DNA barcoding and phylogeny reconstruction

DNA barcoding and evolutionary relationships in
Accipiter Brisson,1760 (Aves,
Falconiformes: Accipitridae) with a focus on
African and Eurasian representatives
• A. gentilis: Nl; havik, Eng; gosshawk, Fr;
Autour des palombes
• A. nisus: Nl; sperwer, Eng; sparrowhawk,
Fr; epervier d’Europe
• Buteo buteo: Nl; buizerd, Eng; Common
buzzard Fr; Buse variable
• Buteo albicaudatus; Nl; witstaart buizerd,
Eng; white tailed hawk, Fr; Buse à queue
• DNA barcoding a short
– General DNA barcoding
– Mini barcodes
– 10X threshold, best compromise
– BCM and BM criteria
– Character based identification
• Coverage
– Sample origins
– The dataset
• Methods
– Character based method (CB)
– The 10 X intraspecific distance
threshold (10 X)
– The Best compromise threshold
– Best match and best close match
• Results for DNA barcoding
– Character based identification
– Different thresholds
• Phylogeny reconstruction
– 291 bp dataset
– 647 bp dartaset
• Discussion
• Conclusion
DNA barcoding
• Identification of species
with an agreed upon (part of
a) gene (COI)
• 5’-3’ part of Cytochrome C
Oxidase subunit I
• ~650bp in length
• Polymerase chain reactions
and Sanger sequencing
Mini barcodes
• Genetic material from museums is often degraded
• Shorter fragments are needed
• E.g. sequences of 250-300bp or, in some cases even
shorter, also work for identification purposes
Sonet et al. in press
Sample coverage and origins
sample distribution through time
number of samples included
• African and European taxa
are well represented
• The new world partially
• Australia and S-E Asia
poorly represented
• Most samples from
RMCA/RBINS (n=83/9)
• Supplemented with
available genbank
sequences (n=28)
30-year periods
The resulting dataset
• 25 European and African Accipiter species with a total of 140
• sequenced for a 291bp (mini) and 647bp (standard) BC
fragment of the COI gene
• three datasets analysed:
– dataset A: 25 species, 291bp
– dataset B: 19 species, 647bp
– dataset C: 19 species, 291bp
Character based indentification
– Easy it is about detecting unique differences
– May be unreliable with insufficient sampling
– No threshold whatsoever is used
10 X intraspecific distances and the barcoding
• Distances
• Arbitrary
• Does not always hold
• However, works well in
The Best compromise threshold (BCTh)
• Based on the intercept
between cumulative intra
and interspecific distances
• Based on the dataset under
study rather than a
predetermined threshold
• Results from one dataset not
applicable to another
Lefébure et al. 2006
BM and Best Close match
• Best Match (BM), assignes the species name of its bestmatching sequence regardless of how similar the query and
reference sequences were. Identification is considered correct
when both sequences were from the same species, incorrect if
the query species differed from the closest reference species or
ambiguous if multiple species were the BM of the query
• Best close match (BCM) also considers the threshold
BM vs. BCM example
• 4 taxa
• 3 species: A. francesiae (n=2) A. badius (n=1) & A. toussenelii (n=1)
• BM would assign A. badius and A. toussenelii to the “ambiguous” or
“incorrect” (dependent on the threshold) categories
• BCM would classify them as impossible when the difference is larger than
the threshold
Results character based identification
Results BCTh vs. 10 X distance threshold
Results BCTh vs. 10 X distance threshold
• Different thresholds
• Partial overlap
• Caused by
– A. nisus/rufiventris
– A. virgatus/gularis
– A. cooperii/gundlachi
A. nisus
• At or just below the
treshold are
– A. badius subsp
– A. gentilis subsp
A. rufiventris
Phylogeny reconstruction
Hypothesis of evolution
Clades are units of supported taxa (related by direct descent)
This dataset is only suited for low taxonomic level inferences
One genegene tree rather than species tree
Comparison with the current taxonomy based on morphology
and morphometrics
Phylogeny based on 291bp
• 10 supported clades
– [tachiro]
• A. tachiro subclades
A. minullus
A. francesiae/ soloensis
A. badius/ brevipes
A. nisus/ striatus/
– A. ovampensis/
– [virgatus] including A.
Phylogeny based on 291bp a closer look
Phylogeny based on 647bp
• 19 supported clades
– [tachiro]
• A. tachiro
• A. toussenelii & subsp
– A. soloensis & A.
francesiae subsp
– [gentilis]
– [cooperii]
– [nisus] (without A.
– [minullus] (without A.
– [virgatus] including A.
Phylogeny based on 647bp a closer look
• Thresholds
– The 10 X threshold would have considered A. brevipes and A. badius,
as well as A. gentilis and A. melanoleucus as belonging to the same
species and
• Species concepts
– Morphological
– Distances based
– Phylogenetic
• Support values
– Models and interpretation remain issues of discussion
• Sampling
– Incomplete or lacking in some cases
• Molecular techniques provide a powerfull method for
reviewing taxonomy
• Museum material works!
• The content of the dataset matters
• COI can identify 19 out of the 25 species included
• COI carries a phylogenetic signal suitable for molecular
taxonomy on a low taxonomic level
• Parts of genus Accipiter may need revision
Thank you for your attention
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