December 16th class notes & report outline

Class notes:

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.

Coral Reef Diversity and Conservation
J.D. Reimer
Assignment Information:
You and your team (2~3 people) must apply for research funding for the following research grant:

The MISE Coral Reef Conservation Fund
This fund provides grants for projects that build private-public partnerships through research that aims to reduce and prevent the degradation of coral refs and associated reef habitats (seagrass beds, mangroves, etc). Projects will be for two years from April 2010.
Part 1 - Application: please submit the following to Science room 353 by 17:00 on Monday, January 25th.
1. Cover page (1 page): Include group name, and project title.
2. Research background and goals (2 pages maximum): provide a background that anyone with science training can understand, clearly state the project goal(s), and explain the necessity of the project.
3. Methods (2 pages maximum): Include detailed explanation of methods used, and a concrete timeline to be followed. If research facilities and equipment are to be used at an institution, include details.
4. Expected results (1 page maximum): Describe concretely what results can be expected, how these results will benefit coral reefs, and how these results will benefit future coral reef conservation research.
5. Team outline (2 pages maximum): Include team member information on skills, relevant work history and experience, and scientific publications (if any). Designate roles for each person. Do NOT include personal private information.
6. Budget (1 page maximum): Divide budget into the following items; 1) travel expenses, 2) equipment (over 100,000 JPY), 3) expendables & items less than 100,000 JPY, 4) other expenses (guides, part-time jobs, rentals, post, etc). Include a budget for 2 years. Budgets may not exceed 30,000,000 JPY in total for 2 years, and cannot be more than 20,000,000 JPY in one year.

Part 2 – Visual presentation: Wednesday, January 27th
All teams will give a 5-minute PowerPoint presentation on their projects on during class. Other students and JDR will vote on their project; this will be 20% of your project mark. There are no rules on this presentation other than the time limit and the use of PowerPoint.
Hints:
a. Your project must be realistic, and must include as many details as possible to make it easy to understand. It can be as simple or large-scale as possible, but ideas must be logical. The project can build on other plans, but these plans must be referenced.
b. Using scientific references increases the amount of knowledge you have access to. On the other hand, using information without references will decrease your score. Use Google Scholar or other sources of information to access scientific literature quickly. Space is limited, so choose references well.
c. Figures and graphs greatly help make ideas easy to understand.
d. For budgets, catalogues may be useful. Please visit JDR’s lab to borrow any catalogue you need.
Marking:
This project is very open-ended. Grades will be assigned based on:
1. originality (new ideas, etc.): 20 points
2. necessity: 20 points
3. realistic possibility: 20 points
4. style & background research: 20 points
5. visual presentation and votes: 20 points
Remember:
 Reports are to be done in groups of 2-3 people.
 Please write which person did what part of the report.
 Due date is January 25th by 17:00.
If you have any questions or problems please contact JDR as soon as you can, either at his office or by e-mail.

December 9th class

NOTICE: Please attend next week; we assign the reports on this day!

Today`s class: Outline
• 1. A new species of whale!
• 2. Atlantic and Pacific corals.
• 3. Four species of COTS.
• 4. Review of Symbiodinium and coral bleaching.



Part 1 - 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 2 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 3 - 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!

Part 4 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.


References:
1. 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.
2. 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.
3. 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

December 2nd class

1. DNA phylogeny introduction, methods.
Vocabulary:
Primer
Alignment
DNA marker
Tree
Bootstrap value
Clade
Monophyletic
Polyphyletic
In order to understand phylogeny we must understand evolution:
The Ågmodern synthesisÅh of evolution is the combination of Darwin's and Mendel's theories.
The theory underlying the modern synthesis has three major aspects:
The common descent of all organisms from a single ancestor.
全ての生き物は共通の祖先から進化した。
The origin of novel traits in a lineage.
それぞれのグループはそれぞれの特徴を持つ。
Changes cause some traits to persist while others perish.
様々な変化によって、あるグループは生き残り、あるグループは絶滅する。
DNA and 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 ÅglettersÅh: 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。
DNA may be a way to have non-specialists identify species quickly!
So, DNA tree = evolutionary tree (or so we hope)

In a cell, two major types of DNA we will study:
. mitochondrial DNA (mt DNA)
evolves very slow in Cnidaria (Anthozoa), opposite to most animals.
他の動物と違い、刺胞動物で進化が遅い。
b. nuclear DNA
evolves faster in Cnidaria, opposite to most animals.
他の動物と違い、刺胞動物で進化が早い。
Example DNA markers:
COI, cytochrome oxidase subunit 1 - mt DNA, used for many studies, much data available.
16S rDNA - mt DNA, useful in zoanthids! some indels, especially V5 region.

Understanding phylogenetic trees:
Calculation methods:
1. MP - maximum parsimony. Least changes. Character-based.
2. ML - maximum likelihood. Must specify evolution model. Character-based.
3. NJ - neighbour-joining. Simplest method, variable evolutionary rates, distance-based.
4. Bayes - like ML on sets of trees!
Calculation done by software.
Bootstrap values:
Values show possibility that this clade/shape is true.
Values under 50% not used.
Values >70% desirable, above 90% confident.
Bayes >95%!
Trees reflect evolution.
Can make conservation decisions from these, or taxonomic decisions.
“Reverse taxonomy”.
Other notes:
More markers better than few.
Analyses also better with many methods.
Be careful of contamination or misidentification.
Back up with other data.
In the future:
Whole genomes will become cheaper due to 454 and new technology.
Cloning? Examination of extinct species. e.g. Wooly mammoth

Part 2 – examples from zoanthids
“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 3- CReefs trip
•November 8th – December 1st
•Census of Coral Reef Ecosystems (www.creefs.org), part of Census of Marine Life (CoML – www.coml.org).
•Large international effort to understand biodiversity, use data for conservation.
•Many researchers from different institutions, focused on different taxa, many “ignored”.
•I focused on zoanthids (of course)!
Details:
On Heron Island, on southern Great Barrier Reef (GBR). Heron Is. has a resort and a research station. Very beautiful setting.
Heron Island home to seabirds, rails, and important nesting ground for sea turtles.
Research station very new – well set up, running sea water, wet and dry labs.
CReefs, biodiversity of coral reef animals, team consisted of: 1) scientists from all over the world, 2) boat guides/divers, 3) a photographer, 4) a blogger, and 5) a chef. A great team and well-supported.
Many locations of the southern GBR investigated, based on preferences, weather, and distance, etc. Very flexible plan. Boats went out almost every day! Sampling methods varied based on taxa being investigated. Diving rules strictly followed for safety reasons. All permits and paperwork were done to follow all rules local, national, and international. Much sharing of specimens to prevent waste.
I found 270 specimens, at 33 sites, many new species!
http://www.creefs.org/index_h.html