209
The classification of microbial life from species to higher
taxa is largely based on the small subunit ribosomal RNA
(SSU rRNA) gene. Microbial molecular ecology employs
the SSU rRNA gene for cultivation-independent studies of
communities where it serves both as a phylogenetic marker
in sequence based analysis, and at the organismal level as a
target for molecular probes through fluorescence in situ
hybridization (FISH). With the advent of high-throughput
next-generation sequencing (NGS), shotgun metagenomes
and transcriptomes of full communities that sample from all
domains of life can replace PCR-based SSU rRNA amplicon
libraries as the basis for the ‘full cycle rRNA approach’ in
molecular ecology. We have developed phyloFlash – a software tool to rapidly assess the phylogenetic composition of
metagenomic or transcriptomic libraries, without assuming
extensive bioinformatics expertise from its users. It reconstructs long and exact (>1200 bp) SSU rRNA sequences that
are suitable for high-resolution phylogenetics and probe
design, from short-read NGS libraries. The software builds
upon a curated version of the SILVA SSU rRNA database
and generates a taxonomic community profile, taxonomicallyannotated full-length SSU rRNA sequences for bacteria,
archaea and eukaryotes, and interactive plots. Multiple
libraries can also be compared on the basis of their taxonomic composition. We have employed phyloFlash-based
community composition analysis on diverse marine samples
ranging from single-celled protists, minute worms, giant
deep-sea clams and stratified sediments. The phyloFlash
approach quickly unveiled unique patterns of diversity and
symbiotic associations, such as a novel phylum of intracellular bacteria in animals and the plasticity of chemosynthetic
symbiont communities in a single species of small gutless
worms. The combined improvements in library preparation
methods for NGS ‘omics’ and in software tools such as phyloFlash open a new age of discovery for marine environments where most of the biodiversity has to be investigated
with cultivation-independent methods.
5.2.2 Challenges of Amplifying Degraded
Nuclear and Mitochondrial DNA from Historical
Dugong Skulls
Morgan L. McCarthy
1,2*
, Kieren J. Mitchell
3
, Jennifer
M. Seddon
2
, Janet M. Lanyon
1
1
The University of Queensland, School of Biological
Sciences, St. Lucia, Queensland 4072, Australia
2
The University of Queensland, School of Veterinary
Science, Gatton, Queensland 4343, Australia
3
Australian Centre for Ancient DNA, School of Earth and
Environmental Sciences, University of Adelaide, North
Terrace Campus, South Australia 5005, Australia
*corresponding author: m.l.mccarthy@uq.net.au
Keywords: Dugong, Ancient DNA, Population genetics,
Dugong dugon, Queensland
The use of ancient DNA (aDNA), including DNA
extracted from museum samples has been an instrumental
tool in the study of historical ecology. Sequencing nuclear
DNA for microsatellites and mitochondrial DNA (mtDNA)
provides information on genetic diversity and estimates of
historical populations, giving our modern calculations perspective. The nature of using museum samples is that DNA
degrades overtime and that extraction, sequencing and
amplification is inhibited by sequence fragmentation. As the
densest bone in the mammalian body, the periotic bone of the
petrous portion of the temporal lobe is reported to yield the
least degraded DNA. Dentine from human teeth and enamel
from elephant tusks have both yielded aDNA as well. This
study tests the amplification of microsatellite loci and mitochondrial control regions in DNA extracted from periotic,
cheek teeth and permanent incisors (tusks) of dugongs to
determine the best portion of the skull to use when extracting
DNA from museum samples. Such data can be used to calculate a historical effective population size and compare it with
contemporary estimation methods based on shark-netting
bycatch hind casting models in Queensland, Australia.
Hence, its results will lay the framework to investigate
whether a decline in dugong populations from extreme storm
events and a government sponsored shark-netting program
from the 1960s is reflected as a loss in present day genetic
diversity when compared with present day DNA samples.
5.2.3 On the Way for Detecting and Quantifying
Elusive Species in the Sea: The Octopus vulgaris
Case Study
Quentin Mauvisseau
1*
, Marina Parrondo
1
, María del Pino
Fernández
2
, Lucía García
2
, Jose Luis Martínez
3
, Eva GarcíaVázquez
1
, Yaisel Juan Borrell
1
1
Department of Functional Biology, University of Oviedo,
Calle Julián Clavería S/N, 33006, Spain
2
Centro de Experimentación Pesquera, Dirección de
Pesca Marítima, Gobierno Del Principado De Asturias,
Gijon, Spain
3
Sequencing Unit, Edificio Severo Ochoa, C/Julian
Claveria S/N, University of Oviedo, Oviedo 33006, Spain
*corresponding author: Q.Mauvisseau@derby.ac.uk
Keywords: Environmental DNA, Detection, Fisheries,
Octopus vulgaris, Quantification
Environmental DNA (eDNA) can be a powerful method
for assessing the presence and the distribution of aquatic species. We used this tool in order to detect and quantify eDNA
from the elusive species Octopus vulgaris, using qPCRs
(SybrGreen protocol). We designed species-specific primers,
and set up an experimental aquarium approach to validate the
new molecular tool in different controlled conditions. Field
validation was conducted from sea water samples taken from
8 locations within an octopus fishery area in the Cantabrian
Sea during February–March 2016. A significant positive corAppendices
The classification of microbial life from species to higher
taxa is largely based on the small subunit ribosomal RNA
(SSU rRNA) gene. Microbial molecular ecology employs
the SSU rRNA gene for cultivation-independent studies of
communities where it serves both as a phylogenetic marker
in sequence based analysis, and at the organismal level as a
target for molecular probes through fluorescence in situ
hybridization (FISH). With the advent of high-throughput
next-generation sequencing (NGS), shotgun metagenomes
and transcriptomes of full communities that sample from all
domains of life can replace PCR-based SSU rRNA amplicon
libraries as the basis for the ‘full cycle rRNA approach’ in
molecular ecology. We have developed phyloFlash – a software tool to rapidly assess the phylogenetic composition of
metagenomic or transcriptomic libraries, without assuming
extensive bioinformatics expertise from its users. It reconstructs long and exact (>1200 bp) SSU rRNA sequences that
are suitable for high-resolution phylogenetics and probe
design, from short-read NGS libraries. The software builds
upon a curated version of the SILVA SSU rRNA database
and generates a taxonomic community profile, taxonomicallyannotated full-length SSU rRNA sequences for bacteria,
archaea and eukaryotes, and interactive plots. Multiple
libraries can also be compared on the basis of their taxonomic composition. We have employed phyloFlash-based
community composition analysis on diverse marine samples
ranging from single-celled protists, minute worms, giant
deep-sea clams and stratified sediments. The phyloFlash
approach quickly unveiled unique patterns of diversity and
symbiotic associations, such as a novel phylum of intracellular bacteria in animals and the plasticity of chemosynthetic
symbiont communities in a single species of small gutless
worms. The combined improvements in library preparation
methods for NGS ‘omics’ and in software tools such as phyloFlash open a new age of discovery for marine environments where most of the biodiversity has to be investigated
with cultivation-independent methods.
5.2.2 Challenges of Amplifying Degraded
Nuclear and Mitochondrial DNA from Historical
Dugong Skulls
Morgan L. McCarthy
1,2*
, Kieren J. Mitchell
3
, Jennifer
M. Seddon
2
, Janet M. Lanyon
1
1
The University of Queensland, School of Biological
Sciences, St. Lucia, Queensland 4072, Australia
2
The University of Queensland, School of Veterinary
Science, Gatton, Queensland 4343, Australia
3
Australian Centre for Ancient DNA, School of Earth and
Environmental Sciences, University of Adelaide, North
Terrace Campus, South Australia 5005, Australia
*corresponding author: m.l.mccarthy@uq.net.au
Keywords: Dugong, Ancient DNA, Population genetics,
Dugong dugon, Queensland
The use of ancient DNA (aDNA), including DNA
extracted from museum samples has been an instrumental
tool in the study of historical ecology. Sequencing nuclear
DNA for microsatellites and mitochondrial DNA (mtDNA)
provides information on genetic diversity and estimates of
historical populations, giving our modern calculations perspective. The nature of using museum samples is that DNA
degrades overtime and that extraction, sequencing and
amplification is inhibited by sequence fragmentation. As the
densest bone in the mammalian body, the periotic bone of the
petrous portion of the temporal lobe is reported to yield the
least degraded DNA. Dentine from human teeth and enamel
from elephant tusks have both yielded aDNA as well. This
study tests the amplification of microsatellite loci and mitochondrial control regions in DNA extracted from periotic,
cheek teeth and permanent incisors (tusks) of dugongs to
determine the best portion of the skull to use when extracting
DNA from museum samples. Such data can be used to calculate a historical effective population size and compare it with
contemporary estimation methods based on shark-netting
bycatch hind casting models in Queensland, Australia.
Hence, its results will lay the framework to investigate
whether a decline in dugong populations from extreme storm
events and a government sponsored shark-netting program
from the 1960s is reflected as a loss in present day genetic
diversity when compared with present day DNA samples.
5.2.3 On the Way for Detecting and Quantifying
Elusive Species in the Sea: The Octopus vulgaris
Case Study
Quentin Mauvisseau
1*
, Marina Parrondo
1
, María del Pino
Fernández
2
, Lucía García
2
, Jose Luis Martínez
3
, Eva GarcíaVázquez
1
, Yaisel Juan Borrell
1
1
Department of Functional Biology, University of Oviedo,
Calle Julián Clavería S/N, 33006, Spain
2
Centro de Experimentación Pesquera, Dirección de
Pesca Marítima, Gobierno Del Principado De Asturias,
Gijon, Spain
3
Sequencing Unit, Edificio Severo Ochoa, C/Julian
Claveria S/N, University of Oviedo, Oviedo 33006, Spain
*corresponding author: Q.Mauvisseau@derby.ac.uk
Keywords: Environmental DNA, Detection, Fisheries,
Octopus vulgaris, Quantification
Environmental DNA (eDNA) can be a powerful method
for assessing the presence and the distribution of aquatic species. We used this tool in order to detect and quantify eDNA
from the elusive species Octopus vulgaris, using qPCRs
(SybrGreen protocol). We designed species-specific primers,
and set up an experimental aquarium approach to validate the
new molecular tool in different controlled conditions. Field
validation was conducted from sea water samples taken from
8 locations within an octopus fishery area in the Cantabrian
Sea during February–March 2016. A significant positive corAppendices