dna barcoding lt cichlids of the rocky...
TRANSCRIPT
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DNA barcoding LT cichlids of the rocky shores
Breman FC1, Van Steenberge M2, Jordaens K1 & Snoeks J2
1 Royal Museum for Central Africa 2 Royal Museum for Central Africa and Catholic University of Leuven
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DNA barcoding of LT cichlids of the rocky shores
• Introduction to the topic • Methods
– Collection – Specimens – Methods
• Specimens • Data and library setup • Sequence based • Species based
– Analysis • Sequence based identification • Species based identification
– Results • Sequence based • Species based • Species complexes • Example of a complex and a newly described species
– Testing OTU´s
• Discussion • Questions?
Tropheus duboisi
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Introduction
• Fish DNA barcoding successful so far
• Most species can be identified
• Different methods can be used
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Introduction
• Habitat – rocky shores of Lake
Tanganyika – Alkaline environment
(average pH 8,4) – Highly diverse habitats – Highly specialized
• Breeding strategies – Mouth brooders – Shell brooders – Substrate brooders
• Feeding strategies – Predation – Algae scraping
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Introduction
• 200+ recognized species of cichlids
– Dozens remain to be described
– >95% endemic
• Model organisms for evolution and speciation
• Economic importance with aquarium enthusiasts
• 75 non cichlid fishes are also present in LT
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Lake Tanganyika
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Collection sites
• 676 km from N-S and average 50 km across
• Average depth 570m (max 1470m)
• 4 countries
– Tanzania
– Congo
– Burundi
– Zambia
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Collection sites
• 15 sites
• 3 expeditions (1992, 1995, 2010)
• 1000s of specimens
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Lepdiolamprologus elongatus
Tropheus brichardi
Lobochilotus labiatus
Gnatohochromis pfefferi
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Methods Specimens
• Covering 11 tribii and 37 genera
• Library A) 78 (98 with singletons) OTUs
• Library B) 70 (91 with singletons) OTUs
• Library C) 52 (66 with singletons) OTUs (11 complexes)
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Methods Data setup
• Three groups
– A) all taxonomic, behavioural and distributional knowledge (published and unpublished) were used to assign a name to a specimen
– B) only currently recognized species used for assigning a name to a specimen
– C) groups with known difficulties in evolutionary history (hybridization, incomplete lineage sorting) and taxonomy are grouped in species clusters
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Methods Sequence based
– BM/BCM method
– Software compares each sequence to all the others and the chosen threshold for the dataset
– Returns a statement on the sequence/specimen with regard to threshold and presence of same/related species
– Returns a success percentage in terms of sequences
– Influenced by dataset properties
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Methods Species based
– Uses the sequences assigned in the sequence based method, but now classified according to threshold per species
– Species identifiable or not
– Returns success percentage in terms of species
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Analysis Species based BM/BCM
• Categories
– True Negative (TN) Best match above threshold and allospecific
– True Positive (TP) Best match is same species below threshold
– False Positive (FP) Closest match is below threshold but is an allospecific
– False Negative (FN) Intraspecific distances are above threshold and conspecific
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Threshold
• Fixed or based on dataset, in our case threshold was determined via an R script
• Obtained via two curves of intra and inter specific distances
• The optimum is then chosen as threshold
FN FP
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Analysis Species based using a NJ tree
• Using a NJ tree
• Counting species in distinct clusters
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Results Sequence based
(A)morphospecies (B)accepted names (C) species complexes
optimal threshold 1.91% 2.37% 2.20%
Sequences: 398 398 398
Correct id's according to "Best Match": 276 (69.34%) 320 (80.4%) 352 (88.44%)
Ambiguous according to "Best Match": 80 (20.1%) 36 (9.04%) 21 (5.27%)
Incorrect id's according to "Best Match": 42 (10.55%) 42 (10.55%) 25 (6.28%)
Correct id's according to "Best Close Match": 274 (68.84%) 318 (79.89%) 349 (87.68%)
Ambiguous according to "Best Close Match": 80 (20.1%) 36 (9.04%) 21 (5.27%)
Incorrect id's according to "Best Close Match": 31 (7.78%) 34 (8.54%) 17 (4.27%)
Sequences without any match closer than threshold 13 (3.26%) 10 (2.51%) 11 (2.76%)
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Results Species based
Library # species TP FP TN FN percentage ID
success A 78 55 14 5 4 0.71 B 70 46 21 2 1 0.59 C 52 40 3 6 3 0.51
Relative ID error Precision Overall ID error Accuracy A 0.20 0.80 0.23 0.77
B 0.31 0.69 0.31 0.69 C 0.07 0.93 0.12 0.88
% success NJ
0.74 0.74 0.81
-Marginal improvement when species complexes in success %, -but an increase in accuracy and precision
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Results Species complexes
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Results Species complex and new species
• Examples of species complexes with unresolved clusters
• Eretmodus cyanostictus specimens have recently been described as Eretmodus marksmithi
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Results • Identification of putative new species with
DNA barcodes
putative species based on expert opinion
# sequences in dataset
intrasp. dist. < intersp dist. to nearest other species (y/n)
dist. to nearest other species
average generic distance +/- se
putative species y/n (difference > generic average +/- se)
Chalinochromis spbifrenatus 1 NA 2.33 1.84 +/- 0.42 y Ectodus cfdescampsi 3 y 1.7 1.45 +/- 0.33 n Neolamprologus cfpetricola 1 NA 0.61 5.70 +/- 0.55 n Neolamprologus speseki 3 y 1.07 5.70 +/- 0.55 n Petrochromis cfmacrognathus 2 y 0.15 2.45 +/- 0.36 n Petrochromis spephippiumsouth 2 y 1.39 2.45 +/- 0.36 n Petrochromis sppolyodonelongate 3 y 0.76 2.45 +/- 0.36 n Petrochromis sppolyodonhigh 2 n 0.15 2.45 +/- 0.36 n Tropheus cfannectens 8 y 1.54 1.55 +/- 0.26 n Tropheus ikola 3 n 0 1.55 +/- 0.26 n Tropheus mpimbwe 51 n 0 1.55 +/- 0.26 n
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Discussion
• LT cichlids well studied
– Genetically and morphologically
• They are examples of ongoing speciation
– mtDNA evolution is slower than morphological evolution
• Hybridisation is common
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Discussion
• Incomplete taxonomy • Unbalanced dataset, influence of sequence
composition • Success percentages not high compared to other
fish groups, however still ok for a complex group such as this.
• Distance method not very useful for detecting potential new species
• Single sequences cannot be evaluated with either method
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Questions?
Thank you for your attention
感谢您的关注
Foto credits: Royal Museum for Central Africa Maarten Van Steenberge
Dimitri Geelhand de Merxem