Mastigoteuthis microlucens, a new species of the squid family Mastigoteuthidae (Mollusca: Cephalopoda)

Abstract

We describe a new species, Mastigoteuthis microlucens, of the family Mastigoteuthidae from the tropical North Pacific. Its most distinctive morphological character is the presence of microscopic photophores in the integument. Morphological data indicate that its closest relative is the Atlantic Ocean species Mastigoteuthis magna. Molecular data from three genes, based on four species for which molecular data is available, indicates that Mastigoteuthis microlucens differs from its closest relative, M. magna, by a sufficient degree to substantiate its separate species status.

Full text

Mastigoteuthis microlucens, a new species of the squid family
Mastigoteuthidae (Mollusca: Cephalopoda)
R. E. Young*, A. Lindgren, and M. Vecchione
(REY) Department of Oceanography, University of Hawaii, Honolulu, Hawaii 96822, U.S.A.,
e-mail: ryoung@hawaii.edu;
(AL) Department of Evolution, Ecology and Organismal Biology, The Ohio State University,
Columbus, Ohio 43212, email: e-mail: lindgren.11@osu.edu;
(MV) Systematics Laboratory, National Marine Fisheries Service, National Museum of Natural
History, Washington, D.C. 20013-7012, U.S.A., e-mail: vecchiom@si.edu
Abstract.—We describe a new species, Mastigoteuthis microlucens,ofthe
family Mastigoteuthidae from the tropical North Pacific. Its most distinctive
morphological character is the presence of microscopic photophores in the
integument. Morphological data indicate that its closest relative is the
Atlantic Ocean species Mastigoteuthis magna. Molecular data from three
genes, based on four species for which molecular data is available, indicates
that Mastigoteuthis microlucens differs from its closest relative, M. magna,
by a sufficient degree to substantiate its separate species status.
The squid of the Mastigoteuthidae are
deep-living pelagic squid that are fragile
and easily damaged during capture. As a
result, although these squid are relatively
common, the systematics of the group is
unstable in spite of a recent review
(Vecchione et al. 2007). This family, with
the addition of the new species described
herein, consists of 13 nominal species and
a peculiar type whose specific status is
uncertain (Vecchione et al. 2007). The
new species described herein is the most
common species of the genus around the
main Hawaiian Islands. Its most distinc-
tive morphological feature is the presence
of numerous tiny photophores that lie
beneath an outer layer of integumental
chromatophores. The photophores are so
small that they cannot be recognized as
photophores without the aid of a micro-
scope. In this paper, we describe the
morphology of the new species and
present molecular data that confirms its
validity as a distinct species. Additional
details of the description can be found
on the Internet at: http://tolweb.org/
Mastigoteuthis_microlucens/65304.
Materials and Methods
Cephalopods for the morphological
study were collected during a variety of
cruises in the vicinity of the Hawaiian
Archipelago and as far south as the
Equator from 1974 to 2001 aboard the
University of Hawaii ships R/V Teritu,
R/V Kana Keoki, and R/V Moana Wave,
and the Hokkaido University ship FTS
Hokusei Maru. In total, 42 specimens
were captured, fixed in 7%formalin and
preserved in 50%isopropanol.
Only one specimen of the new species
was available for molecular analysis. This
individual was sucked up alive through a
deep, sea-water pipe from a depth of
about 925 m at Keahole Pt., Island of
Hawaii and was frozen in seawater after it
died (paratype SBMNH 422889). Upon
thawing, tissue samples from the gill and
fin were fixed in 100%ethanol. In order
* Corresponding author.
PROCEEDINGS OF THE BIOLOGICAL SOCIETY OF WASHINGTON
121(2):276–282. 2008.

to determine if the new species was, in
fact, a genetically distinct species, one
morphologically similar species (M.
magna Joubin, 1913) as well as two more
morphologically distinct species [M. agas-
sizii Verrill, 1881 and M. hjorti (Chun,
1913)] were included in this study. In
order to account for population-level
variation, multiple individuals within
these latter species were included. An
outgroup taxon, Joubiniteuthis portieri
Joubin, 1916, a member of the chiro-
teuthid-group of families to which the
Mastigoteuthidae belongs, was also in-
cluded to establish polarity.
DNA was extracted from mantle tissue
of 11 individuals, representing 4 known
species from a variety of localities and the
single specimen of the new species (Ap-
pendix) using the Qiagen Dneasy Tissue
Kit (Qiagen, Valencia, CA). PCR ampli-
fication was carried out in 50 mL reaction
volumes with ,5 ng DNA, 2.5 mLof103
Buffer A, [200 mL] dNTP, 10 pmol of
each primer, 3 mLofMg
++
and 1 unit of
Taq polymerase (Fisher Scientific, Fair
Lawn, NJ). Data from three mitochon-
drial loci (Appendix), 16S rRNA
(528 bp), 12S rRNA (404 bp), and COI
(658 bp), were collected in order to
determine if the new species is genetically
distinct from M. magna and to establish a
range and consistency of sequence diver-
gence across loci with varying rates of
evolution. Annealing temperatures and
additional primer information is available
in Nishiguchi et al. (2004). Amplified
regions were sequenced on an ABI 3100
Sequencer (Foster City, CA). The for-
ward and reverse chromatograms were
edited and assembled using Sequencher v.
4.6 (Gene Codes, Ann Arbor, MI).
Percent sequence divergence (Table 1)
was calculated by hand prior to alignment
in Sequencher.
In order to examine relationships
among the different morphological forms,
the molecular data were subjected to a
cladistic analysis. Edited sequences were
compiled and formatted using BBEdit
Table 1.—Measurements (mm) of selected specimens. ND 5no data.
Specimen
Holotype
USNM
1111879
Paratype
USNM
11118800
Paratype
SBMNH
422886
Paratype
SBMNH
422887
Paratype
SBMNH
422888
Paratype
SBMNH
422889
Paratype
SBMNH
422890
Sex
Immature
female Unknown Unknown
Immature
female
Unknown–head
only
Immature
female
Mature
male
Mantle length 79 37 77 138 ND 135 215
Tail length 14 8.5 ND ND ND ND ND
Mantle width 21 9 ND ND ND 25 ND
Fin length 54 22 54 ND ND 90 ND
Fin width 57 25 68 ND ND 115 ND
Head width 22 9 ND ND ND ND ND
Head length 16* 10 ND ND ND 34 ND
Eye diameter 16** ND ND ND ND ND ND
Arm I, length 34 11 44+87 90+105 125
Arm II,
length
49 17 56 105+139 126 145
Arm III,
length
36 10 43 94 111 105 137
Arm IV,
length
79 35 92 110+174 174 ND
Tentacle
length
ND 46 158 ND ND ND ND
Club length ND 30 130 ? ND ND ND ND
* Measured laterally from olfactory organ to tentacle.
** Calculated from lens diameter (56 mm).
VOLUME 121, NUMBER 2 277

(Bare Bones Software, Bedford, MA) and
aligned using default parameters in the
alignment program Muscle (Edgar 2004).
Aligned sequences were visualized and
formatted for analysis in MacClade v.
4.06 (Maddison & Maddison 2003).
Individual and combined data sets were
analyzed using multiple 600 replicate
Ratchet searches in NONA (Goloboff
1998) as implemented in WinClada
(Nixon 2002). Support was assessed via
1000 jackknife searches in WinClada.
Systematics
Family Mastigoteuthidae Verrill, 1881
Monogeneric; see diagnosis below.
Genus Mastigoteuthis, Verrill, 1881
Diagnosis.—Arms IV longest, thickest
and with broad lateral membranes which
form tentacular sheaths. Tentacular clubs
slender, whip-like; clubs very elongate
with small suckers in many irregular
series (30 or more in some species); clubs
without keel, locking apparatus and
terminal pad. Funnel locking-apparatus
oval with various knobs (tragus, antitra-
gus) affecting the shape of the depression
in the funnel component which varies
among species.
Mastigoteuthis microlucens, new species
Figs. 1, 2, Tables 1, 2
Diagnosis.—A mastigoteuthid with
flask-shaped funnel locking-apparatus;
tentacular club suckers small (0.05 mm),
of uniform size throughout; funnel pocket
absent; integumental tubercles absent;
large arm suckers generally with closely-
set, rounded teeth on the distal margin;
numerous microscopic integumental pho-
tophores present.
Description.—Arms III moderate in
length, 46%of mantle length (ML), 69%
of arm II length (Fig. 1A). Largest arm
suckers tend to be located mid-arm but
pattern highly variable. Large arm suck-
ers generally with closely-set, rounded
teeth on the distal margin, occasionally
fused; sometimes smaller, proximal teeth
with narrower, more distinctly separated
teeth (Fig. 2A, B). Club length more than
80%of tentacle length. Club suckers
numerous, extremely small (ca. 0.05 mm
in diameter) (Fig. 1B). Club suckers lack
enlarged lateral pegs on outer rings and
have smooth inner rings (Fig. 2C). Base
of club with long taper. Protective mem-
branes present but very low (height
comparable to that of individual suckers),
without detectable trabeculae. Tentacular
stalk not completely surrounded by club
even at tip. Olfactory organ short, broad
papilla with reduced head. Lower beak
without lateral-wall fold but with anteri-
or, solid lateral-wall ridge (Fig. 1I). Fun-
nel pocket between bridles absent
(Fig. 1F). Funnel component of funnel/
mantle locking-apparatus flask-shaped
with narrow stem (Fig. 1G). Mantle
component without nostril (i.e., posterior
bulge not overlapping posterior base)
(Fig. 1H). Fin length approximately
equal to fin width. Skin tubercles absent.
Arms with dense chromatophores aboral-
ly. Head, funnel, mantle and fins with
dense chromatophores. Most pigment
beyond oral region in chromatophores.
Minute (‘‘lens’’ diameter less than
0.1 mm) integumental photophores pre-
sent but unrecognizable to naked eye due
to small size and structure (Fig. 1C–E).
Integumental photophores present on
aboral surfaces of all arms, on dorsal
and ventral surfaces of head, mantle and
fins, on ventral surface of funnel. Integu-
mental photophores lie beneath surface
layer of chromatophores. Eyelid photo-
phores absent. Measurements of selected
specimens presented in Table 1.
Paralarvae were described by Young
(1991) as the young of Mastigoteuthis
inermis. The vertical distribution was
described by Young (1978) as that of
Mastigoteuthis inermis.
Molecular results.—In the molecular
analysis, M. microlucens was found to be
sister to an M. magna clade (96%
278 PROCEEDINGS OF THE BIOLOGICAL SOCIETY OF WASHINGTON

Fig. 1. A, Side view of a living M. microlucens, 135 mm ML. Photograph by Michael Darden; copyright
with West Hawaii Today newspaper; B, Side view of mid-region of tentacular club of M. microlucens. Inset
shows small suckers and small protective membrane; C, Ventral view of portion of head of M. microlucens,
145 mm ML. Much of outer layer of chromatophores lost during capture. Two areas enlarged in Fig. D & E.
Bottom insert shows enlargement of outward directed photophore with small, round chromatophore above it;
D, Enlargement of region mostly devoid of outer layer of chromatophores. Arrows point to few of many
exposed photophores. Inset shows enlargement of one, anteriorally directed, photophore and its associated
chromatophore; E, Enlargement of region where outer layer of chromatophores is mostly intact. Arrows
point to chromatophores surrounded by space without chromatophores. Each of these appears to be
associated with deeper photophore; F, Ventral viewoffunnel and posterior head of preserved M. microlucens,
79 mm ML, showing absence of funnel pocket between funnel bridles and numerous tiny photophores on
head (small dots); G, Funnel component of funnel/mantle locking-apparatus of M. microlucens, 79 mm ML.
Skin of posterior margin (bottom) folded forward, obscuring part of broad groove; H, Mantle component of
funnel/mantle locking-apparatus of M. microlucens, 79 mm ML; I, Side view of lower beak ofM. microlucens,
female, 95 mm ML; J, Side view of upper beak of M. microlucens, female, 95 mm ML.
VOLUME 121, NUMBER 2 279

support), with M. hjorti as the next closest
species for all (individual and combined)
data sets (Fig. 3). Mastigoteuthis agassizii
formed the basal-most clade, with 100%
support for a monophyletic Mastigo-
teuthidae. Mastigoteuthis microlucens
and M. magna were also found to be the
most genetically similar (Table 2), with a
9.89%difference in COI. Due to the high
level of sequence difference between M.
microlucens and M. magna across all
genes, they cannot be considered the same
species (Hebert et al. 2003).
Etymology.—The trivial name ‘‘micro-
lucens’’ refers to the small size of the
photophores of this species.
Holotype.—Immature female (mea-
surements in Table 1) deposited in the
National Museum of Natural History,
Smithsonian Institution (USNM 1111879).
Paratypes.—Specimens of males, fe-
males, and undetermined sex (Table 1)
deposited in the National Museum of
Natural History, Smithsonian Institution
(USNM 1111880) and Santa Barbara
Museum of Natural History (SBMNH,
422886–422890).
Discussion
The distinctness of M. microlucens as a
new species of squid is confirmed by both
morphological and molecular data. Mas-
tigoteuthis microlucens uniquely shares
with M. magna the distinctive flask-shape
of the funnel locking-apparatus and the
very small size of the tentacular suckers.
The two species also share the unique
combination of absence of a funnel
pocket, absence of eyelid, ocular and
large, integumental photophores, and
absence of integumental tubercles. As a
result we place these two species in a
species group, the M. magna group,
following Vecchione et al. (2007). The
two species differ in the microscopic
integumental photophores which are pres-
ent in M. microlucens but not M. magna,
the dentition of the arm sucker-rings
which are generally toothed in M. micro-
lucens but always smooth in M. magna,
the shape of the funnel locking-apparatus
which is slender in M. microlucens but
rather broad in M. magna and the form of
the lower beak which has a lateral-wall
ridge in M. microlucens but a lateral-wall
fold in M. magna.
Due to high levels of sequence diver-
gence in the three genes evaluated,
molecular data confirm that M. micro-
Fig. 2. A, Oral view of large sucker from arm III of M. microlucens, sex unknown, 140 mm ML
(estimate), light photograph; B, Oral view of large sucker from arm III of M. microlucens, sex unknown,
140 mm ML (estimate). Scanning electron micrograph (SEM); C, Oral view of two tentacular club suckers
of M. microlucens, sex unknown, 77 mm ML (SEM). Scale bars: B 5500 mm; C 520 mm.
Table 2.—Percent sequence divergence across 3
loci for M. microlucens and M. magna (with more
distantly-related M. hjorti for comparison).
Locus M. magna M. hjorti
M. microlucens COI 9.89%11.70%
M. microlucens 16S 3.40%6.02%
M. microlucens 12S 5.13%7.00%
280 PROCEEDINGS OF THE BIOLOGICAL SOCIETY OF WASHINGTON

lucens is a separate species from M.
magna. Hebert et al. (2003) hypothesized
a2%rate of divergence in the COI locus
as a general guideline for identifying
distinct species, here the rate of diver-
gence between M. microlucens and the
morphologically similar M. magna is
almost 10%. Interestingly, the more
morphologically distinct M. hjorti had a
COI sequence divergence rate 12%with
M. microlucens, further emphasizing the
surprising result of 10%difference seen
with M. magna.
Relatively large integtumental photo-
phores are characteristic of members of
the M. agassizii species group (i.e., M.
agassizii,M. psychrophila Nesis, 1977,
and M. dentata Hoyle, 1904) and M.
pyrodes Young, 1972. Superficially, the
photophores of M. microlucens, in addi-
tion to their small size, bear little resem-
blance to those of other mastigoteuthids.
We failed to recognize the presence of
photophores in M. microlucens for many
years because of their extremely small
size. We identify these structures as
photophores on the basis of a dark,
pigmented ‘‘cup’’ and a spherical, whitish
‘‘lens’’ combined with their distribution as
a separate layer beneath most chromato-
phores. No bioluminescence has been
observed or attempted to be observed
from these organs.
Acknowledgments
We thank employees, and especially
Jan War, at the Natural Energy Labora-
tory of Hawaii at Keahole Pt. on the
island of Hawaii for finding, maintaining
and freezing the specimen that allowed us
to examine the molecular features of this
squid and to Chris Kelley, University of
Hawaii, for transporting it to Oahu.
Literature Cited
Edgar, R. 2004. MUSCLE: multiple sequence
alignment with high accuracy and high
throughput.—Nucleic Acids Research 32:
1792–97.
Goloboff, P. A. 1998. Nona, ver. 2.0. Program
available at www.cladistics.org.
Hebert, P. D., S. Ratnasingham, & J. R. de Waard.
2003. Barcoding animal life: cytochrome c
oxidase subunit I divergences among closely
related species.—Proceedings of the Royal
Society of London B. 270:S96–S99.
Maddison, D. R., & W. P. Maddison. 2003.
MacClade 4: Interactive Analysis of Phylog-
eny and Character Evolution. Version 4.06.
Sinauer Associates.
Fig. 3. Phylogenetic analysis of the combined data (COI, 16S, 12S) yielded single tree (L 5515, CI 589,
RI 590). Numbers in bold above branches indicate jackknife support values above 50%.
VOLUME 121, NUMBER 2 281

Nishiguchi, M. K., J. E. Lopez, & S. V. Boletzky.
2004. Enlightenment of old ideas from new
investigations: The evolution of bacteriogenic
light organs in squids.—Evolution and De-
velopment 6:41–49.
Nixon, K. C. 2002. Winclada. Ver. 1.00.08. Program
available at http://www.cladistics.com.
Vecchione, M., R. E. Young, & A. Lindgren. 2007.
Mastigoteuthidae. Tree of Life web pages:
http://tolweb.org/Mastigoteuthis.
Young, R. E. 1978. Vertical distribution and
photosensitive vesicles of pelagic cephalopods
from Hawaiian waters.—Fisheries Bulletin
76:583–615.
———. 1991. Chiroteuthid and related paralarvae
from Hawaiian waters.—Bulletin of Marine
Science 49:162–185.
Associate Editor: Rick Hochberg
Appendix
Accession numbers and collection information for specimens included in the molecular analyses. All voucher
specimens are located at the National Museum of Natural History, Smithsonian Institution (USNM).
Species COI 16S 12S Collection Station Approx. Location ID
J. portieri EU201163 EU201153 EU201142 DE0304 (Stat. 14) 40uN, 67uWMV
M. microlucens EU201161 EU201150 EU201139 Keahole Pt., Hawaii 20uN, 156uWRY
M. magna EU201164 EU201155 EU201144 DE0506 (Stat. 1) 40uN, 67uWMV
M. magna EU201168 EU201156 EU201145 DE0506 (Stat. 19) 40uN, 67uWMV
M. magna EU201167 EU201152 EU201141 Mar-Eco (44:369) 43uN, 30uWMV
M. hjorti EU201169 EU201159 EU201148 DE0506 (Stat. 5) 40uN, 67uWMV
M. hjorti EU201170 EU201160 EU201149 DE0304 (Stat. 14) 40uN, 67uWMV
M. agassizii EU201162 EU201151 EU201140 DE0409 (Stat. 12) 40uN, 67uWMV
M. agassizii EU201166 EU201157 EU201146 DE0409 (Stat. 12) 40uN, 67uWMV
M. agassizii — EU201158 EU201147 DE0409 (Stat. 5) 40uN, 67uWMV
Mastigoteuthis sp. EU201165 EU201154 EU201143 Mar-Eco (42:368) 43uN, 30uWMV
282 PROCEEDINGS OF THE BIOLOGICAL SOCIETY OF WASHINGTON