January 27, 2015 supplementary class II

Species (again): linking it all together


Introduction: What is a species?
Biological species: Fish species complex
OTU & barcodes: ARMS and estimating coral reef biodiversity.

Part 1: Introduction: What is a species?

A species can be different, depending on who you talk to, or what meaning is implied.

Before we discuss species, we must be clear about what species concept we are discussing.

Three types are:

A. taxonomic species

B. biological species

C. OTU/barcode species

Taxonomic species

A species with a scientific name.

Names are important for science & conservation.

Accuracy and authorship important

Problems:

Naming species takes time & money.

Few experts.

Biological species

A species proven to reproduce only with itself.

Reproductive isolation.

Species concept important.

Problems:

Experiments take time & money.

Hybrids? Evolution is messy.

OTU/barcode species

A species defined by genetic differences.

Implies a threshold value.

Quick and easy.

Problems:

Sequencing species takes time & money.

Limited by previous knowledge.

Not always “true”, barcoding gap is problematic.

Part 2: Biological species

Fish species complex in the Pacific (Leray et al. 2010)

This study deals with:

Biological species

Can be detected through observation of reproduction.

Also, reproductive experiments.

But these require extensive field work. Recently, molecular analyses usual, using phylogeny.

Background: Genus Dascyllus

Complex has 4 species:

D. trimaculatus (E. Africa to Central Pacific): 3 spots

D. albisella (Hawaii): white flanks

D. strasburgi (Marquesas Islands): gray

D. auripinnis (Line, Phoenix Islands): yellow fins

Each species has its own ecology and preferred habitats. Figure from Leray et al. 2010.

Specimens (n=563) from across the Indo-Pacific.

Microsatellite DNA data examined (data from previous research included).

Phylogenetic analyses and genotype assignment.

From Leray et al. (2010).

Previous research showed 5 clades, not 4 (Bernadi et al. 2003).

This research showed 7 (!) groups of Dascyllus, not all with clear morphology.

Appears ancient history of Indo-Pacific combined with recent evolutionary events drives speciation.
Hybridization appears to be occurring.
NOTE: unknown species are not yet valid species, or new species. They are undescribed species.

Summary: Using biological species data, we can now go and revise taxonomic species.

Biological species
Results could lead to “finding” undescribed species or groups

This leads to....

Taxonomic species

Describing a species.

Only way to give a scientific name.

New species.

Part 3: OTU/barcode species

ARMS and crustaceans

Plaisance et al. 2011;

Brainard et al. 2010

ARMS = Autonomous Reef Monitoring Systems.

Built cheaply, mass produced. In the image of dead coral head. Can use to estimate total biodiversity of a reef.

Hand picking larger specimens + DNA barcoding of all things!

Can estimate how well biodiversity is being found by a saturation curve.

OTU/barcode species

Results could lead to “finding” undescribed species or groups

This leads to...

Biological species

Results could lead to “finding” undescribed species or groups

This leads to....

Taxonomic species

Describing a species.

Only way to give a scientific name.

New species.

Test information

•Wednesday, February 4th; 8:30-10:00, Science Room 327

•10 questions, choose 6. Also bonus question.

•Open book, bring anything you want.

•Must pass to pass class.

•Arrive after 9 a.m. – out.

•Advice: study! Without studying, impossible to pass...

January 28, 2015 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 Zoantharia
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 Zoantharia
(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.

Conclusions
• In the future, more reticulate evolution will be found.
• This will impact conservation and our understanding of species.
• 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.
7. Shiroma and Reimer 2010. Zoological Studies.

January 27, 2015 supplemental class I

Outline
1. Introduction: Problems facing taxonomy and diversity.
2. Accelerating “taxonomy”: DNA barcoding.
3. Promoting taxonomy: Census of Marine Life.
4. Images from one CoML Project (Creefs).
5. Overview of PCoRIE Palau biodiversity project.
1. Introduction: Problems facing taxonomy and diversity.
Problems facing taxonomy 1
Too many species! Diversity confounds our best efforts to examine it.
Keep finding new species.
Extinction rates increasing.
Problems facing taxonomy 2
Not enough taxonomists.
Pay poor, work long.
Everyone says “important” but not considered essential.
Many groups have no active workers.
Potential solutions 1
Increasing technology and information available.
Global information systems.
Molecular experiment techniques.
Potential solutions 2
Increasing international research collaboration.
Growing awareness of biodiversity and importance.


2. Accelerating “taxonomy”: 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
A sequence/marker used to barcode should:
be easy to amplify
not possess paralogues
have conserved regions to design primers efficiently for a broad taxonomic sampling
be variable enough to distinguish species
but conserved enough within species
Choosing the correct DNA marker is critical.
Point:
Barcoding does not aim to solve phylogeny!
Reasons for DNA barcoding
1. Works with fragments.
2. Works with all stages of life: Can link male/females. Different stages of same organism. E.g. Amphipods (White & Reimer 2012)
3. Cryptic species detection. E.g. Astraptes
4. Reduces ambiguity (set DNA code).
5. Makes expertise go further.
6. Democratizes access to data. E.g. Barcode of Life project
7. Opens the way for handheld barcoders.
8. Finds new diversity.
9. Demonstrates value of museum collections. Sequencing of collections vital.
10. Speeds up discovery of new species.
Additional strong point
Does not need expert knowledge.
Weak points
1. DNA (specifically COI) does not always work for each group of organisms.
2. Handheld technology has not succeeded, despite many advances.
3. Different taxa have different DNA protocols, so standardization is difficult.
4. The “barcoding gap”
Barcoding implies that the level of DNA divergence between and within species is different.
But evolution not neat - hybrids, incomplete lineage sorting, etc.
This gap is not always present – so taxonomy comes back to “judgement”.

Results
DNA barcoding proposed in 2003 as a “solution” to taxonomy.
Two large projects: Barcode of Life and Ocean Genome Legacy.
Encyclopedia of Life on the internet. 
Common method of identification.
Gaining acceptance as a practical method to obtain much data.
But has not solved taxonomy, instead a new approach or “sub-field”.

For zoanthids (and corals), > 1 DNA marker is needed.
mt DNA evolves very slowly.
Still, better than no experts at all!

3. Promoting taxonomy: Census of Marine Life.

Scientific Framework
What has lived in the oceans?
What does live in the oceans?
What will live in the oceans?
The Census consisted of four major component programs organized around these questions.
1. Investigating the Past
Census researchers undertook the challenge of constructing the history of marine animal populations since human predation became important, roughly the last 500 years. This program component was called HMAP.
Teams of fisheries scientists, historians, economists and others conducted case studies in southern Africa, Australia, and approximately a dozen other regions. 
Together, these case studies created the first reliable picture of life in the oceans before fishing. 
The long historical records of marine populations help distinguish the contributions of natural fluctuations in the environment from the effects of human activities.
2. Assessing the Present
The largest component of the Census involved investigating what now lives in the world's oceans through 14 field projects. 
Each sampled important kinds of biota in one of six realms of the global oceans using a range of technologies.
This included CReefs.
3. Forecasting the Future
To speak about what will live in the oceans required numerical modeling and simulation. This component program is the Future of Marine Animal Populations (FMAP). 
Integrating data from many different sources and creating new statistical and analytical tools to predict marine populations and composition of ecosystems in the future
4. Living Legacy
Such a global initiative required a state-of-the-art data assimilation framework, and this is:Ocean Biogeographic Information System (OBIS). 

Numbers
2,700 scientists 
80+ nations 
540 expeditions 
US$ 650 million 
2,600+ scientific publications 
6,000+ potential new species 
30 million distribution records and counting

4. Example: CReefs:
Heron Island Trip
November 8th – December 1st, 2010
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)!
Location:
Heron Island
Many seabirds nest here. Always noisy, and dangerous to walk without a hat!
Rails also live on the island.
The island is an important nesting site for green and loggerhead sea turtles.
Heron Island Research Station is run by the University of Queensland. Most of the station is very new.
There are excellent facilities for experiments, sample collection, and analyses, indoors and outside.
Scientists from all over the world, most based in Australia, but others from USA, Japan, Iceland, etc.
Most scientists had much field experience, and were good divers. Many also had boat licenses and first aid training.
There were also dive officers, who acted as boat captains, guides, diving assistants, etc.
Outreach included a professional photographer, and a professional blogger with stories and images posted every day.
A professional chef ensured everyone was well-fed. The food was amazing!
Every night at dinner, based on everyone’s ideas, weather conditions, and tides, the next day’s schedule was decided.
Boats were launched from the harbor, 3-10 people per boat. Often boats were out for over 7 hours.
Diving was always done under the buddy system, with very strict guidelines on diving protocol.
Special permits were obtained to allow specimen collection. Work underwater was intense and focused.
I was able to collect 270 specimens in 33 dives. Other groups collected up to >2000 specimens!
Samples were also collected on reef walks, and by snorkeling. Other groups used ARMS and the “carpet of death”.
Some groups found many new species, others new records for the southern GBR. This genus was found in the southern Pacific Ocean for the 1st time, likely a new species.
Another potential new species of zoanthid.
Many people also collected for other groups.
Back in the lab, data were collected, and specimens numbered. All data were given to the CReefs data manager as well.
All sites were assigned numbers, and all had GPS coordinates. 130 sites were visited in 3 weeks!
Samples will be shipped to institutions all over the world and analyzed further.
Of course, work was not 24 hours a day…
Sunset drinks were the one time of day when everyone would relax and take a break.
And the sunsets were amazing…

5. PCoRIE biodiversity in Palau
Palau overview

Independent since 1994
2008 GDP: $164M 
18,000 habitants
466 km2  land, ~30 0km2 reef
As many fish species as in Philippines!

Our surveys:

30 sites selected, covers most of Palau
3つの方法:
Gene flow with population genetics
DNAの集団解析
‘Focused’ surveys on specific taxa
いくつかの生物分類群に注目する
Quantitative surveys of coral rubble
定量的調査：サンゴの瓦礫

Population genetics:

Determining sources & sinks in Palau

How does gene flow & diversity match with MPAs?

Are populations expanding/growing or shrinking/decreasing?

Family Vermetidae

Genus Dendropoma　フタモチヘビガイ類

Very low dispersal, good for such studies

Focused study 1:

Annella, octocoral, can reach 3 m across!

Common on reef drop offs in Palau.

Important habitat for shrimp, seahorses.

Important for tourism.

Diversity not well-studied.

DNA results show 2 species.

Only 1 fits with current definition.

More research needed!

Focused study 2:

Epizoanthus illoricatus

Order Zoantharia

Common on reef drop offs in Palau.

DNA results show 2 species.

Only 1 species known.

More research needed!

Relative biodiversity (quantity)

Describe diversity and spatial community structure, using coral rubble 

Analyze connectivity (intra-species) 

Compare with current design of MPAs

Sampling & sorting ⇒ Description　&

       molecular analyses

Future research

Finish PCoRIE surveys, improve resolution

DNA analyses & training

More ‘focused’ research on other taxa

Other ecosystems (mangroves?)

MPA design & policy recommendations

References cited:
1. CP Meyer, G Paulay. 2005. DNA barcoding: error rates based on comprehensive sampling. PLoS Biology 3(12) e422.
2. Consortium for Barcoding of Life homepage.
3. Census of Marine Life homepage.
4. OBIS homepage.