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Conservation Genet Resour
DOI 10.1007/s12686-017-0896-9
TECHNICAL NOTE
Mitochondrial genome of the critically endangered smalltooth
sawfish Pristis pectinata from Veracruz, Mexico
Píndaro Díaz‑Jaimes1 · Ramón Bonfil2 · Paola Palacios‑Barreto3 ·
Nataly Bolaño‑Martinez3 · Natalia J. Bayona‑Vásquez1,4 Received: 21 September 2017 / Accepted: 13 October 2017
© Springer Science+Business Media B.V. 2017
Abstract Sawfishes, family Pristidae, occur in tropical and
subtropical coastal marine, estuarine and freshwater ecosystems. The smalltooth sawfish (Pristis pectinata) has been
wholly or nearly eliminated from large areas of its former
range in the Atlantic Ocean by fishing pressure (trawl and
inshore netting) and habitat modification. Here, we report
the complete sequence of the mitochondrial genome of a
specimen caught in Barra de Cazones by local fishermen,
Veracruz, Mexico in 2015, where was thought to be almost
extinct and compare it with the previously reported mitochondrial genome from an individual caught in Florida.
The genome structure and composition of both specimens
is almost identical, except for two nucleotide variations
remarking the low variation between regions for this species. The mitochondrial genome has a total length of 16803
nucleotides; in average the base composition was A 32.1%, T
28.9%, C 26.0% and G 13.1%, containing 13 protein-coding
genes, 2 rRNA genes; 22 tRNA genes.
* Píndaro Díaz‑Jaimes
pindaro@cmarl.unam.mx
1
Laboratorio de Genética de Organismos Acuáticos, Instituto
de Ciencias del Mar y Limnología, Universidad Nacional
Autónoma de México, Apdo. Postal 70‑305, 04510 Mexico,
D.F., Mexico
2
Océanos Vivientes A. C. [Living Oceans Civil Association],
Cerrada Monserrat #9, La Candelaria. Coyoacán,
04380 Mexico, Mexico
3
Posgrado en Ciencias del Mar y Limnología, Instituto
de Ciencias del Mar y limnología, Universidad Nacional
Autónoma de México, Apdo. Postal 70‑305, 04510 Mexico,
D.F., Mexico
4
Present Address: Department of Environmental Health
Science, University of Georgia, Athens, GA 30602, USA
Keywords Batoidea · Pristidae · Sawfish · Nucleotide
divergence · Genome structure
Abbreviations
rRNARibosomal RNA
tRNATransfer RNA
D-loopControl region
Sawfishes (Family Pristidae) are viviparous benthic batoids
inhabiting warm temperate and tropical shallow coastal
areas and inland waters connected to the sea (Last et al.
2016). They are considered among the most threatened
marine fish taxa in the world due to the strong reduction
on their range and population declines driven by incidental
catch in fisheries, habitat loss and/or degradation (Dulvy
et al. 2016). The distribution of the smalltooth sawfish (Pristis pectinata) was formerly known from New York (USA)
to Rio de la Plata (Argentina) but is now listed as Critically
Endangered throughout its former range by the IUCN Red
List of Threatened Species (Carlson et al. 2014). The only
known current stronghold of smalltooth sawfishes occurs off
Florida (USA; National Marine Fisheries Service 2009). In
the Gulf of Mexico and Caribbean, a recent interview-based
survey indicates that the species is nearly extirpated in this
area, with only one live specimen known to exist (Bonfil
et al. 2017). Therefore, we sequence and annotate the mitochondrial genome of this specimen using NGS techniques.
A sawfish specimen of a P. pectinata juvenile female
1.54 m TL, was caught by local fisherman on January 20th
2015 in Barra de Cazones, Veracruz, Mexico (Bonfil et al.
2017) under Promotion Fishing Permit PPF/DGOPA-091/15
and collection permit SGPA/DGVS/07618/15. The specimen
is alive and safeguarded in the Veracruz Aquarium. A tissue
biopsy from the second dorsal fin was obtained and stored
13
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in the Laboratorio de Genética de Organismos Acuáticos at
the Instituto de Ciencias del Mar y Limnología (ICMyL),
Universidad Nacional Autónoma de México (UNAM).
Genomic DNA (gDNA) was isolated using a standard
phenol:chloroform protocol on 200 mg of fin tissue. The
DNA pellet was washed with 200 μl of 70% Ethanol. After
drying, the pellet was resuspended in 50 μl TE buffer 1× (pH
8) and stored at 4 °C.
Conservation Genet Resour
For library preparation the isolated gDNA was sheared
by sonication with ­Bioruptor® by using five cycles of 30 s
sonicating and 30 s without sonication, to obtain optimal
fragment size (≤ 500 bp). The KAPA ­B IOSYSTEMS ®
Hyper Prep Kit (KR0961—v4.15) was used for library
preparation protocol. After end reparation and A-tailing,
the DNA was ligated to stubs and libraries were amplified through PCR using Illumina universal iTru primers
Fig. 1 Mitochondrial genome of the unique known live specimen of small tooth sawfish P. pectinata (in the picture) caught off Mexico coasts
and kept in the Veracruz aquarium
13
Conservation Genet Resour
containing custom nucleotide indexes for both reads (Glenn
et al. 2016). Amplified fragments were purified and size
selected in a ~ 250–450 pb range with the use of SpeedBeads
(Rohland and Reich 2012). This library was normalized and
pooled with other iTru libraries from other projects. Library
sequencing was carried in an Illumina™ HiSeq 3000 to produce paired-end 150 nucleotide reads at Oklahoma Medical
Research Foundation Clinical Genomics Center.
The total reads were assembled in G
­ eneious® 10.0.9 using
as reference the mtDNA genome of a P. pectinata individual
from Florida (Accession KP400584, Chen et al. 2016). We
used BBMap plugin with normal sensitivity. The consensus
sequenced was obtained using the highest quality threshold,
which only calls a base when the total quality for that base
exceeds the 60% of the total quality for all bases. Those variants present in the new assembled mitochondrial genome
were checked and the new genome was annotated using
the web interface MitoAnnotattor (Iwasaki et al. 2013).
We aligned the assembled mtDNA genome from the Gulf
of Mexico with the reported in GenBank using the MUSCLE plugin within Geneious v. 10.0.9 including mtDNA
sequences of Pristis clavata (NC022821) and Anoxypristis
cuspidata (KP233202). A Bayesian inference (BI) was conducted for phylogeny reconstruction using MrBayes 3.2.1
(Ronquist et al. 2012) using sequences of Rhynchobatus australiae (KU746824) and Rhina ancylostoma (KU721837) as
out-group. We used partition by gene previously determined
using the best fitting nucleotide substitution model assessed
through the Bayesian Information Criterion (BIC) approximation in jModeltest 2.1.7 (Darriba et al. 2012). Analysis
in MrBayes consisted of four simultaneous runs and four
Markov chains using 10,000,000 generations, with a burn-in
of 25% of the initial trees.
We recovered a total of 994,666 paired reads, which
after assembled resulted in the complete genome sequence
containing 16,803 nucleotides (GenBank Accession No.
MF682494), which is one nucleotide larger than its Florida
counterpart (Chen et al. 2016). The P. pectinata mitogenome
from the Gulf of Mexico is equivalent in size and structure
to that from Florida containing 13 protein coding genes, two
rRNA genes and 22 tRNAs and the control region of 1103 nt
(Dloop) (Fig. 1). Showing a nucleotide base composition of
A: 32.1%, T: 28.9%, C: 26.0% and G: 13.1%.
The pairwise alignment between both P. pectinata specimens reveals that the genomes present a pairwise identity of
99.99% (DXY = 0.005%) showing only two polymorphisms,
a G -> A substitution at base 4506, and one A nucleotide
insertion at position 15,642, within the D-loop region. However when comparing with P. clavata, a congeneric species
distributed in Australia, the mean nucleotide divergence was
9.5%. This result highlights the low polymorphism contained
in the mtDNA genome for the species since both individuals
most probably belong to different populations as they were
caught at rivers separated by about 1733 km and the species is known not to perform long migrations (Carlson et al.
2014). This fact, besides the scarce registers of the species
on the main tributaries from the Gulf of Mexico, should
call the attention on implementing stronger measures to successfully protect the species’ population. The phylogenetic
tree grouped clearly the Pristis species in a clade separated
from A. cuspidata with a mean nucleotide divergence of 13%
whereas within the clade P. pectinate was separated from
P. clavata by a mean nucleotide divergence of 9.5%. All
Fig. 2 Bayesian phylogenetic
tree showing the relationship
between P. pectinata and P.
clavata using R. australiae and
R. ancylostoma as out-groups.
Values in branches are the
nodes support. MX Mexico, FL
Florida
13
Pristidae species separated with a mean divergence of 15.7
and 16.9% from R. asutraliae and R. ancylostoma respectively (Fig. 2).
Acknowledgements Special thanks go to the fishermen Constantino Correa Santes, Sabino Diego Bautista, and Senovio Guerrero
Domínguez, and to Lic. Saaid Hernández Osorio all from Barra de
Cazones, Veracruz, for rescuing the fish alive and facilitating collection
of tissue samples. Financial aid for collection was provided by Océanos
Vivientes, A.C. the Save Our Seas Foundation and Comisión Nacional
de Áreas Naturales Protegidas (CONANP). We thank the Comisión
Nacional de Pesca (CONAPESCA) and Dirección General de Vida
Silvestre (DGVS) for research and collection permits. Genetic work
was supported by PDJ Institutional Grant 611.
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Conservation Genet Resour
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