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The Giant Squid Consists of One Global Species
Lexie Penwell
Capturing the Giant Squid live on camera was one of the greatest feats of marine biology.
Little is known about the Giant Squid, Architeuthis, most specimens are dead when found or
even partially digested in the stomachs of whales. Therefore little is known about the Giant
Squid, including how many species of Architeuthis there are. The paper by Winkelmann et al.
(2013), attempts to solve the question of the number of species of Giant Squid by comparing the
mitogenome of 43 different Architeuthis samples.
When the Giant Squid was first discovered, 21 different species were described, although
the descriptions were based solely on location, meaning that many of the described “species” are
likely the same. Recent opinions on the topic ranged from as many as eight or as little as one
species of Architeuthis. Giant Squid inhabit all oceans excluding the Polar Regions. However
carbon isotope profiles from their beaks show that they inhabit small, well defined areas where
food resources have a constant carbon isotope composition. In one study the stability of the
carbon and nitrogen isotope composition in four Giant Squid beaks collected from the Bay of
Biscay and Namibian waters was recorded. The table below describes the condition of and gives
information about each sample.
No. Locality
1932
ML
BW
S
MS
UHL
LRL
Comments and source
Carrandi (Bay
150 104 F Ma 60.4 15.1 Fresh stranded. Date: 23 October 2001.
of Biscay)
González et al. (2002) and Guerra et al. (2004)
1964 Carrandi
152 105 F Ma 59.6 15.2 Fresh stranded. Date: 15 September 2003.
González et al. (2002) and Guerra et al. (2004)
1963 Carrandi
153 140 F Ma 70.2 15.2 Fresh stranded. Date: 13 September 2003.
González et al. (2002) and Guerra et al. (2004)
1427 Namibia
105 47 F Im 56.2 14.2 Caught by trawler. Date: 09 February 1990
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No., specimen number at Ecobiomar Research Group's Archive (Instituto de
Investigaciones Marinas, Consejo Superior de Investigaciones Científicas, Vigo); ML,
mantle length (cm); BW, body weight (kg); S, sex (F, female); MS, maturity stage (Ma,
maturing; Im, immature); UHL, upper hood length (mm); LRL, lower rostral length
(mm).
The two graphs below show the carbon and nitrogen stable isotope composition of the four
specimens relative to the distance in mm from the rostral tip of the beak. It was found that
carbon 13 and nitrogen 15 profiles over the lifespan of adult Giant Squid remain relatively stable,
suggesting that they inhabit a very specific niche where their food sources all have a constant
carbon and nitrogen isotope composition (Guerra et al., 2010). However it was found that
composition varies in younger squid, as shown by the steeper curves at short distances, likely
due to either the differences in isotope stability among smaller prey that the young squid eat or
because Giant Squid are more migratory early in life (Guerra et al., 2010).
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In the study by Winkelmann et al. (2013), 37 complete and 6 partial mitochondrial
genomes were taken from samples around the world from the locations shown in the figure
above. The samples consisted of remains of Giant Squid carcasses or those caught as accidental
by-catch of fishermen. DNA was extracted from each of the tissue samples, which is a difficult
process in Giant Squid because they have high concentrations of mucopolysaccharides that coseparate with DNA during extraction and can end up interfering with later enzymatic processes.
They also have ammonia in their muscle tissues. The quality of all extracts was determined by
electrophoresis and by fluorometric quantitation. Fluorometric quantitation is a process that
reveals the concentrations of nucleic acids and proteins in a tissue sample.
The unpublished Architeuthis mitogenome contains two duplicated regions with 99.9%
similarity. Many cephalopod mitogenomes, such as those from W. scintillans and T. pacificas,
contain duplicated regions that are thought to be maintained by recombination (Yokobori et al.,
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2004). The table below shows nucleotide sequence similarities between duplicated regions of W.
scintillans and T. pacificas mitochondrial genomes found in the study by Yokobori et al. (2004).
Wsc vs. Tpa
Gene
Long
NCR
cox3
cox1
cox2
trnD
atp8
atp6
Wsc Copy 1 vs.
Wsc Copy 2 vs.
Wsc Copy 1 vs. Tpa Copy 1 vs. Tpa Copy Tpa copy Tpa Copy Tpa Copy
2
2
1
2
1
2
ac
bc
99.8
99.6
66.8
66.7
66.8
66.8
1st 100
2nd 99.6a
3rd 100
1st 100
2nd 100
3rd 100
1st 100
2nd 99.6a
3rd 100
100
1st 100
2nd 98.1a
3rd 100
1st 100
2nd 99.6a
3rd 100
100
100
99.6a
100
100
100
100
100
100
100
100
100
100
99.6a
100
100
89.6
97.3
55.4
92.2
99.4
50.5
90.8
98.7
57.0
84.8
82.7
88.5
63.5
85.3
97.0
61.9
89.6
97.3
55.0
92.2
99.4
50.5
90.8
98.7
57.0
84.8
82.7
88.5
63.5
85.7
97.0
61.9
89.6
97.7
55.4
92.2
99.4
50.5
90.8
99.1
57.0
84.8
82.7
90.4
63.5
85.3
96.5
61.9
89.6
97.7
55.0
92.2
99.4
50.5
90.8
99.1
57.0
84.8
82.7
90.4
63.5
85.7
96.5
61.9
These duplicated mt gene regions are thought to be a result of concerted evolutionary processes
and the homogeneity of the duplicated genes is maintained by recombination. There is also the
possibility of parallel evolutionary processes that lead to the recent duplication of mt genes in
both species, although that would require that several duplication events and gene-loss events
would have had to occur in both species in the same order, which is unlikely.
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“Supplementary Figure 6: Mitochondrial gene arrangement of the giant squid,
GenBank NC_011581. Duplications are highlighted with blue and green colouring.
Grey lines demonstrate the two ways in which each mitogenome was divided into two
halves for sequencing, depending on which sets of primers were used” (Winkelmann et
al., 2013).
The figure above is a representation of the mitochondrial gene arrangement in
Architeuthis. The blue and green regions highlight the duplicated genes. In this experiment the
accuracy of the similarity of these regions was confirmed using four pairs of primers designed to
amplify each duplicated region in four samples that represent the spread of the population of
Architeuthis. Then mitogenomes were generated using long-range PCR coupled to sequencing.
For the 5 DNA samples that were not of sufficient quality to undergo PCR amplification, a target
capture technique was used instead in which the DNA extracts were directly converted into
sequencing libraries, then denatured and incubated with biotinylated ‘bait’ generated from longrange Architeuthis mtDNA amplicons derived from the higher-quality samples. Later capture of
the biotinylated bait, now hybridized to the mitochondrial sequences from the library, enabled
target sequencing of the mitogenomes. After sequencing the different datasets were processed
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and verified and the matching halves of each individual mitogenome were joined together to
construct the complete mitogenome sequence.
region
length π
s.d.
Hd s.d. Tajima's D stat. sig.
whole mitogenomes 20 331 0.00066 0.00005 1
0.006 −2.51828 p < 0.001
all genes
14 976 0.00068 0.00005 1
0.006 −2.49263 p < 0.01
control region A
554
0.00166 0.00034 0.613 0.087 −1.66738 0.10 > p > 0.05
The data above shows the nucleotide diversity index (π) and the haplotype diversity (Hd)
for the entire set of mitogenomes. Initial data analysis indicated that the level of nucleotide
variation was too low to allow generation of a well-supported phylogenetic tree of the
mitogenomes. There were only 181 segregating sites (Winkelmann et al., 2013). This was the
first indication that Architeuthis consists of only one species. Instead of a phylogenetic tree a
haplotype network was generated, based on the protein coding genes from the 38 complete
mitogenomes. A haplotype network is similar to a phylogenetic tree except instead of comparing
species, the different haploid genotypes within a species are outlined. It showed that haplotype
diversity was high, with each specimen having a unique haplotype. No phylogeographic
structure was shown in the data, meaning that no relationship could be found between the
geographic location of Architeuthis and its mitogenome duplications. There was, however,
strong evidence that Architeuthis has undergone a population expansion. The low mutation rate
of the Architeuthis mitogenome could be due to the fact that many marine species have a low
rate of mitochondrial DNA evolution, or because the duplication in part of the mitogenome
keeps mutations from occurring, or because the Giant Squid population suffered a recent
bottleneck followed by expansion. Assuming that the low mitochondrial diversity is indicative
of variation at the nuclear genome level in Architeuthis, then the data strongly suggest that
globally only a single species of Architeuthis exists.
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Works Cited
Guerra A, Rodriguez-Navarro AB, Gonzalez AF, Romanek CS, Alvarez-Lloret P, Pierce GJ.
2010. Life-history traits of the giant squid Architeuthis dux revealed from stable isotope
signature recorded in beaks.
http://icesjms.oxfordjournals.org/content/67/7/1425.abstract?ijkey=4d4555a83154e6903d
90a419731844b6a95d3274&keytype2=tf_ipsecsha
Winkelmann I, Campos PF, Strugnell J, Cherel Y, Smith PJ, Kubodera T, Allcock L, Kampmann
ML, Schroeder H, Guerra A, Norman M, Finn J, Ingrao D, Clarke M, Gilbert MT. 2013.
Mitochondrial genome diversity and population structure of the giant squid Architeuthis:
genetics sheds new light on one of the most enigmatic marine species.
http://www.ncbi.nlm.nih.gov/pubmed/?term=mitochondrial+genome+structure+and+gen
etic+diversity+of+the+giant+squid
Yokobori S, Fukuda N, Nakamura M, Aoyama T, and Oshima T. 2004. Long-Term
Conservation of Six Duplicated Structural Genes in Cephalopod Mitochondrial Genomes.
http://mbe.oxfordjournals.org/content/21/11/2034.full