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