Class 12 - DNA barcoding and the Tree of Life + TEST OUTLINE!

Outline
• 1. Introduction to DNA barcoding.
• 2. Barcoding example 1: Sharks.
• 3. Barcoding example 2: Zoanthids.
• 4. Tree of Life overview.
• 5. Conclusions.
• 6. Test.
Part 1: Introduction to DNA Barcoding
What is “DNA barcoding”?
遺伝子バーコードというのは？
A DNA barcode is a short sequence, taken from standardized portions of the genome,used to identify species.
遺伝子バーコードとはひとつの配列を利用して、全生物の種類区別を行うこと。
If a genome project is deep and narrow, DNA barcoding is broad and shallow.
Genome projectは深くて、狭いが、遺伝子バーコードは浅くて、広い。
Requirements of a
DNA barcoding marker
遺伝子バーコードの配列の必要な特徴
A sequence/marker used to barcode should:
• be easy to amplify
• 取りやすい、増えやすい。
• not possess paralogues
• ひとつだけのコピー。
• have conserved regions to design primers efficiently for a broad taxonomic sampling
• プライマーが作りやすい配列。
• be variable enough to distinguish species
• 配列の変化レートは種間の区別ができる。
• but conserved enough within species
• 一方、種内の変化は無いほうがよい。
Barcoding does not aim to solve phylogeny!
遺伝子バーコードの目標は分類だけ。関係などを調べるためではない。
Potential applications
1) Facilitating identification and recognition of named (described) species:
linking life history stages, genders.
雌、雄のリンク。
differentiating cryptic species.
cryptic speciesの区別。
traceability of commercialized species.
食べ物などの産地。
identifying gut contents.
生き物の餌の分類。
2) Surveying and inventorying biodiversity;
Identifying new species.
新種の分類。
Range of species.
種類の分布。

Potential applications
1) Facilitating identification and recognition of named (described) species:
Differentiating cryptic species:
Cryptic speciesの区別:
Astraptes属]
mt COI sequence divergence
among North American birds
北米の鳥類のmt COI遺伝子：種内と属内の変異
Barcode of Life project - information available on the internet.


Strengths:
• Offers an alternative taxonomic identification tool for situations in which morphology is inconclusive.
• Focusing on one or a small number of genes enhances efficiency of effort and application.
• Once a reference database is established it can be applied by non-specialist.
• The cost of DNA sequencing is dropping rapidly due to technical advances.
• Potential capacity for high throughput and processing large numbers of samples.
“barcoding gap” exists for many groups of animals, but not all. One key example are benthic cnidarians.
Other weaknesses include old samples (type specimens) in formalin, no “field DNA barcoder” available yet, and the “barcoding gap” issue.
Part 2 - Using genes to identify shark body parts
Shivji et al. 2002
Background
• Sharks threatened due to shark fin demand.
• Need species by species conservation.
• Difficulties in identification make data questionable.
• Genetic identification needed.
Results
• Investigated ITS-2 with new primer sets.
• Used dried fins from Asia & Mediterranean (n=lots!).
• Identification worked on a global scale to species level.

• This system can be used on many taxa that are hunted or need conservation.
• Coral reef applications: sea turtles, red coral, other CITES species.
Part 3 - barcoding zoanthids
Why barcode Zoantharia? Often specimens are small and poorly preserved - barcoding may help!
Some species have much variation - color etc., while other species look identical!

Barcoding and Zoantharia
• Some observed types have much variation, and their status is unknown.
• 分類ができていない。種内変異が大きい。
• Not usually entered into biodiversity estimates.
• 今まで、多様性の調査では無視されている。
• Among mitochondrial genes, COI and 16S rDNA have been widely used in Zoantharia.
• 今までの遺伝子の論文で、mt DNAがよく使われている。
• An examination of the entire mt genome shows no region with high rates of evolution.
• mt DNAには、早い進化の配列が無い。
• ITS-rDNA can NOT be used.
• ITS-rDNAなどは複数のコピーがあるので、利用できない。
• Other nuclear genes may show potential, but common primers do not work.
• Nuclear DNAが利用可能だが、全てのスナギンチャク類に反応するプライマーは不可能。
Our zoanthid barcoding experiment
スナギンチャク目の遺伝子バーコード実験
• Collected zoanthids from around the world (4 families, 7 genera, 65 samples).
• 世界中からスナギンチャクのサンプルを採取した (4科、7属、65サンプル)。
• Examined their mt COI and mt 16S rDNA sequences.
• mt COI と mt 16S rDNA配列をとって、解析した。
• Asked - how effective are the sequences at identifying genera and species? Can they be used for barcoding?
• これらの配列をバーコードできますか？属レベルまでの有効性は？種レベルまでの有効性は？
Coral life cycle - zoanthids have never been truly observed in many life stages; barcoding could help.
Mitochondrial cytochrome oxidase subunit I - COI配列
• 長さは500~650 bp
• Advantages:
– no alignment problems.
– 綺麗に並べる。
– huge database.
– GenBankで、たくさん情報がある。
– common primers (HCO, LCO).
– プライマーが使いやすい。
• Weaknesses:
– very conserved, occasionally cannot distinguish between congeners.
– 配列の進化が遅い、たまに別種の区別ができない。
mitochondrial 16S rDNA配列
• 長さは530~1000 bp
• Advantages:
– Indels.
– Can make specific primers.
– それぞれの属用のプライマーが作れる。
• Weaknesses:
– Still low variation.
– 配列の進化がCOIと同様に遅い。
– Indels tough to align.
– 並べることが難しい。
– No universal primers.
– 全てのスナギンチャク用のプライマーが無い。
16S-rDNA V5配列
• 130 bp
• Advantage:
– Useable with degraded DNA (old, formalin fixed)
Conclusions:
• Both markers were very similar in their effectiveness (90~95%) in identifying species.
• 両方のマーカーは同様に有効性がある。
• Both markers could discern all genera.
• 両方のマーカーは属レベルまでは完璧。
• mt COI is appropriate for broad investigations, while mt 16S rDNA is better for specialized research.
• COIはスナギンチャク目全体を調べるときによい。16Sはもっと細かいレベルの研究でよい。
• Search for “good” nuclear markers continues.
• もっとよいマーカーをまだ探し中。
• For now, any information is better than none!
• スナギンチャク目の場合、何のデータでもデータが無いよりはよい。
Potentials:Among mitochondrial genes, if COI is the most used gene, the large ribosomal subunit (16S) shows interesting variations.

In 16S, the presence of INDELS specific to different groups of species could be a good signature to barcode the order.

Pure barcoding can be very useful at the most to differentiate groups of closely related species.
Combined with geographical and ecological characters it can be a key feature in the taxonomy of Zoantharia.
Preliminary results showed that the V5 region of 16S could be interesting for other anthozoan orders, such as black corals and sea anemones.


• Investigate spawning timing.
• 産卵タイミングの研究。
• Investigate cross-breeding.
• 交配実験。
• Continue to explore the world for new samples.
• 世界中の新種や多様性調査を行う。
• Examine museum specimens.
• 博物館の標本の形態を調べる。
Part 4 - Tree of Life project
http://www.tolweb.org
• Goal is to have one webpage for every species and group of organisms.
• Organized to reflect evolution.
• Hundreds of contributors.
• Aid in learning.
• Link to other databases.

• Authors enter information (data; images; text).
• Automatically formatted and linked to the TOL.
• Branches & leaves.
DNA tree of life figure - animals are very small portion of diversity of life!
Part 5 - Conclusions
More Conclusions
• Large, international, internet-linked projects will become bigger and bigger.
• “Net 2.0” may help harness “people power” to help conserve coral reefs and other biodiverse ecosystems.
Thanks! References cited:
1. Herbert et al. 2004a. Identification of birds through DNA barcoding. PLoS Biology 2 (10): e312.
2. Herbet et al. 2004b. Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator. PNAS 101: 14812-14817.
2. Shivji et al. 2001. Genetic identification of pelagic shark body parts for conservation and trade monitoring. Conservation Biol 16: 1036-1047.
3. Sinniger et al. 2008. Potential of DNA sequences to identify zoanthids (Cnidaria: Zoantharia). Zool Sci 25: 1253-1260.
4. Tree of Life Project. http://www.tolweb.org/tree/.
Test overview
• Format: 11 questions; 1 from each class. Answer 7! Questions will be both sentences and multiple choice style.
• Content: Anything from any class; not only slides but also discussion and talk.
• Bonus question from suggested readings and/or video.