Final Marks

Final marks have been decided for all students. If you wish to know your mark, please come see JDR in his office any time.

The average was 78% - good job!

Test results

Today was the final test for this class. The test itself was worth 30% of the final mark. Marked tests are outside my office - Science 353.

Here are some statistics from the test:

Average: 71.4% plus/minus 16.4% (n=48) = lots of variation in the marks!
High mark: 101% - thanks to the bonus question.
Low marks: in the 30s.

Pass for this test, which I had originally set at 60%, was lowered to 50%.

Students who did not pass the test (and a pass is mandatory) have one last chance to pass - please come see me for details.

For your interest, here are the average marks for each question:
Q1: 6.4/10
Q2: 8.4/10
Q3: 8.7/10
Q4: 8.3/10
Q5: 7.3/10
Q6: 7.4/10
Q7: 4.7/10
Q8: 4.6/10
Q9: 5.4/10
Q10: 5.1/10
Q11: 8.7/10
Bonus: 2.9 extra points

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.

Class 11 - Conservation II

Happy New Year!
今年も宜しくお願いします。

Outline
• 1. Centers of endemism.
• 2. How to stop bleaching (?).
• 3. The importance of fish and mangroves to coral reefs.
• 4. Not just coral reefs: how other ecosystems are connected.
• 5. Conclusions.
• 6. Video: Coral reef organisms.
Part 1 - Centers of endemism
Roberts et al. 2002
• Examined 3235 species of fish, corals, snails, lobsters.
• Ranges in 1 X 1 degree squares (cells).
• 7.2 to 53.6% have restricted ranges; vulnerable to extinction.
• Looked for centers of endemism.

• 10 richest centers cover 15.8% of reefs; 0.012% of oceans.
• Contain 44.8 to 54.2% of restricted range species.
• Major biodiversity hotspots.

Hotspots threatened by human activity.
Conservation should focus on these areas.

• Many threats linked to land.
• Many hotspots next to land hotspots.
• Integrating conservation may be effective.
Part 2 -
How to stop bleaching (?)
West & Salm 2003
• What factors help corals against bleaching?
• Reviewed all research up until 2003.

• Many examples of resistance to or recovery from bleaching.

• Many factors contribute to resistance.
• Can be included in management plans.
• Cumulative stresses worse than one stressor.
Many factors contribute to resilience.
Can be included in management plans.
Healthy diverse reefs more stable than reefs under threat.


Mumby et al. 2004
• Reef fish often use mangroves as nurseries.
• But can use other environments, not confined.
• Also, despite deforestation, other pressures (fishing, larval supply) likely larger.
Examined biomass and numbers of grazing fish at reefs with and without connecting mangrove forests.
Consistently shown despite higher fishing pressure reefs with mangroves have more fish!
Reefs that have lost mangrove have extinctions.


• Management should include connected habitats, not islands of each type.
• Future destruction of mangroves will have negative influence on reef.
Mumby et al. 2007
• Caribbean reefs have damage from loss of Diadema antillarum and two species of coral.
• “Sick” reefs characterized by macroalgae.
• Can macroalgae be reversed? Or is it a stable state?

• Used computer modeling and simulation.
• Showed reefs can easily change to other states once D. antillarum died off.
• With only parrotfish as grazers, small negative change in parrotfish numbers results in macroalgae blooms.
• Coral becomes unstable state with low grazing.

• Regular impact of hurricanes worsens with lack of grazers (fish and urchins).
• Modeling useful for conservation targets.
Part 4 - Not just coral reefs: how other ecosystems are connected
Baum et al. 2003
• Examined shark populations in NW Atlantic.
• Sharks caught in large numbers by longline fishing nets.
• No data until this study.


• All species examined showed rapid declines over last 15 years.
• Most species declined over 50%.
• Three species declined over 75%.

• Recommends marine conservation areas AND reduction in fishing.
• This is because sharks have large ranges and slow rates of population growth.
• Sharks worldwide in danger.
James et al. 2005
• Examined leatherback turtle migration and ranges using satellite telemetry (n=38).
• Tagged off E. Canada.
• Leatherback turtle now critically endangered (IUCN) despite worldwide distribution.

• Turtles migrated south to Caribbean and back within 1 year.
• Most turtles avoided areas of protection; and most spent time in areas of longline fishing.

• Turtles vulnerable to 1) northern coastal and 2) shelf water fisheries.
• Action must be taken to prevent; current conservation is not enough.
Part 5 - Conclusions
1. Coral reefs are linked to other ecosystems, both marine and on land.
2. Conservation plans must protect linked areas and ecosystems, and not “islands”.
3. Protected areas should be decided based on endemism, geographic features, neighboring ecosystems, etc.
4. We must do MORE!

Thanks! References cited:
1. Roberts et al. 2002. Marine biodiversity hotspots and conservation priorities for tropical reefs. Science 295: 1280-1284.
2. West & Salm. 2003. Resistance and resilience to coral bleaching. Conservation Biol 17: 956-967.
3. Mumby et al. 2004. Mangroves enhance the biomass of coral reef fish communities in the Caribbean. Nature 427: 533-536.
4. Mumby et al. 2007. Thresholds and the resilience of Caribbean coral reefs. Nature 450: 98-101.
5. Baum et al. 2003. Collapse and conservation of shark populations in the northwest Atlantic. Science 299: 389-392.
6. James et al. 2005. Identification of high-use habitat and threats to leatherback sea turtles in northern waters: new directions for conservation. Ecol Letters 8: 195-201.

Class 10 - Conservation I

Outline
• 1. Review of link between diversity and conservation.
• 2. Lionfish invading the Atlantic.
• 3. Red coral in the Mediterranean.
• 4. Coral reef conservation - unique problems.
• 5. Community conservation in the Philippines.
• 6. Conclusions.
Part 1 - Review of biodiversity and conservation
Biodiversity
• Biodiversity = Number of taxa (species, genera), or ecosystem types, etc.
• Biodiversity = bioresources.
• Bioresources = long-term economic well-being.
• Conserving biodiversity is important; we need to understand baseline biodiversity.
• Many “neglected taxa” remain.
保全と多様性のリンク
• Species diversity (# of species) for many groups of animals and plants unknown - lack of taxonomy.
• 分類学の研究が足りないせいで、色々な生物の集団の種類多様性（種の数）がほとんど知れていない状態。
• 99.5% of species go extinct before we even describe them.
• 99.5%の種類は、分類する前に絶滅になってしまう。
• Without knowledge of species, how can we protect them?
• 種類の分類が無いと、保全ができない。
• Therefore, taxonomy and diversity VERY important.
• 分類学や多様性の理解が重要な研究。
• BUT…
Dangers facing coral reefs
• Global warming is raising the temperature of the ocean; this kills corals - “coral bleaching”.
• Also, as the oceans become more acidic, it is more difficult for corals to make their skeletons.
• Perhaps 90% of coral reefs will be dead by 2050.


• Crown-of-thorns starfish outbreaks

• Dynamite and cyanide fishing

Part 2 - Lionfish invading the Atlantic

• Lionfish known from the Indo-Pacific.
• Mainly eat reef fish, and often larvae or juveniles.
• Popular in the aquarium trade despite poison.

• Marine fish introductions less common.
• Most introductions due to purposeful introduction for fisheries, or released aquarium fish.
• Success often investigated.

• Whitfield et al. (2002) document several sightings (n=19) of Pterois volitans along E. Atlantic.
• Four specimens collected, numerous juveniles sighted, two collected.
• First introduction of Pacific fish to Atlantic.


• Likely limited by cold waters, but surviving.
• Can spread to Bermuda and Caribbean.
• Similar fish in this region overfished, niche is available perhaps!

Introduction?
• Introduction method; 2 possibilities.
• Ballast water possible, but no reports thus far.
• Aquaria very likely. Specimens known to have been released occasionally.
• Morphology appears to be typical of aquaria types.
Effects?
• No fish in region used to lionfish.
• No predators.
• Need genetic and temperature studies.
• Modeling needed.


Spreading populations
• Since sightings in 2000, lionfish have spread.
• Now known (Snyder&Burgess 2006) from Bahamas.
• Apparently spreading throughout Caribbean.
• Easy to document spread.
Genetic studies
• Since Whitfield et al (2002), more studies.
• Hamner et al. (2007) used mt DNA to examine specimens.
• Two markers (cyt B, 16S rDNA) previously used on lionfish in native ranges.

• Found two species of lionfish; P. volitans (93%) and P. miles (7%).
• Very reduced genetic diversity!
Minimum-spanning network analyses - P. volitans
• Atlantic specimens likely from Indonesia.

• P. miles source unknown.

• Reduced genetic diversity clear.
• Founder effect! Minimum of 3 P. volitans and 1 P. miles established populations.
• Invasions may be rapid and irreversible.
• Education needed.
Part 3 - Red coral in the Mediterranean
Red coral
• Corallium rubrum is a precious coral in the Mediterranean.
• Found 10 -250 m.
• Harvested for long time, over-exploited.
• Harvest reduced 66% in last 15 years.

Population structure
• Two population types, large deep colonies and shallow small colonies.
• Large drop off in shallow water at age 4, due to sponges and collection.

• Genetic distance becomes significant at 100s of kms.
• Thus, preservation of numerous populations needed.
• Management on regional scale needed.
• Must avoid local extinctions.
Conservation recommendations
• Must be managed at national and international scales.
• Only policy that works for such species.
• Set minimum colony sizes, maximum yield per area, harvesting seasons.
Part 4 - Conservation problems unique to coral reefs

• On land, biodiversity hotspots arise from small ranges and endemism.
• Examples include the Galapagos and Ryukyu Islands.

Hughes et al. 2002
• Coral reefs are different.
• Central Indo-Pacific (coral triangle) has very high biodiveristy.
• Arises from many species with large ranges having overlapping ranges in CIP.

• No correlation between numbers of coral endemics and reef fish endemics at locations, even though total numbers related.
• Endemism does not contribute much to high biodiversity of coral reefs.

• Centers of high biodiversity and endemism are separate!
• Two part approach to conservation needed.

Implications for conservation
• 8% of the CIP has 83% of coral species and 58% of fish species.
• CIP protection is cost effective.
• Endemics at peripheral locations (e.g. Red Sea, Madagascar).
• Such locations have lower biodiversity, higher risk for extinction.
Recommendations
• MPAs now too small in size and number, too far apart.
• Focus on fish and mega-fauna, also coral.
• Small and cryptic taxa ignored.
• Need to work more on these taxa.
Part 5 - Community conservation of coral reefs
History
• Philippines consist of 7000+ islands.
• Centuries have used reefs for livelihood.
• Since 1970s, threatened by over-exploitation and destructive fishing methods.

• Conservation started in 1974. Many projects failed.
• Politics tied to conservation.
• Local governments have authority but not knowledge or budget.
• To be successful, combination of local and national people.
• Within local group, must include users of reef; fishermen, resort owners, coastal residents, scuba divers.
Start of conservation
• MDCP started in 1986 on three islands (62-166 households); Apo, Pamilacan, Balicasag.
• All had less fish catch, increasing destruction and poverty.

MCDP plan
• Marine reserves with buffer areas to increase number and diversity fish.
• Development of local knowledge and alternative work.
• Community center.
• Outreach and replication program.
MCDP steps
• Integration into community.
• Education - marine ecology and resource management.
• Group building, formalizing, strengthening.
Results
• Apo & Pamilacan remain strong.
• Balicasag protection groups somewhat weakened due to large PTA resort and less local “ownership”.
• PTA has good points too.
• All islands have stronger municipal laws now.
Results
• Local fisherman believe sanctuary has helped.
• Comparison of 1985-86 data with 1992 shows increases in fish, stable coral cover.
Conclusions
• MPAs work on small islands by preventing destructive fishing and making locals understand value of conservation.
• Small islands easier to implement plans.
• Immediate benefits must be seen.
• Baseline data necessary.
• Local fishermen help with MPA location decisions.
Conclusions
• Locals must understand how problem and answer related.
• Management groups must have respected members.
• Link with all potentially helpful groups.
• All plans vulnerable to politics and outside groups.
Part 6 - Conclusions

Conclusions 2
• In the future, more conservation plans will be implemented.
• The gap between well protected areas and those not protected will widen.

Conclusion 3
• Very few non-protected reefs will survive.
References cited:
1. Whitfield et al. 2002. Biological invasion of the Indo-Pacific lionfish Pterois volitans along the Atlantic coast of North America. Mar Ecol Prog Ser 235: 289-297.
2. Snyder & Burgess. 2007. The Indo-Pacific red lionfish, Pterois volitans (Pisces: Scorpaenidae), new to Bahamian ichthyofauna. Coral Reefs 26: 175.
3. Hamner et al. 2007. Mitochondrial cytochrome b analysis reveals two invasive lionfish species with strong founder effects in the western Atlantic. J Fish Biol 71: 214-222.
4. Santangelo & Abbiati. 2001. Red coral: conservation and management of an over-exploited Mediterranean species. Aquatic Conserv Mar Freshwater Ecosys 11: 253-259.
5. Hughes et al. 2002. Biodiversity hotspots, centres of endemicity, and the conservation of coral reefs. Ecol Let 5: 775-784.
6. White & Vogt. 2000. Philippine coral reefs under threat: lessons learned after 25 years of community-based reef conservation. Mar Poll Bull 40: 537-550.

Class 9 - Reticulate Evolution

Outline
• 1. Review of evolution.
• 2. Introduction to reticulate evolution.
• 3. Examples from plants and fish.
• 4. Examples from corals.
• 5. Examples from zoanthids.
• 6. Conclusions
Part 1 - Evolution

Genetic Diversity
• Required to adapt to change in environment.
• Many methods of measurement.
• Large populations of naturally breeding animals have high genetic diversity.
• Reduced populations are concern.
Cnidaria DNA
刺胞動物の遺伝子
mitochondrial DNA (mt DNA)
• evolves very slow in Cnidaria, opposite to most animals.
• 他の動物と違い、刺胞動物で進化が遅い。
DNA amd phylogenetics: All cells contain DNA - the code or blueprint of life.
全ての細胞には遺伝子が入っている。遺伝子は生き物の設計図。
This code has only four different “letters”: A, G, C, T.
遺伝子は４つのコードしかない。
Usual length 105 to 1010 base pairs.
生き物のひとつの細胞にある遺伝子の長さは105 to 1010 。
Genome projects read everything in one organism, but takes time and expensive.
全ての遺伝子を読むことは時間とお金の無駄。
Many studies use one or a few “markers” to investigate relations.
遺伝子の短い部分だけでも系統関係が解析できる。

• By collecting the same marker from different samples and then analyzing them, we can make a tree.
• いくつかのサンプルから同じマーカーを読んで、並べてから、解析し系統樹を作る。
• It is thought/hoped a tree is similar to how evolution occurred.
• 系統樹から進化が見えると思われる。
Part 2 -
Reticulate Evolution
What is evolution?
進化というのは？
• The descent of all organisms from a common ancestor.
• 全生物は共通の祖先から。
• The development of unique traits in response to environment, etc.
• 環境の変化などのせいで、それぞれのグループがユニークな特徴を持つ。
• Groups gradually “drift” away from each other.
• それぞれのグループが他のグループからだんだん離れる。
• But…
Some problems…
いくつかの問題点がある
• How can “mega”-diversity arise?
• 非常に高い多様性はどうやって進化した？
• Even allowing for rapid evolution, there are cases of “mega”-diversity in very new and small environments, with many species adapted to very specific niches (plants, cichlids etc.).
• 時として、新しい環境で、種の数が想像以上に多い。
• Often hard to accurately explain “species” over large geographic scales.
• large geographic scaleで、種の説明や分類が困難になる場合がある。
• How can hybridization between species be explained?
• 別種のhybridizationも説明がしにくい。
Theory of evolution over time
• Evolution is evolving.
• Darwin - classic model.
• Currently, reticulate evolution is a “rare nuisance”.
• Likely our ideas will develop into an even more complex model.
Reticulate evolution?
網状進化とは？
• The pattern of evolution resulting from recombinational speciation.
• 種類Aと種類Bのハイブリッドによる進化。
• Not generally expected to be a common occurrence, but can explain “mega-diversity” in new environments and unexpected genetic results.
• 普通の進化より珍しいが、新しい環境などでは起こる可能性がある。
• Results in retainment of ancestral patterns in the genome, with “repackaging”.
• 遺伝子の配列は進化（変異）しない。ただ新しい組み合わせができるだけ。
• Believed to occur in many plant groups, and cichlids (fish).
• 植物やアフリカの池の魚類で起こっていると思われている。
Evidence of reticulate evolution
網状進化の証拠
• Without laboratory experiments very hard to infer, but some ways:
• 研究室の実験以外で網状進化をどうやって見つける？
• Shared sequence portions between or within species.
• 種内、また種間の配列を見て、同じ部分があるかどうか？
• Differences between mitochondrial and nuclear DNA.
• ミトコンドリアDNAと核DNAの解析結果が違うかどうか？
Part 3 - Examples of Reticulate Evolution: Plants and Fishes
Example 1: peony flowers
(Sang et al. 1995)
• Sequenced ITS-rDNA of 33 species of Paeonia from Europe and Asia.
• Shrubs and herbs in northern hemisphere.
• Spotty distribution.
Results
• Examined ITS-1 sequences.
• Many species showed additive patterns.
• Subsequent evolution has taken place in some species.

• Many hybrid species Asian.
• Parents of these hybrid species European.
• Suggests hybridization occurred in past.
Conclusions
• Can see historical patterns, useful in species with no fossil history.
• This type of evolution may be common in plants.
• In such cases must be careful with phylogenetics.
Another example:
Cameroonian crater
lake cichlid fish
• Megadiverse group of fish with monophyletic origin.
• Much research shows reticulate evolution may occur when nuclear and mt DNA phylogenies do not match.
• Invasion of new environments could trigger hybridization between species.
Background
• Do hybrid swarms result from large areas with different environments or not?
• Cichlid fish provide great test case!
Barombi Mbo Lake
• 2.5 km in diameter.
• 110 m deep, only oxygen to 40 m.
• Four endemic genera; seven species.
• All on IUCN Red List - critically endangered.
• Evolved over 10000 years.
Materials and methods
• Two mt DNA markers and 2 nuclear markers.
• All types of fish from lake sampled; specimens deposited in museums.
Results
• Differences in mt DNA and nuclear DNA.
• Secondary hybridization after evolution.
• Two ancient lineages formed new species; Pungu madareni.
Conclusions
• Hybrid speciation can make complex species assemblages even without prior hybridization.
Part 4 - Examples of Reticulate Evolution: Corals
Reticulate Evolution in Cnidaria?
刺胞動物門は網状進化する？
• Several studies hint at reticulate evolution in Cnidaria, particularly corals and related groups.
• 特に花虫綱で網状進化の可能性がある。
• Marine environments where coral reefs are found are generally “new”.
• サンゴ礁の環境は比較的新しい。
• Centers of “mega-diversity” with “hyper-evolution” to micro-niches.
• 狭い地域で、多様性が非常に高い。
Acropora spp.
(Odorico & Miller 1997)
• Acropora very diverse, much morphological variation.
• Hybridization known from lab tests.
• ITS-rDNA shown to be a useful tool to detect this.

• Six colonies from five species.
• 18S rDNA and 28S rDNA obtained as well as ITS-rDNA.
Results
• Acropora ITS rDNA very short.
• Unexpected patterns of diversity, even within individuals!
• Such patterns consistent with ongoing reticulate evolution.
Conclusions
• Much more diversity than seen in plant ITS-rDNA.
• Could be due to more hybridization over longer ranges.
• Hybridization may occur over biological (not geological) time scales.
More corals
(Vollmer & Palumbi 2002)
• Examined all three Caribbean Acropora spp.
• Examined 2 nuclear and one mt DNA marker.
Results
• A. cervicornis and A. palmata distinct species.
• A. prolifera are F1 hybrids.
• Shape of A. prolifera depends on which species provided egg.
Conclusions
• F1 hybrids are immortal mules that may occasionally hybridize.
• Hybrids may be common in corals.
Part 5 -
Reticulate evolution in zoanthids
網状進化とスナギンチャク

Zoanthus spp. according to mt COI DNA
mt COIの結果による、マメスナギンチャク属の多様性
• Three species found with varying distribution. All ecologically similar to hard corals.
• ３つの種。生態はイシサンゴと似ている。
• Clear morphological variation between all three species.
• それぞれの種を区別できるようになった。
• This appears to be normal evolution.
• このデータから、普通の進化が推測できる。
核遺伝子(ITS-rDNA)配列結果
• All Z. kuroshio and Z. gigantus sequenced as expected.
• Z. kuroshio と Z. gigantusの結果はそれぞれが単系統。
• Z. sansibaricus had unusual results.
• 一方、 Z. sansibaricusの結果は単系統ではなかった！
• Some (2/3) samples gave expected sequences.
• 2/3のサンプルの配列（sansi）はmt DNAでの系統的位置と同様だったが、
• Some samples had both expected sequences and unknown “B” sequences.
• いくつかのZ. sansibaricus は不思議な “B”配列と普通の配列（sansi） 、両方を持つ。
• Some samples had only “B” sequences.
• 残りのZ. sansibaricus は不思議な “B”配列しか持っていない。
• B is closely related but different than Z. gigantus.
• “B”はZ. gigantus と近縁である。


Zoanthus undergoing reticulate evolution?
マメスナギンチャク属の網状進化？
• Samples with normal sequences and with normal/B, or just B have normal Z. sansibaricus morphology.
• 全てのZ. sansibaricusの形態が同じだった。
• Could B-only be F2 - resulting from backcrossing or F1 x F1 crossing?
• “B”配列しか持っていないサンプルはF2？
• Z. sansibaricus mass spawns, same as coral. No distribution barriers.
• マメスナギンチャク類はサンゴの様に同時に産卵する可能性がある。
• COI and morphology suggests NOT incomplete lineage sorting.
• 形態の結果やmt DNA配列を見ると、 incomplete lineage sortingじゃないと思うことができる。


Possible scenario for Zoanthus evolution
Zoanthus類の進化の説明
• Ancestor of Z.gigantus/B underwent one way hybridization (male B X female sansi), introducing B allele into Z. sansibaricus species.
• Z.gigantus/Bの精子（nuclear DNA）がZ. sansibaricus 種内に入ってきた。
• Modern-day Z. sansibaricus has both B and sansi alleles, ancestral B/giga evolved into modern Z. gigantus.
• 現在のZ. sansibaricusはsansiもBも持っている。
• 現在のZ. gigantusは昔のZ.gigantus/Bから進化した。

More zoanthids
(Reimer et al. 2007b)
• Investigated Palythoa spp. in Japan.
• Thought to be two genera, but mt DNA shows one genus.
• P. tuberculosa and P. mutuki very closely related.

Results
• ITS-rDNA shows two species (P. tuberculosa & P. mutuki) very closely related.
• Some specimens with intermediate morphology also apparently intermediate in phylogeny.
Results (2)
• Alignment of ITS-rDNA shows “reticulate” patterns between intermediates of two species.
• Appears as if some P. tuberculosa DNA has entered into P. mutuki population.

Conclusion 2
• In the future, more reticulate evolution will be found.
• This will impact conservation and our understanding of species.

Conclusion 3
• This will lead to better understanding of other related evolutionary events, such as lateral gene transfer (LGT).
References cited:
1. Sang et al. 1995. Documentation of reticulate evolution in peonies (Paeonia) using internal transcribed spacer sequences of nuclear ribosomal DNA: Implications for biogeography and concerted evolution. PNAS USA 92: 6813-6817.
2. Schliewen & Klee. 2005. Reticulate sympatric speciation in Cameroonian crater lake cichlids. Frontiers Zool 1:5.
3. Odorico & Miller. 1997. Variation in the ribosomal internal transcribed spacers and 5.8S rDNA among five species of Acropora (Cnidaria; Scleractinia): Patterns of variation consistent with reticulate evolution. Mol Biol Evol 14: 465-473.
4. Vollmer & Palumbi. 2002. Hybridization and the evolution of reef coral diversity. Science 296: 2023-2025.
5. Reimer et al. 2007a. Molecular evidence suggesting interspecific hybridization in Zoanthus spp. (Anthozoa: Hexacorallia). Zool Sci 24: 346-359.
6. Reimer et al. 2007b. Diversity and evolution in the zoanthid genus Palythoa (Cnidaria: Hexacorallia) based on nuclear ITS-rDNA. Coral Reefs 26: 399-410.

Class 8 - Disease

Note: Class 7 was an explanation of the report assignment.


Outline
• 1. Quick introduction to diseases.
• 2. Common coral reef diseases.
• 3. Why are diseases becoming common?
• 4. How do diseases affect conservation?
• 5. Conclusions
Part 1: Disease
Example 1: Plague in humans

• Plagues have struck humans many times.
• Often kill 10-50% of population.
• Caused by an influenza virus.
• Two most infamous cases are 13th century Black Plague, and 1919-1920 Spanish Influenza.
• No one knows where plagues came from.
• Spread through common routes of trade.
• Spread faster in modern cases.
• Often affects young adults worse due to “cytokine storms”.
Spanish Influenza
• In some countries fatalities were as high as 50%.
• Killed more people than WWI.

How does this happen?
• New mutation in influenza virus that most humans do not have capability to respond to.
• Genetic variation provides resistance.
• SARS is a more recent case.


Example 2:
Introduction of a new disease into an isolated area
Elm trees common in North America and Eurasia.
Preyed upon by two species of bark beetles.
Beginning in the 1910s, some elms began to die.
Die-offs became rapid in 1960s.
Bark beetles somehow involved in the disease.

Survival of elms close to 0%.
• The causative agents of DED are ascomycete microfungi.
• Carried by the elm bark beetles.

• Three species are now recognized: Ophiostoma ulmi, which afflicted Europe in 1910, reaching North America on imported timber in 1928, Ophiostoma himal-ulmi, a species endemic to the western Himalaya, and the extremely virulent species, Ophiostoma novo-ulmi, which was first described in Europe and North America in the 1940s and has devastated elms in both areas since the late 1960s.
• The origin of O. novo-ulmi remains unknown but may have arisen as a hybrid between O. ulmi and O. himal-ulmi.
Part 2: Common coral reef diseases
Introduction to
coral reef diseases
• Bacteria observed in corals in early 1900s.
• Diseases noticed in 1970s, seemingly increasing over last 30 years.
• 34 mass events, affecting sponges, seagrasses, cetaceans, urchins, fish, molluscs, corals.
• Have changed composition of reefs.

Diseases affecting Scleractinia
• Many diseases named, but very little known.
• Most pathogens still unknown.
• Most common in Atlantic (Green & Bruckner).
• Not to be confused with coral bleaching.
Green & Bruckner 2000
Black Band Disease (BBD) Caused by numerous cyanobacteria (500 spp.) as a microbial mat.
Mat makes the colored band.
First observed in 1973.
Moves 3mm to 1cm/day.
Found in 42 spp. of coral.

Kuta & Richardson 2002
• BBD correlates strongly with depth, temperature, nitrites.
• Also correlates with diversity and orthophosphate.
White band disease: Pathogen unknown, may be bacteria. Noticed in 1981.
Tissue loss from base to tip.
Affects two species, Acropora cervicornis and A. palmata.
Moves 3mm to 1cm/day.
• WBD has drastically altered Caribbean reefs.
• Shifts in coral species.
• Loss of overall coral cover; algae increasing.

• Both species now “threatened”.
• Losses of over 98% of A. cervicornis. Locally extinct.
White plague: Affects many species, but no acroporoids.
Caused by Aurantimonas bacteria.
First observed in 1977.

Aspergillosis: Caused by terrestrial fungi.
Affect mainly Atlantic gorgonians.
Also affects waterfowl.
Noted in 1997.

Tumors: Similar to cancer.
Affects mainly A. palmata.
Irregular growth, no zooxanthellae.
Noted in 1960s and 1970s.

Other diseases: Many other diseases.
Mostly known from Atlantic.
Yellow band disease, yellow spot disease, white pox disease, brown band disease.
Most noted for first time in last 20 years.
Pathogens usually unknown.

Part 3: Why are diseases becoming common?
1. Global warming?
• Many people blame global warming.
• But likely much more complex.
2. Nutrient enrichment - Bruno et al. 2003
• Experiments done with YBD and Aspergillosis.
• Controls were disease only, experimental with added nitrogen and phosphorus.
Results - Aspergillosis
• Nutrients increased severity of disease in sea fans.
Results - YBD
• Presence of nutrients increased rate at which YBD developed in two species of coral.
3. Dust? -
Garrison et al. 2003
• Airborne dust from Africa and Asia carries many contaminants to reefs.
• Global warming and desertification increasing dust, therefore increasing contaminants.

Part 4: How do diseases affect conservation?
Effects are widespread
Many studies have documented widespread coral decline in almost ALL coral species.
Porter et al. 2001 showed many declines 1996-1998 NOT due to coral bleaching but disease.

• Porter et al. 2001 cont
• Green & Bruckner 2000

• Green & Bruckner 2000
Many examples of diseases spreading, many examples of reef degradation (show many photos).

Overview of disease
• All diseases have negative effects.
• Only WBD has changed communities drastically.
• Pacific 15 years behind Atlantic.
• Compounded negative influences more severe for coral reefs.
Part 5: Conclusions.
Conclusion 1
• Disease more widespread on reefs in Caribbean.
• More research? Partially.
• Monitoring in Pacific very critical.
Conclusion 2
• Only one disease has permanently changed community structure (WBD).
• Other diseases locally important.
Conclusion 3
• Very few studies have investigated in detail mortality rates.
• Monitoring of individual colonies needed.
Conclusion 4
• Diseases increasing.
• Bleaching appears to be more critical, but two problems appear related.
Conclusion 5
• Diseases not well understood.
• Many diseases affect many species; possibly more or less diseases.
• Pathogens need to be investigated.
Conclusion 6
• While bleaching currently more serious, foolish to ignore diseases.
• May be “indicator” of serious problems, similar to amphibians.
What needs to be done
• <3% of reefs in danger have low human impact.
• More research needed on human influences and pathogens.
• Management and conservation then follow.
References:
1. Green & Bruckner. 2000. The significance of coral disease epizootiology for coral reef conservation. Biological Conservation 96: 347-361.
2. Aronson & Precht. 2001. White-band disease and the changing face of Caribbean coral reefs. Hydrobiologia 460: 25-38.
3. Garrison et al. 2003. African and Asian dust: from desert soils to coral reefs. BioScience 53: 469-481.
4. Bruno et al. 2003. Nutrient enrichment can increase the severity of coral diseases. Ecology Letters 6: 1056-1061.
5. Kuta & Richardson. 2002. Ecological aspects of black band disease of corals: relationships between disease incidence and environmental factors. Coral Reefs 21: 393-398.
6. Porter et al. 2001. Patterns of spread of disease in the Florida Keys. Hydrobiologia 460: 1-24.

Class 6 - Symbiodinium

 Outline
 1. Review of Symbiodinium and coral bleaching.
 2. Investigating diversity of Symbiodinium: past to present.

 Part 1: Review of Symbiodinium and bleaching.
 Dangers facing coral reefs:
 Global warming is raising the temperature of the ocean; this kills corals - “coral bleaching”.
 Also, as the oceans become more acidic, it is more difficult for corals to make their skeletons.
 Perhaps 90% of coral reefs will be dead by 2050.

 Diagram of iving tissue

 Numbers of zooxanthellate genera over time, increase in ZX genera of corals.
 More diverse than ever, showing benefits of symbioses.
 Believed to have started approximately 60 million years ago.
 Symbiodinium spp. in invertebrates
holobiont=host+symbiont(s)
 Corals and symbionts
 Many shallow water corals get their energy from symbiotic zooxanthellae.
 These small animals make it possible for corals to live in the warm oceans.
 But, these symbionts are sensitive to hot ocean temperatures.
 What turns the coral white?
- As a stress response, corals expel the symbiotic zooxanthellae from their tissues
- The coral tissue is clear, so you see the white limestone skeleton underneath
 What can stress a coral?
 High light or UV levels
 Cold temperatures
 Low salinity and high turbidity from coastal runoff events or heavy rain
 Exposure to air during very low tides
 Major: high water temperatures
 Thermal stress
 Corals live close to their thermal maximum limit
 If water temperature gets 1 or 2°C higher than the summer average in many parts of the world, corals may get stressed and bleach
 NOAA satellites measure global ocean temperature and thermal stress
 How warm is warm?
 How hot do you think the ocean has to get before corals start to bleach?
 GLOBAL WARMING
 Glaciers and Sea Ice are melting
 World map showing levels of
coral bleaching. Source: ReefBase
 Can corals recover?
 Yes, if the stress doesn’t last too long
 Some corals can eat more zooplankton to help survive the lack of zooxanthellae
 Some species are more resistant to bleaching, and more able to recover
 Can corals recover?
 Corals may eventually regain color by repopulating their zooxanthellae
 Algae may come from the water column
 Or they may come from reproduction of the few cells that remain in the coral
 Can corals recover?
 Corals can begin to recover after a few weeks
 Does bleaching kill corals?
 Yes, if the stress is severe
 Some of the polyps in a colony might die
 If the bleaching is really severe, whole colonies might die
 Bleaching in Puerto Rico killed an 800-year-old star coral colony in 2005
 What else can stress do to corals?
 Question: what is something that happens to people when they are highly stressed?
 What else can stress do to corals?
 Question: what is something that happens to people when they are highly stressed?
 Bleaching and coral disease
 Coral diseases are found around the world

 High temperatures and bleaching can leave corals more vulnerable to disease

 Can quickly kill part or all of the coral colony
 Bleaching and bioerosion
 We have seen that bleaching can kill part or all of a coral colony
 Areas of dead coral are more vulnerable to bioerosion (when animals wear away the coral reef’s limestone structure)
 Storms & coral bleaching
 The same warm water that causes corals to bleach can also lead to strong storms.
 Storms: a mixed blessing
 Storms: a mixed blessing
 Each passing hurricane in 2005 cooled the water in the Florida Keys.


 Part 2: Investigating diversity of Symbiodinium: past to present.
 What are zooxanthellae?
 Algae that live in the coral polyp’s surface layer
 Algae get nutrients and a safe place to grow
 Corals get oxygen and help with waste removal
 Corals also get most of their food from the algae
 Symbiosis overview
 Genus Symbiodinium
 Described in 1962 by H. Freudenthal.
 Within dinoflagellates.
 Was though there was one single species worldwide.

 Morphology & life cycle
 Host species
 Cnidaria (corals, jellyfish, anemone, zoanthids, octocorals).
 Mollusca (clams, snails).
 Platyhelminthes (flatworms).
 Porifera (sponges).
 Protista (forams).
 First genetic studies
 Rowan & Powers 1991.
 Utlized 18S ribosomal DNA.
 Sampled from corals & anemones.
 Found unexpected diversity!
 Recommended further genetic studies.

 Second wave of studies
 Used faster evolving DNA markers.
 Particularly ITS-rDNA.
 Even more diversity!
 Zooxanthellae clade
DNA analyses
Clade: A group composed of all the species descended from a single common ancestor
 Diversity
 Eight major clades known.
 Within each clade many subclades.
 Do not know what taxonomic level clades are equal to.
 Evolution and biogeography
 Many studies have catalogued diversity.
 Can now understand on many scales.
 Can predict evolution.
 Specific types
 Many subclades or types associate with similar hosts.
 Could be co-evolution.
 Symbiodinium in Zoanthus sansibaricus
 We sampled the same species from 4 locations.
 Each host colony was shown to associate with one subclade of Symbiodinium.
 Subclade C1/C3 was common in the north, and subclade A1 was dominant in the south.
 C1/C3 has been shown to be a dominant Indo-Pacific “generalist”, with C15 common in Porites spp., and A1 a shallow-water specialist.
 Modes of transmission & flexibility
 2 major types; a) vertical and b) horizontal.
 Vertical should result in more co-evolution and less flexibility.
 Also, in horizontal, ZX from environment still rare.
 Changes in ZX
over time?
 Changes have been seen over time in content of ZX within coral colonies!
 Particularly after bleaching events.
 ZX shuffling?
 Adaptive Bleaching Hypothesis (ABH).
 Very controversial, large conservation implications.
 Two ways this occurs.
 Diversity within colonies
 Same colony may have different ZX at different locations!
 Differences in types
 Since we know diversity, we can experiment with different conditions.
 Many ZX are easy to culture.
 Control light, temperature, nutrients, etc.

 Can also then experiment in situ.
 Symbiodinium spp. characters
 Believed to alternate between a free-living stage with flagella, and a non-motile stage with chlorophyll.
 Believed to sexually reproduce, although this has not been observed.
 Overall morphological condition can degrade based on non-optimal environmental conditions, in particular low (<15 º C) and high (>30ºC) sustained ocean temperatures.
 “Adaptive bleaching” hypothesis
 Bleaching may enable corals to adopt different classes of zooxanthellae, better suited for a new environment. By:
 ‘symbiont switching’ (a new clade from exogenous sources) or
 ‘symbiont shuffling’ (host contains multiple clades and a shift in dominance occurs).

 Can we protect corals from bleaching?

 Marine invertebrate - Symbiodinium spp. symbioses overview
 Symbiodinium spp. found in many clonal cnidarians (and other invertebrates) in tropical and sub-tropical oceans. Symbiodinium are the main reason coral reefs exist and have large levels of diversity.
 Symbiodinium is now divided into 8 “clades” labelled A-H (of unknown taxonomic level) with many “subclades” (designated by numbers) within each clade (see various works by Pochon et al., and LaJeunesse et al.)
 Host species’ association with various clades and subclades of Symbiodinium (often more than one) may be at least partially responsible for differences in bleaching patterns seen during bleaching events (i.e. ENSO event of 2001, etc.).
 Also, some host species have been shown to have flexible associations with Symbiodinium over biogeographical ranges (depth, latitude, etc.) or time (summer versus winter, etc.). This is part of the Adaptive Bleaching Hypothesis (ABH) (Buddemier and Fautin 2004; Baker 2001), and is very contentious.
 Need to understand Symbiodinium diversity within zoanthids before any discussion of symbiotic zoanthid ecology can be conducted.
References:
1. Rowan & Powers. 1991. Molecular genetic identification of symbiotic dinoflagellates (zooxanthellae). Marine Ecology Progress Series 71: 65-73.
2. Stat et al. 2006. The evolutionary history of Symbiodinium and scleractinian hosts - Symbiosis, diversity, and the effect of climate change. Plant Ecology, Evolution and Systematics 8: 23-43.
3. LaJeunesse 2005. ‘Species’ radiations of symbiotic dinoflagellates in the Atlantic and Indo-Pacific since the Miocene-Pliocene transition. Molecular Biology and Evolution 22: 570-581.
4. Pochon et al. 2004. Biogeographic partitioning and host specialization among foramineferan dinoflagellate symbionts (Symbiodinium; Dinophyta). Marine Biology 139: 17-27.

Class 5 - Reverse taxonomy - DNA and classification

LFrom last week:
Understanding phylogenetic trees
• Branch length:
• 1. Vertical height has no important meaning.
• 2. Horizontal length is very important, tells the genetic distance of each sequence!
Calculation done by software.

Today`s class: Outline
• 1. Examples of reverse taxonomy from zoanthids (my research).
• 2. A new species of whale!
• 3. Atlantic and Pacific corals.
• 4. Four species of COTS.

Part 1
“Reverse taxonomy” = using DNA to find species; then describing morphology:
Zoanthids (Cnidaria: Anthozoa: Hexacorallia)
• Order Zoantharia (=Zoanthidea, Zoanthiniaria)
• Sand-encrusted, colonial
• Found in most marine environments
• Often symbiotic or parasitic
• Morphologically challenging, taxonomically neglected
• Often ignored in biodiversity surveys, non-CITES
Example: specimens in the Pacific:
Specimens 0-50 m, some but not as many as there should be, very few from coral triangle.
Specimens 50-1000 m, much much less.
Specimens >1000 m, only three!


Zoanthus spp. diversity in Japan
日本のマメスナギンチャク属の多様性
• Using genetics, backed up with morphology, currently we can accurately identify three Zoanthus spp. in Japan.
• 遺伝子解析で、綺麗に三つの種類に分かれた。
• Markers used are 16S, COI (both mt DNA) and ITS-rDNA (nuclear).
• Many presumed species not true species.
• 今まで４つの種類と思われていたものは、ひとつの種類だった。
• Oral disk color not a characteristic of species.
• 色は分類ができる特徴ではない。
• Not one morphological characteristic clearly defines each species.
• 一つだけの形態的特徴で分類できない。



Shallow water sampling & research
• Evidence of reticulate evolution, intraspecific variation.
• Many new families, genera and species await description. Unexpected findings.
• Current studies often limited to specimens from Japan.
Large gaps in our knowledge
• Almost complete lack of examination in regions between Japan and Australia. Formalin specimens and lack of modern examination in Australia.
• Lack of trained taxonomists.
• Ignored in almost all biodiversity surveys.
• The deeper we go, less knowledge.
• Biogeography impossible.
Investigating Deep-sea Zoanthids
深海のスナギンチャク類

What about deep-sea zoanthids?
深海のスナギンチャクというのは？
• All described deep-sea zoanthids are placed in Epizoanthidae despite morphological and ecological differences.
• 今まで、全ての深海スナギンチャクはヤドリスナギンチャク科に分類されていた。
• No deep-sea zoanthids formally described from the Pacific.
• 太平洋の深海スナギンチャクは全く分類されていない。
• None described from limited environments.
• 極限環境（化学合成環境）のスナギンチャクの報告はあるが、サンプルや論文も無い。
• However, data literature suggests deep sea zoanthids may be quite common - underreported? Theorized to be worldwide is distribution - almost always found when specifically searched for.
• おそらく、珍しくはない。
Potential new deep sea zoanthid
謎の深海スナギンチャク？
• During Shinkai 6500 dive #884 (June 2005), several unidentified zoanthid-like samples “accidentally” collected off Muroto, Nankai Trough, depth=approx. 3300 m.
• 高知県の室戸の近くにある南海トラフで、2005年に間違えて、謎のスナギンチャクらしき生き物が採取された。水深は約3300m、冷水の極限環境。
• Back checks of images show that the sample organism is apparently quite common at the dive site.
• 画像をチェックすると、この生き物が非常に多い。
• Lives on mudstone but not loose sediment.
• 固い泥岩の上に存在、泥上には存在しない。
• No high-resolution in situ images exist.
• 綺麗な画像が無い。
• Only 12 polyps collected.
• ポリプは12個しか採取されなかった。


Deep-sea specimens
• Very limited thus far, but specimens divergent.
• Use of ROVs and manned submersibles have resulted in 1 new family, 2 new genera in Japan, several new species (3 missions).
• Found on other benthos, found in limited environments.
• Below 1000m very few samples.
External morphology
外側の形態について
• Samples appeared to be zoanthid-like based on: sand encrustation and polyp shape. No tentacle data available.
• スナギンチャクと同様に、砂を取り込んでいる。ポリプが閉じている。
• However, samples have several unique features: free-living and inhabited a deep sea methane cold seep. Morphology and ecology do not fit with any known zoanthid families.
• 単体性、極限環境の初めてのスナギンチャク。

Internal morphology?
内部の形態について？
• As expected, cross section using normal (wax-embedded) methods gave poor results.
• パラフィン切片での結果はあまりよくない。
• Attempted to set sample in epoxy resin, cut a section, and polish to necessary thickness but failed.
• レジンでの切片も無理。
• Another possibility is digestion of outer surface of polyp.
• フ酸での切片は可能だが、非常に危ない。
• Could obtain mesentery count number from rough cross-sections (19-22).
• 状態が悪い切片で、約19〜22隔膜を確認できたが、形など観察できなかった。
Genetic results
遺伝子解析の結果
• Obtained mt COI, mt16S rDNA, and 5.8S rDNA sequences confirm samples are zoanthid, but divergent from all known zoanthid families.
• 今回のサンプルはスナギンチャク目に入っているが、今まで知られているスナギンチャクと離れている。
• Particularly, divergent from all known groups of deep-sea zoanthids described.
• 特に、今までの深海のスナギンチャクと違う。
• Bootstrap support for monophyly 100% (all methods, all markers).
• 遺伝子解析の結果の確率が非常に高い。
Abyssoanthus nankaiensis n. fam, n. gen. et n. sp.
Abyssoanthus nankaiensis 新科、新属、新種
• Based on external morphology and genetic results, these samples are a new family of zoanthid: Abyssoanthidae.
• 形態、生態、遺伝子解析を含めて、今回のサンプルは新科、新属、新種。
• However, several questions remain regarding ecology and reproduction of this new family.
• 今後、日本周辺の深海で調査を行う予定。

Part 2 -
A new species of whale!
Dalebout et al. 2002. A new species of beaked whale Mesoplodon perrini sp. n. (Cetacea: Ziphiidae) discovered through mitochondrial DNA sequences. Marine Mammal Science 18: 577-608.
Introduction
• Beaked whales are rare, with cryptic lifestyles. Most never observed alive.
• 12 species described in last 100 years!
• Mesoplodon hectori common in southeast Pacific.
Materials & Methods
• 5 specimens of beaked whale stranded in California, 1977-1995.
• Thought to be M. hectori based on morphology.
• Researchers then examined 2 mt DNA markers…
Results
• Results surprisingly show five specimens not M. hectori.
• New species!
• Re-examination shows morphological differences as well.
Discussion
• Authors suggest genetic voucher material for all taxa.
• Also state there are likely 40 marine mammal species still unknown!
• Cookiecutter sharks feed on M. perrini.

• Who knows what species await description?
Part 3 -
Atlantic & Pacific corals
Fukami et al. 2008. Mitochondrial and nuclear genes suggest that stony corals are monophyletic but most families of stony corals are not (Order Scleractinia, Class Anthozoa, Phylum Cnidaria). PLoS One 3:9: e3222

• Coral phylogeny has been in flux for 10+ years.
• Perhaps corallimorphs within hard corals.
• Here examine 127 species, 75 genera, 17 families.
• Four markers; 2 nuclear, 2 mitochondrial.

• Corals monophyletic.
• 11/16 families not monophyletic.
• Corresponding morphological characters found.
• Corallimorphs not part of stony corals.

• Many Atlantic corals are very unique, and should be conserved.
• Some clades vulnerable to extinction (II, V, VI, XV, XVIII+XX).
• Ability to conserve depends on knowing what to conserve.

• Re-organize based on DNA, re-examine morphology.
• Atlantic corals must be protected more strongly.
• Basic ideas need to be re-examined (e.g. favids).
Part 4 - Crown-of-thorns
Vogler et al. 2008. A threat to coral reefs multiplied? Four species of crown-of-thorns starfish. Biology Letters doi:1-.1098/rsbl.2008.0454

• Acanthaster planci outbreaks threaten coral reefs.
• Causes of outbreaks not clear.
• Species has long-lived larvae, but apparent population structure.
• Here used COI sequences from 237 samples.

• Four clades found, 8.8-10.6% divergent.
• Diverged 1.95-3.65 mya.
• Species show geographical partitioning. Due to sea level changes.
• All populations expanding.

• Four species, SIO, NIO, Red Sea, and Pacific.
• Outbreaks mainly seen in Pacific - could this be a species difference?
• Clearly more research needed, critical for coral reef management.

Overall conclusions:
1. Genetics already impacting our understanding of diversity.
2. Expect more surprises in the future.
3. Massive revision of all coral reef organisms!

References:
1. Reimer et al. 2004-2008. Various papers on zoanthid phylogeny.
2. Dalebout et al. 2002. A new species of beaked whale Mesoplodon perrini sp. n. (Cetacea: Ziphiidae) discovered through phylogenetic analyses of mitochondrial DNA sequences. Marine Mammal Science 18: 577-608.
3. Fukami et al. 2008. Mitochondrial and nuclear genes suggest that stony corals are monophyletic but most families of stony corals are not (Order Scleractinia, Class Anthozoa, Phylum Cnidaria). PLoS One 3:9: e3222.
4. Vogler et al. 2008. A threat to coral reefs multiplied? Four species of crown-of-thorns starfish. Biology Letters doi:1-.1098/rsbl.2008.0454