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Octopus americanus: a cryptic species of the O. vulgaris
species complex redescribed from the Caribbean
Otilio Avendan
˜o.A
´lvaro Roura .Celso Edmundo Cedillo-Robles .
A
´ngel F. Gonza
´lez .Rossanna Rodrı
´guez-Canul .Iva
´n Vela
´zquez-Abunader .
A
´ngel Guerra
Received: 20 March 2020 / Accepted: 17 June 2020
ÓSpringer Nature B.V. 2020
Abstract The common octopus Octopus vulgaris
Cuvier, 1797, once considered a cosmopolitan species,
is a species complex composed by six species: O.
tetricus, O. cf tetricus and O. sinensis in the Pacific;
type I and II, in the West Atlantic; and type III in the
Indian Ocean around South Africa. The tropical
western central Atlantic is an important octopus
fishing ground targeting O. maya,O. insularis, and a
cryptic species considered to be O. vulgaris type I. In
order to clarify the identification of this octopod,
phylogenetic analyses were carried out with mito-
chondrial (COI and 16S) and nuclear (rhodopsin)
genes, together with morphological analyses of 16
specimens caught in the northeastern continental shelf
of Yucatan (Mexico). The main morphological traits
differing from O.vulgaris were the presence, position
and size of enlarged suckers and hectocotylus sucker
number in males. Genetic distances and haplotype
networks of the species complex were estimated using
285 COI sequences of nine Octopus species from 14
different locations around the world. The octopod
sequences from Yucatan clustered within a mono-
phyletic group that included sequences of O. vulgaris
type II for the three genes analyzed. Phylogenetic
distances with other members of the complex ranged
between 2.71 and 3.89% using COI data. These
Handling Editor: Te
´lesphore Sime-Ngando.
Electronic supplementary material The online version of
this article (https://doi.org/10.1007/s10452-020-09778-6) con-
tains supplementary material, which is available to authorized
users.
O. Avendan
˜oR. Rodrı
´guez-Canul
I. Vela
´zquez-Abunader
Centro de Investigacio
´n y de Estudios Avanzados del IPN,
CP 97310 Me
´rida, Yucata
´n, Mexico
O. Avendan
˜o
Universidad de Ciencias y Artes de Chiapas,
C.P. 30500 Tonala
´, Chiapas, Mexico
A
´. Roura (&)A
´. F. Gonza
´lez A
´. Guerra
ECOBIOMAR, Instituto de Investigaciones Marinas
(IIM-CSIC), C.P. 36208 Vigo, Spain
e-mail: aroura@iim.csic.es
C. E. Cedillo-Robles
Laboratorio de Ecologı
´a, Departamento de Zoologı
´a,
Escuela Nacional de Ciencias Biolo
´gicas del IPN,
C.P. 11350 Ciudad de Me
´xico, Mexico
123
Aquat Ecol
https://doi.org/10.1007/s10452-020-09778-6(0123456789().,-volV)(0123456789().,-volV)
genetic results support the presence of Octopus
americanus Monfort, 1802 (formerly known as O.
vulgaris type II) along the Yucatan continental shelf, a
new octopod extending from the north of Argentina to
the northwest coast of the USA.
Keywords Octopus americanus O. vulgaris
complex Genetic analysis Morphometry Cryptic
species Caribbean Sea
Introduction
The common octopus, Octopus vulgaris, was origi-
nally described by Cuvier in 1797. Most probably this
species description was based on specimens collected
from the Mediterranean Sea, but no type specimen was
designated. Despite preliminary works by Mangold
and Hochberg (1991) to describe the main character-
istics of this taxon and to designate a neotype, both
tasks have still not been done (Guerra 1992; Norman
et al. 2016). It is currently accepted that Octopus
vulgaris sensu stricto (ss) occurs in the Mediterranean
Sea as well as the central and northeast Atlantic Ocean
(Amor et al. 2017). This species has a paralarval stage
with a coastal-oceanic dispersal pattern (Roura et al.
2019), giving it considerable dispersal and coloniza-
tion potential, features once related to the cosmopoli-
tan distribution of the species and a source of
controversy since the early 1990s (Hochberg et al.
1992; Voigt 1994). However, based on its morpho-
logical and meristic characters, this species name has
also been applied to at least four additional types with
disjointed geographical distributions across temper-
ate, subtropical, and tropical waters worldwide,
known as the O. vulgaris species complex (Norman
et al. 2016).
Among this species complex, O. vulgaris type I
would be distributed along the tropical western central
Atlantic Ocean (Caribbean Sea and North America);
type II would inhabit subtropical western South
Atlantic waters along the coast of Brazil; type III
would occupy the temperate eastern South Atlantic
and the Indian Ocean (along the coast of South
Africa); and type IV has been recently classified as
Octopus sinensis d’Orbigny, 1841 by Gleadall (2016),
which is the valid species name for the commercially
valuable East Asian octopus. In addition, a genomic
study based on 604 genome-wide double-digest
restriction-site-associated DNA sequencing (ddRAD-
seq) loci confirmed the existence of six species within
the O. vulgaris species complex: O. vulgaris sensu
stricto (Northeastern Atlantic and Mediterranean), O.
vulgaris type II (Southern Brazil), O. vulgaris type III
(South Africa), O. sinensis,O. tetricus, and O. cf.
tetricus (Amor et al. 2019). Remarkably, this genomic
study showed significant phylogenetic discordance
between Cytochrome Oxidase subunit I (COI) data
and nuclear markers because mitochondrial DNA
failed to distinguish between O. vulgaris ss and O.
vulgaris type III as distinct species. This study did not
include samples from the Caribbean Sea, which is a
hot spot of marine diversity. Recently, two new
species have been described in the Colombian
Caribbean: Octopus tayrona and Octopus taganga
(Guerrero-Kommritz and Camelo-Guarin 2016).
According to its distribution, O. taganga could be
considered a member of the O. vulgaris type I
(Norman et al. 2016), but phylogenetically, O.
taganga showed a closer relationship with species in
Caribbean (e.g., Octopus insularis and Octopus maya)
and Pacific (e.g., Octopus mimus) than with other
members of the O. vulgaris species complex
(Ritschard et al. 2019). On the other hand, molecular
analysis revealed that O. tayrona was in fact O.
insularis despite some morphological differences
(Ritschard et al. 2019).
In the last decades, several studies using both
morphological and molecular tools revealed cryptic
species that had been traditionally called O. vulgaris.
The common octopus of the Chilean-Peruvian Pacific
coast was identified as O. mimus Gould, 1852 (Guerra
et al. 1999;So
¨ller et al. 2000; Warnke et al.
2000,2004) Leite et al. (2008) described O. insularis
from oceanic islands and the continental coast of
Brazil, whose distribution has since found to extend to
the mid-Atlantic islands of Ascension and Saint
Helena (Amor et al. 2015) and the Caribbean coast
of Mexico (Lima et al. 2017; Flores-Valle et al. 2018;
Gonza
´lez-Go
´mez et al. 2018). Different genetic stud-
ies also confirmed the monophyletic status of O.
vulgaris type II caught in Brazilian waters and the
tropical Northwestern Atlantic relative to those of
other areas around the world (Europe and Asia) (De
123
Aquat Ecol
Luna-Sales et al. 2013,2019; Amor et al. 2017; Lima
et al. 2017; Mellis et al. 2018).
Besides the above-mentioned issues, the case of
Octopus americanus (Montfort 1802) in the West
Atlantic, whose present status is taxon inquirendum or
unresolved (Bouchet 2020), should be highlighted.
Monfort (1802) referenced ‘‘le poulpe americaine’’ but
indicated that it was Backer (1758) who described it
for the first time under the name of O. americanus.
Baecker realized that the octopus he examined (a
mature male) was different from the common octopus
from the Mediterranean and the Northwestern Atlan-
tic. Monfort (1802) reviewed all existing bibliography
and verified that all the O. americanus-like specimens
preserved in European museums had been collected
from Caribbean Sea islands (e.g. Barbados, Jamaica).
Bouchet (2020) found some species to be synonymous
with O. americanus. One of these species was Octopus
bakerii d’Orbigny 1826, a species probably described
with specimens from Brazil (Rio de Janeiro) which
d’Orbigny visited around 1826. Another synonymous
species that was found in the National Museum of
Natural History in Paris was Octopus eudora Gray
1849, described with a copy from Jamaica and
deposited in the Natural History Museum of London.
The final synonymous species was Octopus geryonea
Gray 1849, described with specimen(s) collected on
the coast of Brazil in the Catalogue of the Mollusca in
the collection of the British Museum. These three
species have been considered synonymous with O.
vulgaris Cuvier, 1797 (Norman and Hochberg 2005).
Consequently, there is a species described more than
200 years ago whose taxonomic identity is still
unsolved, ranging from the coasts of Brazil to the
Caribbean Sea, a distribution found to be overlapping
with that of O. vulgaris types I and II (Norman et al.
2016).
The octopus fisheries in the Western Central
Atlantic are centered in Mexico and Venezuela, with
three quarters of all Latin American octopus landings
caught in the Gulf of Mexico (DOF 2016; Norman
et al. 2016). This fishery yields up to 2 910
4
tonnes
annually, with catches comprising mainly Octopus
maya and O. vulgaris from the Campeche Bank and to
a lesser extent O. insularis, in the southeastern Gulf of
Mexico (Norman et al. 2016; DOF 2016; Lima et al.
2017; Flores-Valle et al. 2018; Gonza
´lez-Go
´mez et al.
2018). Improved taxonomy and identification of
cryptic species boundaries is required to improve the
reliability of catch estimates and inform sustainable
management strategies for the different Caribbean
octopods.
The main aim of the present paper was to identify
the O. vulgaris-like species captured by the fishing
fleet in the Yucatan shelf (Mexico). A combined
approach of morphological, meristic, and molecular
analysis was carried out to compare it with other
commercially exploited species in the area. Phyloge-
netic analyses were carried out to confirm whether this
species belongs to the O. vulgaris species complex and
evaluate if it could be considered as type I or II.
Finally, we will be able to answer the following
questions: Could O. americanus be a valid species and
a cryptic member of the O. vulgaris species complex,
and if so, what is its distribution range?
Materials and methods
Sample collection
In November 2018, 16 specimens (14 males and two
females), tentatively identified as O. vulgaris type I,
were caught in the northeastern area of the Yucatan
continental shelf within the continental shelf of the
Campeche Bank. The capture area was located
between 87°and 87.5°W and from 20 to 30 m depth
(Fig. 1). Sampling was undertaken aboard the vessel
San Rafael VII from the semi-industrial fleet (20 m of
length) based on Puerto Progreso, Yucatan. The
method of capture was that used by the local fishing
known as ‘‘gareteo,’’ which consists of small vessels
drifting from which the catches are made with line and
bait (Diplectrum sp. and Haemulon sp.) (Salas et al.
2008).
Approximately 1 cm
3
of muscle tissue was
removed from the third pair of arms and preserved in
absolute alcohol for subsequent DNA extraction. The
rest of the body of each organism was fixed in 4%
formalin in saline solution. Subsequently, 12 speci-
mens (11 males and one females) octopuses were
preserved in the Marine and Estuarine Fish and
Invertebrate Collection (‘‘CPIME’’ by its acronym in
Spanish) of the National School of Biological
Sciences at the National Polytechnic Institute of
Me
´xico, with catalogue numbers CPIME-8564 and
CPIME-8555 for the neotype and paratype,
respectively.
123
Aquat Ecol
Morphometric and meristic analysis
Morphometric indices and meristic characters were
obtained and calculated accordingly to recommenda-
tions from Roper and Voss (1983) and Norman and
Sweeney (1997) (Supp. mat. 1). Figure 2a illustrates a
male specimen of O. americanus and the distal tip of
its hectocotilized arm, respectively. Also, upper and
lower beak along with radula, digestive tract, and male
and female reproductive system were obtained,
described, and illustrated.
Molecular analysis
DNA was extracted with a QIAamp DNA Micro Kit
(QIAGEN) following the manufacturer’s instructions.
The mitochondrial genes 16S rDNA and COI were
amplified with the primers 16Sar–16Sbr (Palumbi
et al. 1991) and HCO2198–LCO1490 (Folmer et al.
1994). Moreover, the nuclear gene rhodopsin (Rho)
was amplified with the primers described in Allcock
et al. (2008).
PCR was set up on a total volume of 25 ll with 1 lL
of each forward and reverse primer (10 lM), 12.5 ll
Thermo Scientific TM Phusion TM High-Fidelity
PCR Master Mix with HF Buffer (Thermo Fisher
Scientific Inc.), 1 ll of DNA (20 ng/ll), and 9.5 ll
H
2
O. PCR conditions were as follows: COI gene—
initial denaturation at 94 °C for 1 min, followed by 39
cycles of denaturation at 94 °C for 15 s, annealing at
48 °C for 30 s and extension at 72 °C for 45 s, with a
final elongation at 72 °C for 7 min; 16S gene—2 min
at 94 °C for denaturation, followed by 30 cycles of
30 s at 94 °C, 1 min at 51 °C for annealing, 2 min at
72 °C for extension, and 7 min at 72 °C for the final
extension; Rho gene—denaturation at 94 °C for
2 min, 35 cycles of 94 °C for 40 s, 50 °C for 40 s,
and 72 °C for 90 s, and final extension at 72 °C for
10 min. Then, 2 ll of each PCR product was checked
on 1.5% agarose gels. PCR samples were cleaned
using USB
Ò
ExoSAP-IT
Ò
PCR Product Cleanup
(Affymetrix, Inc. USA) following the manufacturer’s
protocol and sequenced by Sanger sequencing (Stab
Vida, Portugal). Overall, 42 sequences were obtained.
Phylogenetic analysis
The DNA sequences were aligned using the ClustalW
multiple alignment tool implemented in MEGA7
Fig. 1 The square area shows the place where the specimens were caught in the east of the continental shelf of Yucatan
123
Aquat Ecol
(Kumar et al. 2016). After automatic alignment, each
sequence was inspected visually for the correction of
possible edition errors. Sequence data were compared
against those held in publicly available databases
(GenBank) using the BLASTn algorithm. In order to
evaluate the phylogenetic position of the octopods
collected, sequences of O. vulgaris present in
GenBank for the three genes were downloaded
together with sequences of Caribbean members of
the genus Octopus (Supp. mat. 2). The neighbor-
ioining (NJ) method of phylogenetic inference was
used to construct the phylogenetic trees, and evolu-
tionary distances were computed using the Tamura-
Nei method (Tamura and Nei 1993). All positions
containing gaps and missing data were eliminated. The
strength of support for internal nodes of the tree was
measured using 1000 bootstrap replicates.
Estimates of evolutionary divergence between O.
vulgaris types/species and the Yucatan octopods were
conducted using the Tamura-Nei model (Tamura and
Nei 1993). Bootstrap support was estimated using
1000 iterations. Prior to the analysis, sequences were
trimmed at the 5and 3ends, in order to have the same
genetic fragment. The analysis involved 285
sequences of 451 bp for COI, 107 sequences of
392 bp for 16SrRNA, and 25 sequences of 129 bp
for Rho. Evolutionary analyses were conducted in
MEGA7.
To evaluate the genetic diversity of the O. vulgaris
species complex and its relation with other Caribbean
octopods, an haplotype network was calculated with
COI data according to their location using Nexus
software (www.fluxus-engineering.com). A final
alignment with 285 COI sequences of 451 bp was used
to build the median joining haplotype networks. This
type of analysis allowed visualization of the genetic
structure of the different species/populations accord-
ing to the number of mutations present in the DNA.
Results
Morphological analysis
Diagnostic features: Moderate to large (dorsal mantel
length: 80–132 mm), muscular species (Fig. 2a).
Mantle broadly oval to saccular. Arms long (4–5
times mantle length), robust, and taper to narrow
rounded tips. Male third right arm hectocotylized,
shorter than the opposite arm (opposite arm length
index (OALI): 70–94%); ligula small to minute (ligule
length index: 1.2–1.7%), and moderate calamus
(calamus length index: 45–57%) (Fig. 2b). Arms with
two rows of suckers; number of suckers in normal non-
hectocotilized arms of larger animals range from 224
to 258, and suckers in the hectocotylized arm range
from 138 to 158. Mature males have three enlarged
suckers on arms II and III at the level of 7th and 8th
proximal suckers. Webs of moderate depth (deepest
15–20% of longest arm). Web formula, typically
C[B[D[E[A, highly variable but sector A
always shallowest. Gills with 7–8 lamellae per outer
demibranch. Funnel organ ‘‘W’’ shaped. Ink sac and
anal flaps present. Mature eggs around 2 mm long and
1 mm wide. No false ocelli present. Color in life
whitish with brown spots. Skin texture of nearly
regular patch and groove system with four large
erectile papillae in rhombus arrangement on the dorsal
mantle. One supraocular papilla present over each eye
and two secondary papillae in each eye.
Fig. 2 a Habitus and bdistal tip of the hectocotylized arm of
Octopus americanus collected east of the continental shelf of
Yucatan
123
Aquat Ecol
Description: Moderate to large octopuses (total
length: 790 mm) (Fig. 2a). The mantle is thick and
wide, but longer than wide (mantel width index:
50–80%), with a saccular shape and rounded tip. Head
narrow to slightly wide and always narrower than the
mantle (head width index: 30–77%), and the eyes are
somehow prominent. The funnel is tubular and solid;
the form of the funnel organ is a well-defined in ‘‘W’’
shape. The arms are thick and long (mantel arm index:
4–5). The most typical formula of the right arms is
4B2\3\1 and that of the left is 2 \3\4\1.
Mature males have two to three enlarged suckers in the
7th and 8th proximal pair in arms II and III that are
easily distinguished from the others (normal sucker
diameter index: 10–10.8% and enlarged sucker diam-
eter index: 13.6–15.4%). In males, the third right arm
is hectocotylized and short in relation to the length of
the opposite arm (OALI: 70–94%) (Fig. 2a). The
spermatic groove (Fig. 2b), located along the hecto-
cotylus, is well-defined and thick and ends in a
moderate calamus (calamus length index: 45–57%),
with a pyramidal shape and a slightly sharp point. The
ligule is tiny (ligule length index: 1.2–1.7%), and the
folds that line the calamus free the tip of the ligule that
is slightly acute. The number of suckers in normal
arms range from 224 to 258, and the number of suckers
in the hectocotylus range from 138–158. The depth of
the web is moderate (umbrella depth index: 14.3%);
lateral sectors are the deepest and the ventral and
dorsal sectors the shallowest. The web formula is:
C[B[D[E[A. Gills are characterized by
seven to eight lamellae per demibranch.
The new species has a typical Octopus digestive
tract (Fig. 3a); a large buccal mass with one pair of
anterior salivary glands that are flattened and moderate
in size (1/3 length of buccal mass), joined by salivary
ducts to the posterior portion of the buccal mass.
Paired posterior salivary glands are triangular and
almost as wide as the buccal mass. The crop is
fusiform in shape, with distinctive diverticulum. The
tripartite stomach is wider than the cecum (which has
two to three whorls) but similar in length. Intestines
are long, curved, ending in a muscular rectum
associated with one pair of small, conical-shaped anal
flaps. The digestive gland is ovoid and large, two to
three times the length of the buccal mass. Glandular
ducts that join the cecum form a concave region. Ink
sacs embedded or buried in the mid portion of the
digestive gland. The beak has a thick, typical Octopus
shape (Fig. 3b); the upper beak with a short, hooked
rostrum, and the lower beak with a narrow hood and
thick wings. Radula with seven teeth (Fig. 3c);
rachidial teeth with one and two symmetric cusps,
first lateral tooth with one high conical peaks, only one
high cuspid in the second lateral tooth, marginal tooth
long and curved with one cusp.
Testis are large, with a diameter of around 25–30%
mantle length (ML), with narrow, long vas deferens
with numerous turns wrapped in a membranous sac.
The vas deferens opens in a wide, round mucilage
gland followed by a long, thin spermatophore gland.
This gland opens in a hall that connects the accessory
gland and spermatophore sac (Needham sac). Acces-
sory gland has a recurved distal end, narrow respect
the Needham sac. The spermatophore sac is L- shaped,
wider than accessory gland but similar in length, and
Fig. 3 a Digestive tract of Octopus americanus (male): buccal
mass (BM), anterior salivary glands (ASG), posterior salivary
glands (PSG), crop (Cr), stomach (S), cecum (Ca), digestive
gland (DG), intestine (I), rectum (R), and anal flaps (AF);
bbeak, upper beak (UB), lower beak (LB). cradula, rachidial
tooth (RT), first lateral tooth (1LT), second lateral tooth (2LT),
and marginal tooth (MT)
123
Aquat Ecol
the terminal organ is moderately long (terminal organ
length index: 14–22%) with diverticulum (Fig. 4a).
The female reproductive system has a unique and
large ovary, around 50% ML (Fig. 4b), with small
proximal oviducts. Oviductal glands are spherical
shaped, and distal oviducts are moderate in length
(40% ML).
The skin texture is rough with a well-defined
pattern of patches and grooves, with dark and light
patches throughout the body. This pattern has semi-
circular grooves of different sizes that give the
appearance of being crosslinked. The newly captured
specimens have a beige coloration with light and dark
brown spots all over the body. The fixed specimens
have a grayish blue to dark purple color. Four papillae
present on the dorsal mantle form the vertices of a
rhombus, and each eye has a supra ocular primary
papilla and two secondary papillae.
Molecular analysis
Fourteen out of the 16 samples were used in the
subsequent analyses. Overall, five COI, seven 16S and
six Rho haplotypes were obtained from the 14
analyzed individuals. The three markers supported
the monophyly of the Mexican octopus analyzed in
this study, clustering with sequences that correspond
to O. vulgaris type II, which are clearly distant to O.
vulgaris ss. Given that O. vulgaris type II will have to
be considered as a new species within the O. vulgaris
species complex genome-wise (Amor et al. 2019), we
will restore the name O. americanus Montfort, 1802 to
designate the Mexican octopus analyzed herein as well
as other sequences previously considered as O.
vulgaris type II (De Luna-Sales et al. 2013; Lima
et al. 2017).
The phylogenetic tree constructed with COI data
(Fig. 5) showed that O. americanus belongs to the O.
vulgaris species complex and is phylogenetically
distant to other octopuses coexisting in the same
fishing grounds (O. maya and O. insularis). Within the
species complex, four out of five haplotypes were
grouped in a highly supported branch, including O.
vulgaris type II from the Western Atlantic (Brazil,
Mexico, and Virginia) which is a sister group to that of
O. vulgaris ss/type III. Curiously, there is an individ-
ual (number 7) that grouped with sequences corre-
sponding to O. vulgaris ss from the Eastern Atlantic
Fig. 4 a Male reproductive system of Octopus ameri-
canus: testis (T), vas deferens (VD), mucilage gland (MG),
spermatophore gland (SG), accessory gland (AS),
spermatophore sac (SS), diverticulum (D), and terminal organ
(TO); bfemale reproductive system, ovary (O), proximal
oviduct (PO), oviductal gland (OG) and posterior oviduct (PO)
123
Aquat Ecol
Fig. 5 Phylogenetic tree based on COI data of Octopus
vulgaris species complex showing the evolutionary distances
between O. americanus from O. vulgaris sensu stricto. The
analysis involved 285 nucleotide sequences and 451 positions in
the final dataset. Bootstrap values above 50% are shown
123
Aquat Ecol
and the Mediterranean. For the 16S tree (Fig. 6), all
Mexican specimens appeared within a highly sup-
ported clade that includes O. vulgaris type II
sequences from the Western Atlantic (Venezuela and
Brazil), clearly separated from other members of the
O. vulgaris species complex (O. vulgaris ss, O.
vulgaris type III, O. sinensis, and O. tetricus). It is
important to note the location of the specimen number
7 within the O. americanus clade, thus indicating some
discrepancy between the two mitochondrial markers.
Finally, the tree obtained with Rho sequences (Supp.
mat. 3) showed all specimens grouped in a sister clade
to O. vulgaris ss, which are clearly separated from
other Caribbean (O. insularis and O. taganga) and
Pacific octopods (O. bimaculoides).
The genetic distances were estimated considering
the published sequences of O. vulgaris type II (De
Luna-Sales et al. 2013; Lima et al. 2017)asO.
americanus. The different sequences available for O.
vulgaris (Supp. mat. 2) were grouped according to
Fig. 6 Phylogenetic tree based on 16S data of Octopus vulgaris
species complex showing the evolutionary distances between O.
americanus from O. vulgaris sensu stricto. The analysis
involved 110 nucleotide sequences and 386 positions in the
final dataset. Bootstrap values above 50% are shown
123
Aquat Ecol
their location and the species delimited by the
genome-wide study of Amor et al. (2019). The genetic
distances obtained (Tables 1,2,3) confirmed the
differentiation shown by the phylogenetic trees, thus
supporting the taxonomic status of O. americanus as
an independent member of the O. vulgaris species
complex. O. americanus showed genetic divergences
ranging between 2.71 and 3.89% from other members
of the species complex using COI data (Table 1).
Specifically, the divergence of this new species and O.
vulgaris ss was 2.78%. The genetic divergence of O.
americanus is even greater when compared with other
species coexisting in the same habitat such as O.
insularis (14.30%) or O. maya (19.62%). Genetic
divergences calculated with 16S data (Table 2)
showed a higher divergence between O. americanus
and O. vulgaris ss (1.77%), than between O. sinensis
and O. vulgaris ss (1.43%). Finally, the scarcity of
sequences of Rho, together with the lack of common
regions among the sequences available, permit us to
make only limited comparisons (Table 3). Nonethe-
less, 1.6% of divergence was obtained between the
available sequences of O. vulgaris ss and O.
americanus.
The COI haplotype network showed 32 mutations
between the O. vulgaris species complex and other
Caribbean octopods (O. maya,O. insularis, and O.
taganga, Fig. 7), suggesting that these two groups
diverged long time ago. Interestingly, the COI
network illustrates that O. americanus is closer to O.
tetricus (16 mutations) than to O. insularis (45
mutations), despite the fact that O. tetricus inhabits
the Eastern coast of Australia. The Mexican specimen
number 7, shown in the network as haplotype 46,
belonged to the O. vulgaris ss group. This specimen
together with haplotype 47—OvuPA 184 that was
retrieved from the stomach of a red snapper collected
in Para
´(Northeast Brazil) by De Luna-Sales et al.
(2013)—are the only two specimens collected in the
western Atlantic that are closely related with O.
vulgaris ss, potentially indicating the presence of
another cryptic species within the species complex.
However, as noted previously, the 16S gene showed
the Mexican specimen number 7 robustly placed with
the clade of O. americanus, thus revealing two
contrasting phylogenetic interpretations within the
mitochondrial genes.
The genetic structure observed in O. vulgaris ss
showed a star shaped pattern with two main haplo-
types. Haplotype number 1 is only found in the
Mediterranean (yellow color; Fig. 7) and the North-
eastern Atlantic (brown color, Fig. 7). However,
haplotype number 3 is also found in specimens from
South Africa, Tristan da Cunha, and St. Paul and
Amsterdam Islands (indicated in orange). Figure 8
shows an attempt to illustrate the distribution of the
Table 1 Tamura-Nei evolutionary divergences estimated from COI gene, with standard error above the diagonal. The analysis
involved 285 sequences of the O. vulgaris species complex and other Caribbean octopods, with 451 positions in the final dataset
O.
vulgaris
ss
Type
III
O.
americanus
O.
sinensis
(Asia)
O. sinensis
(Kermadec Is)
O.
tetricus
O. cf
tetricus
O.
insularis
O.
taganga
O.
maya
O. vulgaris ss –0.47% 0.69% 0.82% 0.80% 0.94% 0.91% 1.80% 2.01% 2.13%
Type III 1.44% –0.74% 0.85% 0.83% 0.96% 0.94% 1.76% 1.98% 2.09%
O. americanus 2.72% 3.06% –0.83% 0.81% 0.85% 0.94% 1.74% 1.89% 2.07%
O. sinensis
(Japan)
3.55% 3.67% 3.51% –0.23% 0.90% 0.87% 1.83% 2.02% 2.13%
O. sinensis
(Kermadec
Is)
3.18% 3.31% 3.08% 0.77% –0.84% 0.87% 1.80% 2.01% 2.11%
O. tetricus 4.11% 4.24% 3.40% 3.81% 3.25% –0.89% 1.72% 1.84% 2.10%
O. cf tetricus 3.70% 4.05% 3.79% 3.68% 3.32% 3.39% –1.82% 2.03% 2.14%
O. insularis 13.42% 13.08% 12.83% 13.69% 13.05% 13.15% 13.60% –1.23% 1.46%
O. taganga 15.97% 16.06% 14.89% 15.71% 15.16% 14.11% 15.63% 6.52% –1.41%
O. maya 16.73% 16.81% 16.44% 16.86% 16.35% 16.28% 16.07% 8.64% 8.05% –
123
Aquat Ecol
different members of the O. vulgaris species complex
around the world according to the information
obtained in this work.
Discussion
The integrative approach carried out in this study
proved the distinct species status of a suspected cryptic
member of the O. vulgaris species complex inhabiting
the coastal waters of the Yucatan continental shelf.
This species, previously known as O. vulgaris type II
by other authors (De Luna-Sales et al. 2013,2019;
Amor et al. 2017,2019; Lima et al. 2017; Gonza
´lez-
Go
´mez et al. 2018), has been renamed as O. ameri-
canus Montfort, 1802 in this study. We present for the
first time the detailed description of O. americanus,
which is a species distributed along the American
continent from Mar del Plata, Argentina, Brazil,
through the Caribbean Sea, the Gulf of Mexico to
the coast of Virginia. Such distribution of a benthic
organism could only be supported by a planktonic
paralarval stage with high dispersal capacity, as occurs
with O. vulgaris ss (Roura et al. 2016,2019).
Morphology
It is possible to determine and separate Octopus by
using morphological characters with low variation,
such as the number of suckers and the length of the
hectocotylus arm (Amor et al. 2017). However, this
characteristic for O. vulgaris ss is so variable that there
is clear overlapping with other species, which has been
reported as a common feature in members of the O.
vulgaris complex (Amor et al. 2017) and even among
species outside this complex such as O. vulgaris ss and
O. insularis (Table 4) (Leite et al. 2008). Character-
istics such as the presence and position of enlarged
suckers only in mature males, the index of the
diameter of the enlarged suckers, the number of gill
lamellae per demibranch, and the size of the egg in
mature females of O. americanus are characteristics
that clearly differ from O. vulgaris ss (Table 4). The
presence of enlarged suckers is associated with
gonadic maturity and the recognition of any of the
sexes in the mating, where the latter can be specific
(Voight 1991). The absence of these suckers in
females of O. americanus indicates some difference
with respect to O. vulgaris ss. The features that refer to
the male genitals in O. americanus (ILL, ILC, and the
Table 2 Tamura-Nei evolutionary divergences between Octo-
pus species estimated from the 16S gene, with standard error
above the diagonal. The analysis involved 110 nucleotide
sequences of the O. vulgaris species complex and other
Caribbean octopods, with 366 positions in the final dataset
O. vulgaris ss type III O. sinensis (Asia) O. americanus O. tetricus O. insularis
O. vulgaris ss – 0.32% 0.60% 0.68% 0.88% 2.03%
type III 0.69% – 0.70% 0.77% 1.00% 1.86%
O. sinensis (Asia) 1.43% 1.84% – 0.82% 1.09% 2.11%
O. americanus 1.77% 2.18% 2.10% – 0.94% 2.29%
O. tetricus 2.72% 3.14% 3.74% 2.84% – 2.02%
O. insularis 9.56% 8.60% 9.69% 10.80% 10.18% –
Table 3 Tamura-Nei evolutionary divergences between Octopus species estimated from the rhodopsin gene, with standard error
above the diagonal. The analysis involved 25 nucleotide sequences and 129 positions in the final dataset
O. vulgaris ss O. americanus O. insularis O. taganga
O. vulgaris ss – 1.19% 2.22% 1.70%
O. americanus 1.60% – 2.70% 2.21%
O. insularis 5.01% 6.89% – 1.19%
O. taganga 3.24% 5.01% 1.60% –
123
Aquat Ecol
shape of the ligule) appear to have no differences with
respect to O. vulgaris ss, as it happens between O.
sinensis and O. vulgaris ss (Gleadall 2016).
The O. insularis characteristics have already been
discussed by Lima et al. (2017), where they mentioned
that the size and fecundity in mature specimens is
lower in O. insularis than in O. vulgaris type II from
Brazil. Likewise, O. insularis has a shorter interval in
the number of suckers of the hectocotylus, its enlarged
suckers are barely distinguishable (IDVn: 8 to 14 and
IDVe: 10 to 15), and the pattern of patches and
grooves consists of regular tessellated grooves, in
comparison with the irregular pattern of semicircles in
O. americanus. There have been also other morpho-
logical characteristics that support the differentiation
between O. vulgaris ss and O. americanus (formerly
known as O. vulgaris type II) along the Brazilian
coast. For example, Vidal et al. (2010) found marked
differences in the distribution of chromatophores in O.
vulgaris paralarvae from the Northeastern (Galicia,
Spain) and Southwestern Atlantic (Southern Brazil),
reinforcing the findings of the current study.
Genetics
According to Norman et al. (2016), the octopus
present in the tropical Western and Central Atlantic
Ocean (the Caribbean and Gulf of Mexico) was
classified as O. vulgaris type I. However, recent
studies carried out in the Gulf of Mexico indicated that
O. insularis is widely distributed along this area
(Flores-Valle et al. 2018; De Luna-Sales et al. 2019).
From a genetic perspective, this second species would
not belong to the O. vulgaris species complex. Our
analysis revealed that the O. americanus present in the
Campeche bank was not O. vulgaris type I, but rather
Fig. 7 Haplotype COI network estimated from 285 sequences
and 451 bp showing the divergence between the different
haplotypes that are represented by numbers (details about the
sequences can be found in the supplementary material 3). Gray
dots indicate mutations observed in the dataset and red dots
show mutations absent in the dataset. The different colors within
each species correspond to the following geographical areas: O.
vulgaris sensu stricto (yellow: Mediterranean; light brown:
Northeastern Atlantic; orange: South Africa/Madagascar; dark
green: Western Central Atlantic; pink: samples from Turkey
(Keskin and Atar 2011)), O. americanus (dark green: Mexican
specimens corresponding to this study; light green: specimens
from the Northwest Atlantic to the Southwest Atlantic), O.
sinensis (violet: O. sinensis from Asia; purple: O. sinensis from
Kermadec Islands; pink: samples from Turkey (Keskin and Atar
2011))
123
Aquat Ecol
Fig. 8 Worldwide distribution of the different species belonging to the O. vulgaris species complex, including the new species O.
americanus, together with other Octopus from the Caribbean (O. insularis, O. maya and O. taganga)
Table 4 Diagnostic characters of Octopus vulgaris sensu stricto (Mangold 1998; Guerra et al. 2010; Norman et al. 2016); O.
insularis (Leite et al. 2008; Gonza
´lez-Go
´mez et al. 2018), and O. americanus
Trait Taxa
O. americanus (this study) O. vulgaris ss O. insularis
Sucker count in normal arms 224 to 258 220 to 240 220 to 238
Sucker count in the
hectocotylus
138 to 158 140 to 180 96 to 142
Number of gill lamellae per
demibranch
7to8 9to11 8to11
Presence and position of
enlarged suckers
Only males, in the 7th and 8th
proximal row.
Both sexes, in the 15th and 19th
proximal row.
Only males, in the 8th and 9th
proximal row.
Mantle arm index 4 to 5 4 to 5 3 to 4
Ligule length index 1.2 to 1.7 1.2 to 2.1 0.6 to 1.4
Calamus length index 45 to 57 47 to 52 40 to 60
Terminal organ length index 14 to 22 15 to 21 15 to 16
Diameter of normal suckers
index
10 to 10.8 12.5 to 13.5 8 to 14
Diameter of enlarged
suckers index
13 to 15 18.2 to 21.1 10 to 15
Egg size *1.5 mm 2 mm 1.5 mm
Total length 790 mm 1000 mm 530 mm
Depth range 15 to C100 m 100 to 150 m 0 to 45 m
123
Aquat Ecol
O. vulgaris type II, which is in agreement with other
genetic studies (Lima et al. 2017; De Luna-Sales et al.
2013,2019; Van Nieuwenhove et al. 2019). Cryptic
speciation within the Octopodidae is common as
evidenced by the three new species described in the
Caribbean: O. taganga,Callistoctopus Macropus, and
Macrotritopus beatrixi (Ristchard et al. 2019), or the
new genus Lepidoctopus joaquini Haimovici and
Sales, described in the Amazon reef system (De
Luna-Sales et al. 2019).
Other genetic studies in the Caribbean are likely to
determine whether O. vulgaris type I is a different
species, which overlaps its distribution with other
species of the O. vulgaris complex. In fact, the COI
sequence corresponding to the specimen number 7
(Fig. 5), as well as the specimen Ovu PA 184 sampled
by De Luna-Sales et al. (2013) off Para
´(Northeastern
Brazil), revealed a haplogroup closely related to O.
vulgaris ss that could represent the Type I. These
haplotypes might be the result of a recent introduction
of O. vulgaris ss in the Western Atlantic, as was
suggested for the presence of O. vulgaris haplotypes in
South Africa (Teske et al. 2007). However, the 16S
phylogenetic tree, unmistakingly placed specimen
number 7 within the O. americanus clade, while De
Luna-Sales et al. (2013) failed to amplify the 16S gene
for specimen Ovu PA 184.
A recent genomic study within O. vulgaris species
complex found that the phylogenetic signal within the
mitochondrial gene COI failed to identify O. vulgaris
type III as a distinct species within the complex, while
nuclear markers did (Amor et al. 2019). This species is
found along the South African coast and Indian Ocean
and our network analysis clearly showed a haplogroup
constituted by haplotypes 49–57 (Fig. 7) that would
correspond to this type III (haplotypes retrieved from
Teske et al. [2007] and Van Nieuwenhove et al.
[2019]), together with other haplotypes (numbers 3,
26, and 48 in orange) that are closer with O. vulgaris
ss. Indeed, haplotype number 3 includes sequences
obtained from specimens collected in the Southern
Atlantic islands of Tristan de Cunha, South Africa
(Teske et al. 2007) and the Southern Indian Ocean
Islands of Amsterdam and St Paul (Guerra et al. 2010).
In the latter studies, there was genetic support for a
second genetic lineage apart from the O. vulgaris ss
(Teske et al. 2007; Van Nieuwenhove et al. 2019),
together with marked morphological differences such
as a narrower head, smaller funnel, and larger number
of suckers on the hectocotylus (Guerra et al. 2010).
These findings, together with the genomic study of
Amor et al. (2019), support the validity of the cryptic
species O. vulgaris type III, which will have to be
described.
At this point, it is important to note some misiden-
tifications in Genbank within the O. vulgaris ss, which
have made exploring the genetic distances between
groups challenging. Misidentifications are quite com-
mon and difficult to deal with in animals that lack hard
structures. In this work, we noticed that many
sequences obtained from specimens collected along
the southern coast of Turkey (shown in pink color,
Fig. 7), that were classified as O. vulgaris (Keskin and
Atar 2011,2013), actually correspond to three differ-
ent species: O. sinensis, O. vulgaris type III, and O.
vulgaris ss. Similarly, Ritschard et al. (2019) recently
revealed that O. tayrona and O. vulgaris inhabiting the
northeastern Caribbean Islands (Puerto Rico and the
Lesser Antilles) were in fact O. insularis, as suggested
by Lima et al. (2017). Also, our COI haplotype
network revealed that Octopus jollyorum Reid and
Wilson 2015 shared the same haplotype (#74, Fig. 7)
with specimens belonging to O. sinensis (Gleadall
et al. 2016). Reid and Wilson (2015) considered
mitochondrial-based differences to warrant the dis-
tinction of Kermadec Island individuals from O.
vulgaris ss, establishing the name O. jollyorum for
this clade, which also encompassed Asian Type IV O.
vulgaris individuals. The designation of a neotype for
O. sinensis (Gleadall et al. 2016) effectively renames
the clade member taxa and places O. jollyorum in
synonymy with O. sinensis. Amor et al. (2017)
formally synonymize the two species on the basis that
each species description contained the same genetic
‘‘type’’ individual. Nevertheless, further nuclear mark-
ers are needed in order to stablish whether or not they
are the same species.
In the case of the American population, it was
previously believed that O. americanus Montfort 1802
was distributed in this region (Pickford 1945; Norman
and Hochberg 2005). However, despite the efforts to
describe the main characteristics of this taxon (Man-
gold and Hochberg 1991; Voss and Toll 1998; Warnke
et al. 2004), there is no assigned holotype and the
morphological information is insufficient to determine
it (Norman and Hochberg 2005). We deposited the
holotypes of this species in the CPIME of the National
School of Biological Sciences of the National
123
Aquat Ecol
Polytechnic Institute of Mexico. Furthermore, the
genetic data can be found in GenBank under Acces-
sion numbers: MT022413-MT022426 (COI),
MT025987-MT026000 (16S); MT035860-
MT035873 (rhodopsin).
As the value of octopus fisheries continues to
increase, the need for rigorous taxonomic knowledge
is of paramount importance. Ideally, a thorough
knowledge of the systematics of a species is the
required foundation upon which all other biological
and resource management studies must be based. For
example, in the Gulf of Campeche, Mexico, a
traditional fishery was believed to be based on O.
vulgaris Lamarck 1798, a ubiquitous octopus of broad
distribution. In the absence of local studies, knowl-
edge about the biology of O. vulgaris from other seas
was applied to the Campeche octopus for fishery
statistics and management purposes. The discovery
that the octopus was actually a new species, described
as O. maya Voss and Solis, 1966, with very different
life history traits, explained the problems that biolo-
gists had to deal with at the time of studying the fishery
and recommend management measures. This example
augments the necessity to improve the systematic
knowledge of the different species and populations in
order to effectively regulate fisheries (Roper 1983).
This is particularly important in Mexico since it is the
largest American octopus producer (Emery et al. 2016;
Norman and Finn 2016), and it is known that at least
three octopods with different life traits coexist in the
fishing grounds: O. maya,O. insularis, and O.
americanus.
Acknowledgements Thanks to the Federacio
´n de Sociedades
Cooperativas de la Industria Pesquera del Centro y Poniente del
Estado de Yucata
´n, SC de RL for the logistical and the vessel
support to carry out the cruises. Thanks to Jesu
´s Miguel Soto
Va
´zquez, Miguel A
´ngel Cabrera and Luis A
´ngeles Gonza
´lez
(CINVEST-IPN) for their support in the fieldwork, and to
Andrea Ramilo and Javier Tamame (IIM-CSIC) for their
support with the molecular labwork. We would like to thank
the valuable comments of Dr. Michael Amor.
Author Contribution OA and AR contributed equally to the
manuscript.
Funding The results of this study were obtained as part of the
project ‘‘Distribution, reproduction, biomass and movement
patterns of the Octopus vulgaris common octopus, Cuvier 1797,
on the Yucatan coast’’ with funding from the National Council
of Science and Technology of Mexico (CONACyT) (No.
237057). This work was partially supported by the Project
CALECO (CTM2015-69519-R) funded by the Spanish Ministry
of Economy and Competitiveness and FEDER Funds.
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