January 13th class notes

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.

January 6th class notes

Happy New Year!

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.