968)
Syn: Aglaja virgo Rudman, 1968
Syn: Melanochlamys virgo (Rudman, 1968)
Spinoaglaja Ortea,
Moro & Espinosa,
2007
S. petra (Ev. Marcus, 1976) (*) S. aeci (Ortea & Espinosa, 2001)
Type locality: Pernambuco, Brazil S. orientalis (Baba, 1949) (*)
Syn: Aglaja orientalis Baba, 1949
Synonym:
Philinopsis anneae (Ornelas-Gadtula &
Valdés, 2012) new synonym
Spinophallus gen.
nov.
S. coronata (Gosliner, 2011) (*) S. falciphallus (Gosliner, 2011) (*)
Type locality: Mainit Bubbles, Mabini,
Batangas, the Philippines
Syn: Philinopsis falciphallus Gosliner, 2011
Synonym:
Philinopsis coronata Gosliner, 2011
Tubulophilinopsis
gen. nov.
T. pilsbryi (Eliot, 1900) (*) T. gardineri (Eliot, 1903) (*)
Type locality: Rotuma Island, Fiji Syn: Doridium gardineri Eliot, 1903
Syn: Aglaja splendida Marcus, 1965
Synonyms: T. lineolata (H. & A. Adams, 1854) (*)
Doridium nigrum Martens, 1879 Syn: Aglaia lineolata A. & A. Adams, 1854
Philinopsis nigra (Martens, 1879) Syn: Aglaja lineolata Pease, 1860
Doridium alboventralis Bergh, 1897 T. reticulata (Eliot, 1903) (*)
Doridium pilsbryi Eliot, 1900 Syn: Doridium reticulatum Eliot, 1903
Aglaja pilsbryi hawaiiensis Pilsbry, 1920 Syn: Aglaja phaeroreticulata Yonow, 1990
Valid genera and species names are highlighted in bold, and all species used in this study are marked with an asterisk. Authorities of Melanochlamys
fukudai and M. kohi are Cooke et al. (2014). Most synonymies have been taken from WoRMS (http://www.marinespecies.org) and from original
descriptions of some species.
Table 4. Continued
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MOLECULAR PHYLOGENY OF THE AGLAJIDAE SEA SLUGS 33
© 2017 The Linnean Society of London, Zoological Journal of the Linnean Society, 2017, XX, 1–20
Table 5. Synopsis of the most useful morphological characters for diagnosis of the genera of Aglajidae
Character Aglaja Biuve Camachoaglaja Chelidonura Mannesia Mariaglaja Melanochlamys
Body shape Elongated,
broad
Elongated,
narrowed
Elongated, blunt Elongated,
narrowed
Elongated,
narrowed
Elongated,
narrowed
Short, blunt, cylindrical
Dorsal surface
of the body
Smooth Smooth Smooth Smooth Smooth Smooth Smooth
Cephalic shield Anterior edge
bilobed;
lobes slightly
squared; pos-
terior edge
laying over
posterior
shield
Anterior edge
trilobed; lobes
rounded; pos-
terior edge
laying over
posterior
shield
Anterior edge
bilobed; lobes
rounded; pos-
terior edge
laying over
posterior
shield
Anterior edge
tri- or quad-
rilobed; pos-
terior edge
laying over
posterior
shield
Anterior edge
bilobed; lobes
quadrangu-
lar; posterior
edge laying
over posterior
shield
Anterior edge
trilobed,
expanded
outwards;
lobes rounded;
posterior
edge laying
over posterior
shield
Anterior edge trilobed; posterior
edge laying over posterior
shield
Posterior shield Broader, larger
than cephalic
shield
Smaller than
cephalic shield
Nearly equal
in length
than cephalic
shield
Smaller than
cephalic
shield
Smaller than
cephalic
shield
Smaller than
cephalic
shield
Nearly equal in length than
cephalic shield
Caudal lobes: Asymmetrical; Asymmetrical; Asymmetrical; Asymmetrical,
both
acuminated;
Asymmetrical; Asymmetrical; Reduced, only two small symmet-
rical projections of the foot
R: right R: shorter,
rounded
R: rounded,
shorter
R: minute R: shorter R: minute,
triangular,
acuminate
R: inconspicuous
L: left L: acumi-
nated or
flagellum-like
L: acuminated or
flagellum-like
L: larger,
triangular
L: larger L: larger, flat,
tail-like
L: larger,
fleshier
Parapodia: Covering body
dorsally
Reduced Reduced Covering body
dorsally
Reduced Covering body
dorsally
Reduced
Reduced
Covering body dorsally
Eyes Not visible Dorsal; visible Dorsal; visible Not visible Not visible Dorsal; visible Dorsal; visible, sunken
Sensory bristles Reduced, not
visible to
naked eye
Along front side
of the cephalic
shield, large,
visible to
naked eye
Along front side
of cephalic
shield, large,
visible to
naked eye
Along front side
of cephalic
shield, large,
visible to
naked eye
Two clusters
of bristles on
each side of
mouth, visible
to naked eye
Along front side
of cephalic
shield, large,
visible to
naked eye
Reduced, pair of sensory mounds
on head
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34 A. ZAMORA-SILVA AND M. A. E. MALAQUIAS
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Character Aglaja Biuve Camachoaglaja Chelidonura Mannesia Mariaglaja Melanochlamys
Shell shape Convex-dilated
with open
whorl
Spoon-shaped
with prolonged
and semi-wide
open whorl
Spoon-shaped
with outward-
pointed
fringes of pro-
toconch; well-
defined
acuminated
whorl
(a) Spoon-
shaped with
acuminated or
rounded whorl
Spoon-shaped
with pro-
longed and
semi-wide
open whorl
Spoon-shaped
with acumi-
nated or
rounded whorl
Spoon-shaped with inward spiral
whorl
(b) Spoon-
shaped with
prolonged and
semi-wide
open whorl
Colour of the
shell
White;
translucent
Yellowish; white White;
translucent
Reddish; brown Amber; chestnut Reddish; brown White
Buccal bulb Massive, bul-
bous, eversible
Reduced,
bulbous,
non-eversible
Reduced,
bulbous,
non-eversible
Reduced,
bulbous,
non-eversible
Reduced,
bulbous,
non-eversible
Reduced,
bulbous,
non-eversible
Massive, bulbous, non-eversible
Penial accessory
structures
Absent Absent Absent Absent Absent Hooks only in
few species
Absent
Penial papilla Conical, shorter
than the
prostate
Cylindrical,
longer than
the prostate
Conical, shorter
than the
prostate,
anteriorly
bifurcated
Conical, irregu-
lar, shorter
than the
prostate
Cylindrical,
shorter than
the prostate
Conical, shorter
than the
prostate
Conical, larger than the prostate
Prostate One gland not
bifurcated
One gland not
bifurcated
One gland not
bifurcated
One gland not
bifurcated
One gland not
bifurcated
One gland not
bifurcated
One gland not bifurcated
Mucous gland Monolobed,
coiled
Monolobed,
coiled
Monolobed,
coiled
Monolobed,
blunt
Monolobed,
blunt
Monolobed,
coiled
Bilobed; primary lobe coiled; sec-
ondary lobe blunt
Radula Absent Absent Absent Absent Present;
8 × 1.0.1
Absent Absent
Geographic
range
EA + EP IWP (invasive
in the EA and
Mediterranean
Sea)
WA + EA IWP + WA EA IWP IWP + EP +EA
Table 5. Continued
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MOLECULAR PHYLOGENY OF THE AGLAJIDAE SEA SLUGS 35
© 2017 The Linnean Society of London, Zoological Journal of the Linnean Society, 2017, XX, 1–20
Character Aglaja Biuve Camachoaglaja Chelidonura Mannesia Mariaglaja Melanochlamys
Character Nakamigawaia Navanax Niparaya Odontoglaja Philinopsis Spinoaglaja Spinophallus Tubulophilinopsis
Body shape Short, blunt,
cephalic
shield
flattened
Elongated,
narrowed
Elongated, blunt Elongated,
tubular
Rectangular,
wide, blunt
Elongated,
narrowed
Elongated,
narrowed
Elongated,
cylindrical
Dorsal surface
of the body
Smooth Smooth Dorsal tubercles
present
Dorsal tubercles
present
Smooth Smooth Smooth Smooth
Cephalic shield Anterior edge
monolobed,
rounded; pos-
terior edge
laying over
posterior
shield
Anterior edge
trilobed; lobes
extended as
semi-circular
funnels; two
cephalic tenta-
cles; posterior
edge laying
over posterior
shield
Anterior edge
rhomboid;
frontal vel-
lum; posterior
edge laying
over posterior
shield, acumi-
nated, with a
three-pointed
elevated
crown-
like projection
Anterior edge
rounded, tri-
or quadrangu-
lar, flattened;
cephalic
shield not
overlap-
ping poste-
rior shield;
separated
Anterior edge
bilobed; lobes
short, slightly
squared;
posterior end
elevated
Anterior edge
symmetrical;
arrow-like,
acuminated;
posterior
edge laying
over posterior
shield
Anterior edge
quadrangu-
lar; blunt
head; pos-
terior edge
laying over
posterior
shield
Anterior edge
angular;
elevated;
resembling a
differentiated
head; posterior
edge laying
over posterior
shield
Posterior shield Nearly equal
in length
and width
than cephalic
shield
Nearly equal
in length and
width than
cephalic shield;
with backward
extensions
Nearly equal
in length
and width
than cephalic
shield; with
dorsal, bul-
bous tubercles
Nearly equal
in length
and width
than cephalic
shield; with
numerous
tubercles
Nearly equal
in length
and width
than cephalic
shield
Nearly equal
in length
than cephalic
shield
Nearly equal
in length
than
cephalic
shield;
rounded;
posterior
end termi-
nates in
a medial
bulbous
elevated
projection
Nearly equal in
length than
cephalic shield
Caudal lobes: Symmetrical;
rounded, short
Symmetrical;
long,
acuminated
Symmetrical;
rounded,
short,
flattened
Asymmetrical; Symmetrical;
rounded, short
Symmetrical;
rounded, short
Symmetrical;
short, blunt
Symmetrical;
rounded, short
R: right R: minute
L: left L: larger,
slightly
curved
Table 5. Continued
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36 A. ZAMORA-SILVA AND M. A. E. MALAQUIAS
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Character Aglaja Biuve Camachoaglaja Chelidonura Mannesia Mariaglaja Melanochlamys
Parapodia: Reduced Covering body
dorsally
Reduced Covering body
dorsally
Covering body
dorsally
Reduced Reduced Reduced
Reduced
Covering body
dorsally
Eyes Dorsal; visible
on front of
the cephalic
shield.
Inserted in
unpigmented
periocular
areas
Dorsal; vis-
ible on top of
cephalic shield.
Inserted in
unpigmented
periocular
areas
Not visible Not visible Not visible Not visible Not visible Frontal on flat-
tened side of
cephalic shield.
Inserted in
unpigmented
periocular
areas
Sensory bristles Reduced, not
visible to
naked eye
Along front side
of cephalic
shield, large,
visible to
naked eye
Reduced, not
visible to
naked eye
Reduced, not
visible to
naked eye
Reduced, not
visible to
naked eye
Reduced, not
visible to
naked eye
Reduced, not
visible to
naked eye
Reduced, not vis-
ible to naked
eye
Shell shape (a) Convex-
dilated with
open whorl;
(b) spiral
evolute
Convex-dilated
with open
whorl
Concave-
constricted
with moderate
close whorl
Spoon-shaped
with conspic-
uous-broad
and extended
whorl
Convex-
concealed
Spoon-shaped
with extended
whorl and
conspicuous
upward-
pointed
spines (one
or two) on
protochonch
Convex-dilated
with open
whorl
(a) Convex-
dilated with
open whorl
(b) Convex-
concealed
(c) Spoon-shaped
with prolonged
and semi-wide
open whorl
Colour of the
shell
White White;
translucent
White;
translucent
White;
translucent
White;
translucent
White;
translucent
White;
translucent
White;
translucent
Buccal bulb Reduced,
bulbous,
non-eversible
Massive, bul-
bous, eversible
Reduced,
bulbous,
non-eversible?
Reduced,
bulbous,
non-eversible
Massive,
bulbous,
non-eversible
Reduced,
bulbous,
non-eversible
Massive,
bulbous,
non-eversible
Tubular, the
longest in
aglajids,
non-eversible
Penial accessory
structures
Absent Absent With a rounded
distal bulb
Absent Absent Absent Large anterior
and poste-
rior spines
on penial
papilla and
penial sac
Absent
Table 5. Continued
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MOLECULAR PHYLOGENY OF THE AGLAJIDAE SEA SLUGS 37
© 2017 The Linnean Society of London, Zoological Journal of the Linnean Society, 2017, XX, 1–20
Character Aglaja Biuve Camachoaglaja Chelidonura Mannesia Mariaglaja Melanochlamys
Penial papilla Conical, shorter
than prostate
Conical, shorter
than prostates
Tubular, shorter
than pros-
tate, with a
rounded distal
bulb
Conical, shorter
than prostate
Conical, shorter
than prostate
Conical, shorter
than prostate
Conical, broad,
shorter than
prostate
Irregular, mas-
sive, shorter
than prostate
Prostate One gland; long,
tubular, mas-
sively coiled
One gland; bilob-
ulated; main
lobe larger
Two glands;
main gland
larger;
secondary
gland located
besides penial
sheath
One gland; long,
tubular
One gland; long,
tubular
One gland; long,
tubular
One gland;
broad,
pear-shaped
One gland; long,
tubular, not
coiled
Mucous gland ? Monolobed,
coiled
Monolobed,
blunt
Monolobed,
blunt
Bilobed; primary
lobe coiled;
secondary
lobe blunt
Bilobed; primary
lobe coiled;
secondary
lobe blunt
Monolobed,
coiled
Bilobed; primary
lobe coiled;
secondary lobe
blunt
Radula Absent Absent Absent Present;
24–36 × 1.0.1
Absent Absent Absent Absent
Geographic
range
IWP + WA EP + WA + EA IWP + EP IWP IWP + WA + EA WA + IWP IWP IWP
EA, eastern Atlantic; EP, eastern Pacific; IWP, Indo-West Pacific; WA, western Atlantic.
Table 5. Continued
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38 A. ZAMORA-SILVA AND M. A. E. MALAQUIAS
© 2017 The Linnean Society of London, Zoological Journal of the Linnean Society, 2017, XX, 1–20
formerly synonymized with Philinopsis by Camacho-
García et al. (2014), and comprises the WA type species
S. petra and the western Pacific species S. orientalis
(PP/BS = 0.97/100; Tables 4 and 5).
SYSTEMATIC DESCRIPTIONS
class gasTropoda cuvier, 1795
order cephalaspidea fischer, 1883
family aglajidae pilsbry, 1895 (1847)
genus Biuve gen. nov.
(Figs 3B, 5i; Tables 1 and 2)
Type species: Chelidonura fulvipunctata Baba, 1938.
By subsequent designation.
Diagnosis: Live animals up to 30 mm in length.
Body narrow, elongate, flat; cephalic shield larger
than the posterior shield; anterior edge of cephalic
shield trilobed, lobes rounded; posterior shield with
two asymmetrical caudal lobes, right one short,
nearly inconspicuous, and rounded, left prolonged,
thin, flagellum-like. Whitish W-shaped mark on
cephalic region (autapomorphy) (Fig. 3B). Shell
spoon-shaped with prolonged, semi-wide open
whorl, solid, yellowish-white, oldest region brown
(Fig. 5I). Buccal bulb reduced; penial papilla cylin-
drical, longer than prostate; prostate with single
lobe, short and rounded (Baba, 1938; Rudman,
1974; Gosliner, 1987).
Type locality: Kii, Japan.
Etymology: The name stems from the presence of a
W-shape mark on the head region of animals (Lat.
bi = two or double; ‘uve’ Spanish name for letter ‘v’).
genus CamaChoaglaja gen. nov.
(figs 3c, 5J; Tables 1 and 2)
Type species: Chelidonura africana Pruvot-Fol, 1953.
By subsequent designation.
Diagnosis: Live animals up to 10 mm in length.
Body elongate, blunt, flat; cephalic and posterior
shields nearly equal in length; anterior edge of
cephalic shield bilobed, lobes rounded; posterior
shield with two asymmetrical caudal lobes, right
one minute, rounded, left triangular (Fig. 3C). Shell
calcified, white or translucent, apex with external
fringe, projections at protoconch level (probably
an autapomorphy) (Fig. 5J). Buccal bulb reduced;
penial papilla conical, with two frontal lobes; pros-
tate cylindrical, not bifurcated, longer than penis
(Martínez et al., 2002).
Type locality: Tenerife Island, Canary Islands, Spain,
after neotype designation.
Etymology: This genus is named after Yolanda
Camacho-García for her studies on the phylogeny of
Aglajidae gastropods and for being the first to reveal
the existence of this clade of exclusively Atlantic
species.
genus mannesia gen. nov.
(Figs 3E, 5g, 6g; Tables 1 and 2)
Type species: Chelidonura sabadiega Ortea, Moro &
Espinosa, 1996. By subsequent designation.
Diagnosis: Live animals up to 15 mm in length. Body
elongated, narrow; cephalic shield bilobed, lobes
quadrangular with straight sides; posterior shield
about half length of cephalic shield, with two asym-
metrical caudal lobes, the left larger, flat, tail-like, the
right minute, triangular, acuminate (Fig. 3E). Shell of
amber- or chestnut colour, spoon-shaped with pointed
shoulder protruding slightly above the apex (Figs 5G,
6G). Radula present, with few rows of teeth (8 × 1.0.1)
(autapomorphy); one single lateral tooth on each side,
rachidian tooth absent; lateral teeth hook-shaped.
Prostate cylindrical, unilobed (Rudman, 1978; Ortea
et al., 1996).
Type locality: El Hierro Island, Canary Islands, Spain.
Etymology: This genus is dedicated to John Kenneth
Mannes, husband of Andrea Zamora-Silva, and to the
members of the family Mannes.
genus mariaglaja gen. nov.
(Figs 3F, 5i, 6f; Tables 1 and 2)
Type species: Chelidonura alexisi Gosliner, 2015. By
subsequent designation.
Diagnosis: Live animals up to 10 mm in length. Body
elongated-narrow; cephalic shield trilobed, middle lobe
sometimes with small indent, lateral lobes rounded to
triangular, expanded outwards, elongated, comprising
two thirds of body length; posterior shield with asym-
metrical caudal lobes, left one longer and fleshier, right
lobe reduced, nearly invisible (Fig. 3F). Brown-reddish
shell, calcified, spoon-shaped with acuminated shoul-
der or with extended whorl (Figs 5I, 6F). Buccal bulb
reduced, with microscopic labial spines on the lips.
Penial papilla conical, with small chitinous hooks or
spines (Gosliner 1987, 2015).
Type locality: Batangas, The Philippines.
Etymology: This genus is named after María Luisa
Silva Villanueva, mother of Andrea Zamora-Silva.
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MOLECULAR PHYLOGENY OF THE AGLAJIDAE SEA SLUGS 39
© 2017 The Linnean Society of London, Zoological Journal of the Linnean Society, 2017, XX, 1–20
Figure 3. Live images of the type species of all valid genera of Aglajidae. A, Aglaja tricolorata (Southern Portugal, photog-
raphy by M. A. E. Malaquias). B, Biuve fulvipunctata (Kwajalein Atoll, photography by Jeanette Johnson). C, Camachoaglaja
africana (São Miguel I., Azores, photography by M. A. E. Malaquias). D, Chelidonura hirundinina (Réunion Island, photog-
raphy by Philibert Bidgrain). E, Mannesia sabadiega (Tenerife, Canary Is, photography by Manuel Caballer). F, Mariaglaja
alexisi (the Philippines, photography by Sven Kahlbrock). G, Melanochlamys cylindrica (New Zealand, photography by
Simon Franicevic). H, Nakamigawaia spiralis (the Philippines, photography by Philibert Bidgrain). I, Navanax inermis
(California, USA, photography by Douglas Mason). J, Niparaya regiscorona (Maui, Hawaii, photography by Cory Pittman).
K, Odontoglaja guamensis (Guam, photography by Jun Imamoto). L, Philinopsis speciosa (Réunion Island, photography
by Philibert Bidgrain). M, Spinophallus coronata (Indo-West Pacific, photography by Nick Hobgood). N, Spinoaglaja petra
(Bermuda, photography by Linda Ianiello). O, Tubulophilinopsis pilsbryi (Marshall Islands, photography by S. & J. Johnson).
Copyright images are shared under the Creative Commons Attribution 4.0 International Public License.
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40 A. ZAMORA-SILVA AND M. A. E. MALAQUIAS
© 2017 The Linnean Society of London, Zoological Journal of the Linnean Society, 2017, XX, 1–20
genus niparaya gen. nov.
(Figs 3J, 5d; Tables 1 and 2)
Type species: Aglaja regiscorona Bertsch, 1972. By sub-
sequent designation.
Diagnosis: Live animals up to 5 mm in length.
Cephalic shield acuminated, with translucent
velum along the front side (autapomorphy) and a
posterior, small, three-pointed elevated crown-like
projection (autapomorphy); reduced sensory bris-
tles, not visible to the naked eye; posterior shield
with bulbous pustules (autapomorphy); symmetri-
cal caudal lobes, rounded, flat; parapodia reduced
(Fig. 3J). Shell calcified, concave, constricted, with
closed nuclear whorl, with ridges separated by a
slight indentation (Fig. 5D). Small ampulla, slightly
coiled; mucous gland faintly curved; receptaculum
seminis entering the hermaphroditic duct at the
middle of its length. Penial papilla with a rounded
distal bulb (autapomorphy); prostatic region with
two sections varying in shape and texture (autapo-
morphy) (Bertsch, 1972; Gosliner, 1980).
Type locality: Bahía de las Cruces, Baja California,
México.
Etymology: The word ‘niparaya’ means ‘creator of
everything’ in Pericú, the language of the aborigi-
nal inhabitants of the southern part of the Baja
California Peninsula (México), the type locality of
N. regiscorona and is also the name of the main deity
and king of the Pericú mythology. Thus, the genus
name Niparaya is a metaphor for the ‘crowned slug’
N. regiscorona (‘regiscorona’ means king’s crown in
Latin).
genus spinophallus gen. nov.
(figs 3m, 5b; Tables 1 and 2)
Type species: Philinopsis coronata Gosliner, 2011. By
subsequent designation.
Diagnosis: Live animals up to 15 mm in length. Body
elongated, wide; cephalic shield blunt, quadrangular;
reduced sensory bristles, not visible to the naked eye;
posterior shield rounded, terminates in a medial,
elongate, conical or bulbous posterior projection
(autapomorphy); symmetrical caudal lobes, short,
blunt; parapodia short (Fig. 3M). Shell calcified, con-
vex-dilated with open whorl (Fig. 5B). Buccal bulb
large, muscular, bulb-shaped. Penial sac with inter-
nal spines; penial papilla covered by scattered series
of large spines (autapomorphy), or with a ring of
rounded tubercles (autapomorphy) (Rudman, 1972a;
Gosliner, 2011).
Type locality: Batangas, The Philippines.
Etymology: The genus name (Lat. schedo = penis;
spino = spines) refers to the spines present on the penial
papilla or in the penial sac of species in this genus.
genus TuBulophilinopsis gen. nov.
(figs 3o, 4a–d, 5b, e, i; Tables 1 and 2)
Type species: Philinopsis pilsbryi Eliot, 1900. By subse-
quent designation.
Diagnosis: Living animals up to 30 mm in length. Body
elongated, oval, reduced sensory bristles, not visible to
the naked eye; cephalic shield well differentiated from
the body, with frontal elevation, crest-like (autapomor-
phy); eyes frontal, conspicuous, inserted in small cir-
cular unpigmented periocular areas (autapomorphy);
posterior shield with symmetrical, rounded, short cau-
dal lobes (Figs 3O, 4). Shell variable, convex-dilated
with open whorl (Fig. 5B), convex-concealed (Fig. 5E)
or spoon-shaped with prolonged and semi-wide open
whorl (Fig. 5I). Buccal bulb tubular, long, muscu-
lar (autapomorphy). Penial papilla conical; prostate
formed by a long narrow duct, not coiled (autapomor-
phy) (Rudman, 1972a; Gosliner, 1980).
Type locality: Rotuma Island, Fiji.
Etymology: The genus name (Lat. tubule = tube)
refers to the tubular buccal bulb present in all species
included in this new genus.
DISCUSSION
definiTion and monophyly of The family
aglajidae
The family Aglajidae is morphologically defined by the
presence of sensory bristles surrounding the mouth,
a yellow gland in the mantle cavity and a muscular
buccal bulb devoid of both radula and jaws – except
in Odontoglaja and in the new genus Mannesia. Most
aglajids have an elongated body covered with parapo-
dia and divided into cephalic and posterior shields, the
latter ending in two caudal lobes. The mantle cavity
has an internal and reduced shell in all species.
Previous molecular phylogenetic studies of Aglajidae
have included limited coverage of genera and/or spe-
cies (Anthes & Michiels, 2007; Malaquias et al., 2009;
Gonzales & Gosliner, 2014; Oskars et al., 2015). Only
the study by Camacho-García et al. (2014) contained
a broad representation of the generic and specific
diversity of the family. These studies cast doubts on
the monophyly of Aglajidae; for example in Oskars
et al. (2015), Aglajidae received marginal support in
Bayesian analysis (PP = 0.93) and was not supported
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MOLECULAR PHYLOGENY OF THE AGLAJIDAE SEA SLUGS 41
© 2017 The Linnean Society of London, Zoological Journal of the Linnean Society, 2017, XX, 1–20
Figure 5. Shell morphology in Aglajidae. A, bulloid
(adapted from Burn & Thompson, 1998). B, convex-dilated
with open whorl. C, spiral evolute. D, concave-constricted
with moderate close whorl. E, convex-concealed. F, spoon-
shaped with inward spiral whorl. G, spoon-shaped with
acuminated or rounded whorl. H, spoon-shaped with con-
spicuous-broad and extended whorl. I, spoon-shaped with
prolonged and semi-wide open whorl. J, modified version
of the typical spoon-shaped with outward-pointed fringes
of the protoconch and well-defined acuminated whorl.
K, spoon-shaped with extended whorl and conspicuous
upward-pointed spines on the protochonch.
Figure 4. Live images of species of Tubulophilinopsis
gen. nov., with details of the morphology of the anterior
part of the cephalic shield. A, T. lineolata (Thailand, pho-
tography by Nick Chaloklum). B, T. reticulata (Thailand,
photography by Nick Chaloklum), C, T. pilsbryi (Thailand,
photography by Nick Chaloklum). D, E, detail of the
cephalic hump of the type species T. pilsbryi (D, Kwajalein
Atoll, photography by S. Johnson and J. Johnson; E, East
Timor, photography by Brian Francisco). Copyright images
are shared under the Creative Commons Attribution 4.0
International Public License.
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42 A. ZAMORA-SILVA AND M. A. E. MALAQUIAS
© 2017 The Linnean Society of London, Zoological Journal of the Linnean Society, 2017, XX, 1–20
by ML analysis (BS = 51). In the study by Camacho-
García et al. (2014), the family received marginal
support in the ML analysis (BS = 76) and was not
supported when third codon positions of the COI gene
were included (BS = 50). Gonzales & Gosliner (2014) in
a 16S rRNA analysis of philinids and aglajids retrieved
some coloured philinid species nested together with
aglajids, although this relationship was not supported
(BS = 19); however, the relationships between radula-
less coloured philinids and agaljids remain to be thor-
oughly tested as highlighted by Oskars et al. (2015).
Despite these results, the current study based on
larger taxon and genetic sets produced unequivocal
support for the monophyly of Aglajidae (Fig. 2A, B).
The genera of Aglajidae have traditionally been
distinguished based on external morphology, col-
our patterns, shells and features of the reproductive
and digestive systems [Rudman, 1972a (Philinopsis);
1972b (Melanochlamys); 1973 (Chelidonura) ;
1974 (Aglaja, Chelidonura and Navanax); 1978
(Odontoglaja); Gosliner, 1980 (all genera); Ortea et al.,
2007 (Spinoaglaja); Ortea et al., 2014 (Migaya)], but as
highlighted before, some genera such as Chelidonura
and Philinopsis have been rendered paraphyletic in
molecular phylogenetic studies (Anthes & Michiels,
2007; Malaquias et al., 2009; Camacho-García et al.,
2014; Oskars et al., 2015), questioning the validity of
previous morphological-based definitions (discussed
below).
AglAjA renier, 1807 and The new genus
NipArAyA
The type genus of Aglajidae, Aglaja Renier, 1807 (type
species A. tricolorata Renier, 1807; type locality near
Caesarea, Mediterranean coast of Israel; K. Jensen,
pers. comm.) is distributed in the eastern Atlantic,
Mediterranean Sea and eastern Pacific. This genus
was validated by Lemche (1974) after conflicts with
preoccupied names (Lemche, 1972, 1974; Cernohorsky
& Lemche, 1976; ICZN Opinion 1079, 1977). The
genus is represented in our analyses by the type spe-
cies A. tricolorata from the Mediterranean Sea, by the
eastern Pacific species A. ocelligera, and at least five
additional species (Fig. 2A, B; Table 4).
However, a taxon usually ascribed to this genus (A.
regiscorona) did not cluster together with the other
species of Aglaja (Fig. 2A, B; Supporting Information,
Figs S1–S4). Furthermore, Bertsch (1972) and Gosliner
(1980) highlighted the presence of morphological char-
acters distinguishing ‘Aglaja’ regiscorona and raised
concerns about its taxonomic placement in Aglaja.
Representatives of ‘A.’ regiscorona from the Pacific
Ocean are small animals (up to 5 mm) with unique
characters such as light-creamy coloration, numerous
dark-tipped dorsal papillae along the body, cephalic-
anterior velum (Fig. 3J), cephalic-posterior elevation
in crown-like shape and a distinctive penial structure
than the rest of the aglajid genera (see Gosliner, 1980:
figs 1–12 and Marcus & Marcus, 1966: figs 22–27, for
comparisons of the penial papilla and prostate of ‘A.’
regiscorona). In contrast, slugs of the genus Aglaja
are larger (up to 30 mm); devoid of dorsal papilla,
velum, and upward projections of the cephalic/poste-
rior shields; and their penial anatomy resembles that
of Navanax (Rudman, 1974).
Camacho-García et al. (2014) previously hinted that
‘A.’ regiscorona could warrant a different phylogenetic
affiliation, which is here confirmed by our results.
No genus name is available for this new lineage, and
therefore, we introduce the name Niparaya with type
species N. regiscorona (type locality Las Cruces, Baja
California, México) for eastern Pacific specimens with
the aforementioned characteristics. Specimens iden-
tified in our work as ‘Aglaja regiscorona’ collected in
Hawaii and western Pacific localities seem to belong
to two distinct species (Fig. 2A, B). Moreover, available
images from field guides and specialized webpages
clearly hint the possible existence of potential cryptic
diversity in Niparaya regiscorona (Gosliner, 2015: 49;
Pittman & Fiene, 2015a).
ChelidoNurA s.s. a. adams, 1850 and The new
genera Biuve, CAmAChoAglAjA and mAriAglAjA
Chelidonura s.l. has been assembled together based
often on the shared presence of a narrow and elon-
gated body, a tri- or quadrilobed anterior shield, calci-
fied shell, a small non-eversible buccal bulb, a single
lobed mucous gland and a simple penis (Rudman, 1971,
1973, 1974; Gosliner & Williams, 1972; Gosliner, 1981;
Ortea & Martínez, 1997). Rudman (1974) and Gosliner
(1980) discussed the anatomy of several chelidonurids
and disagreed regarding the disposition of the labial
glands and the presence/absence of a genital ganglion,
while agreeing on the uniformity of the reproductive
system of Chelidonura, giving C. hirundinina as a typ-
ification for the whole genus (Gosliner, 1987).
Anthes & Michiels (2007) were the first to hint at
the possible non-monophyly of the genus, a result simi-
larly obtained by Malaquias et al. (2009) and Oskars
et al. (2015). Nevertheless, none of these works aimed
to discuss the systematics of Aglajidae and it was only
with the work by Camacho-García et al. (2014) that
the paraphyletic status of the genus was thoroughly
emphasized and discussed. The latter authors recov-
ered a ‘superclade’ with Aglaja + Navanax + three
sub-clades of Chelidonura and considered the possible
synonymization of one of the clades of Chelidonura
(the one containing the type species C. hirundinina)
and Navanax with Aglaja, but low statistical support
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