Using Biotechnology and Bioinformatics to Track a Marine Invader Based on the work of Dr. Jonathan Geller, Moss Landing Marine Laboratories and Dr. Caren Braby, Monterey Bay Aquarium Research Institute Marine Biotechnology and Bioinformatics Teacher Enhancement Program at Moss Landing Marine Labs

Outline Background Background Invasive species Invasive species Cryptic invaders Cryptic invaders Local mussels Local mussels Hypothesis Hypothesis Materials and methods Materials and methods The molecules The molecules Expected results Expected results Possible conclusions Possible conclusions Carcinus maenas, the European green crab, invader of the Pacific west coast. Photo credit: Jim Carlton

Invasive species Non-native species are organisms introduced into an environment in which they did not evolve Non-native species are organisms introduced into an environment in which they did not evolve Invasive species are non-native species that significantly out-compete native species Invasive species are non-native species that significantly out-compete native species Mostly introduced by human Mostly introduced by humanactions Largest threat to biodiversity Largest threat to biodiversity after habitat loss Estimated at least $137 billion Estimated at least $137 billion in damages annually in the US Most marine invaders probably Most marine invaders probably came as larvae in ballast water Caulerpa taxifolia, an invasive marine alga, in the Mediterranean Sea. Photo credit: U. of Nice, France

The coasts of the world are interconnected by extensively used shipping routes The coasts of the world are interconnected by extensively used shipping routes Ocean-going ships use seawater for ballast (balance) Ocean-going ships use seawater for ballast (balance) Ballast water mages/carte_logistique.gif

Ballast water The water (and plankton) are moved great distances and released The water (and plankton) are moved great distances and released Planktonic organisms after transport are alive, abundant, and diverse Planktonic organisms after transport are alive, abundant, and diverse Reference: Carlton & Geller, 1993, Science Reference: Carlton & Geller, 1993, Science Supplement: S1 “Transport of marine invaders” Supplement: S1 “Transport of marine invaders”

Cryptic invaders Some invasions can be cryptic (obscure) and therefore difficult to track Some invasions can be cryptic (obscure) and therefore difficult to track Many species with world-wide distribution may actually be successful cryptic invaders Many species with world-wide distribution may actually be successful cryptic invaders One example of world-wide distribution in temperate areas is shown above One example of world-wide distribution in temperate areas is shown above Modified from figure in Hilbish, et al. 2000

One cryptic marine invader Before 1988 all “Bay mussels” were considered Mytilus edulis Before 1988 all “Bay mussels” were considered Mytilus edulis Thought to be distributed world-wide in temperate areas Thought to be distributed world-wide in temperate areas eshellfish.com/Pen nCoveMussels.htm

Genetic analysis Genetic studies uncovered three morphologically indistinguishable species Genetic studies uncovered three morphologically indistinguishable species Mytilus edulis Mytilus edulis Mytilus trossulus Mytilus trossulus Mytilus galloprovincialis Mytilus galloprovincialis Only one is a successful Only one is a successfulinvader Genetic markers are Genetic markers are essential for species identification Photo credit: J. Geller

World-wide distribution? Historic (native) ranges for each species were identified Historic (native) ranges for each species were identified Areas of hybridization were found Areas of hybridization were found Invasion events were mapped Invasion events were mapped Only M. galloprovincialis appears to invade, and it has done so repeatedly Only M. galloprovincialis appears to invade, and it has done so repeatedly All done with phylogenetic analysis All done with phylogenetic analysis References: Hilbish, et al., 2000, Marine Biology; Riginos & Cunningham, 2005, Molecular Ecology References: Hilbish, et al., 2000, Marine Biology; Riginos & Cunningham, 2005, Molecular Ecology

Phylogenetic trees A diagram showing evolutionary lineages of organisms A diagram showing evolutionary lineages of organisms DISCUSSION: branches, clades, outgroup DISCUSSION: branches, clades, outgroup A speculatively rooted tree for rRNA genes. search phylogenetic tree

Our native: M. trossulus North Pacific M. trossulus North Pacific M. trossulus Invades North Atlantic after the opening of the Bering Strait approx. 3.5 million years ago Two “natural invasion” events occurred (1 & 2) Reference: Riginos & Cunningham, 2005, Molecular Ecology

Our Native Adapted from Hilbish, et al., 2000

Native

Native Our Invader Adapted from Hilbish, et al., 2000

Native Invader came from Mediterranean Sea Adapted from Hilbish, et al., 2000

Native Invader Possible scenario being studied now Adapted from Hilbish, et al., 2000

Invader displaces native DNA from museum collections shows M. trossulus in southern CA DNA from museum collections shows M. trossulus in southern CA M. galloprovincialis arrived in Southern CA in the 1930's M. galloprovincialis arrived in Southern CA in the 1930's It has progressively spread northward and displaced M. trossulus It has progressively spread northward and displaced M. trossulus Reference: Geller, 1999, Conservation Biology Reference: Geller, 1999, Conservation Biology

Our Location: Monterey Bay Part of the MBNM Sanctuary Part of the MBNM Sanctuary Mixed population of M. trossulus and M. galloprovincialis Mixed population of M. trossulus and M. galloprovincialis

Another California native Mytilus californianus Mytilus californianus Lives on outer coast Lives on outer coast Morphologically distinct Morphologically distinct Genetically distinct Genetically distinct Will use as an outgroup Will use as an outgroup

Why do we care? To identify what makes a good invader To identify what makes a good invader Best to work with closely related species Best to work with closely related species To address major questions in natural selection and biodiversity To address major questions in natural selection and biodiversity How can an invader with limited genetic diversity out compete the genetically diverse native? How can an invader with limited genetic diversity out compete the genetically diverse native? How does an invader displace the native that has been selected for its niche? How does an invader displace the native that has been selected for its niche? How does the invader move up the coast to SF bay when it did not invade there from ballast water? How does the invader move up the coast to SF bay when it did not invade there from ballast water?

Quick Quiz A(n) _________ species is a non-native species that successfully out-competes the native species. a. robust b. invasive c. genetically superior d. cryptic

Quick Quiz A(n) _________ species is a non-native species that successfully out-competes the native species. a. robust b. invasive c. genetically superior d. cryptic

Quick Quiz All invasive species are non-native but not all non-native species are invasive. a. True b. False

Quick Quiz All invasive species are non-native but not all non-native species are invasive. a. True b. False

Quick Quiz Which of the following is an invasive mussel species in California? a. M. galloprovincialis b. M. trossulus c. M. californianus d. M. edulis

Quick Quiz Which of the following is an invasive mussel species in California? a. M. galloprovincialis b. M. trossulus c. M. californianus d. M. edulis

Outline Background Background Invasive species Invasive species Cryptic invaders Cryptic invaders Local mussels Local mussels Hypothesis Hypothesis Materials and methods Materials and methods The molecules The molecules Expected results Expected results Possible conclusions Possible conclusions Carcinus maenas, the European green crab, invader of the Pacific west coast. Photo credit: Jim Carlton

Hypothesis As Mytilus galloprovincialis spreads northward it hybridizes with AND displaces Mytilus trossulus As Mytilus galloprovincialis spreads northward it hybridizes with AND displaces Mytilus trossulus

Materials and Methods Materials: Mussels Materials: Mussels "Mussel" is a common name for bivalves that attach using threads (byssus) "Mussel" is a common name for bivalves that attach using threads (byssus) Supplement: S2 “Know your mussel” Supplement: S2 “Know your mussel” Washington Dept. of Fish and Wildlife

Materials and Methods Methods: Biotechnology: Methods: Biotechnology: DNA extraction -> PCR -> Analysis DNA extraction -> PCR -> Analysis Restriction digestion Restriction digestion Gel electrophoresis Gel electrophoresis DNA sequencing DNA sequencing DISCUSSSION DISCUSSSION

Materials and Methods Methods: Bioinformatics: Methods: Bioinformatics: DNA sequences -> Edit -> Align DNA sequences -> Edit -> Align Search DNA database Search DNA database Build trees Build trees Translate DNA sequences Translate DNA sequences View protein structure View protein structure DISCUSSION DISCUSSION

The molecules For this study we need For this study we need to find molecules that: Share similarities within each species BUT display differences between species Share similarities within each species BUT display differences between species In other words, molecules that are just different enough! In other words, molecules that are just different enough! Also need well supported results Also need well supported results Therefore we will use several (3) molecules Therefore we will use several (3) molecules

The molecules Remember: We will be working with PCR products Remember: We will be working with PCR products Pieces of DNA Pieces of DNA Not whole gene Not whole gene Not just protein- Not just protein- coding regions Supplement: S3 Supplement: S3 “Meet the Molecules”

The molecules 1.ITS = the internal transcribed spacer of the nuclear ribosomal genes  Region has restriction site polymorphism between species cology/Techniques/mt- what_dna.htm DNA RNA Ribosome (RNA + proteins)

HHGGT ITS - Interpreting results GM Lane M: 100 b.p. ladder lane Lanes G: M. galloprovincialis M. galloprovincialis Lanes H: hybrid Lane T: M. trossulus Easy, useful tool to differentiate species Photo credit: C. Braby GG

The molecules 2.Glu = Polyphenolic adhesive protein - nuclear  Number of repeats (and therefore gene length) varies between species protein = repeat

MG Glu - Interpreting results Second tool to validate ITS results Photo credit: C. Kirlin Lane M: 100 b.p. ladder lane Lanes G: M. galloprovincialis M. galloprovincialis 300 & 500 bp bands Lane T: M. trossulus 240 bp band Hybrids would have a combination of the two patterns. GGMTTT

The molecules 3.CO3 = Cytochrome c oxidase subunit III - mitochondrial Species level differences are observed after DNA sequencing Species level differences are observed after DNA sequencing Also, highlights an interesting phenomenon: Also, highlights an interesting phenomenon: Mussel mitochondria don’t play by the rules! Mussel mitochondria don’t play by the rules! Mitochondria are not maternally inherited as they are in mammals Mitochondria are not maternally inherited as they are in mammals database/bioinformatics/mitochondria.gif

Unusual mtDNA inheritance “Doubly Uniparental Inheritance” “Doubly Uniparental Inheritance” Daughters receive maternal mtDNA while sons receive both but only pass on paternal mtDNA Daughters receive maternal mtDNA while sons receive both but only pass on paternal mtDNA How that works we don’t know How that works we don’t know mitochondrial type: daughter son

Two separate mtDNA lineages Two separate mtDNA lineages Same sex mtDNA from different species are more related to each other than opposite sex mtDNA of their own species Same sex mtDNA from different species are more related to each other than opposite sex mtDNA of their own species Remember the previous tree? Remember the previous tree? Reference: Geller, 1999, Conservation Biology Reference: Geller, 1999, Conservation Biology First seen with Bioinformatics

Why CO3? CO3 gene fragments need to be CO3 gene fragments need to be purified and sequenced. This will… Support mussel species identification Support mussel species identification Identify the mtDNA type in hybrids Identify the mtDNA type in hybrids Provide novel DNA sequences to Genbank Provide novel DNA sequences to Genbank Provide raw material for bioinformatics work Provide raw material for bioinformatics work We will amplify the female genotype from gill since it is found in both sexes We will amplify the female genotype from gill since it is found in both sexes

Quick Quiz M. galloprovincialis displaces M. trossulus by: a. coveting the best substrates b. tolerating more environmental changes c. genetic hybridization d. producing more offspring ??

Quick Quiz M. galloprovincialis will be distinguished from M. trossulus by: a. dissection b. behavior c. shell morphology d. genetic analysis

Quick Quiz M. galloprovincialis will be distinguished from M. trossulus by: a. dissection b. behavior c. shell morphology d. genetic analysis

Biotech Flowchart

Bioinformatics Flowchart

Outline Background Background Invasive species Invasive species Cryptic invaders Cryptic invaders Local mussels Local mussels Hypothesis Hypothesis Materials and methods Materials and methods The molecules The molecules Expected results Expected results Possible conclusions Possible conclusions Carcinus maenas, the European green crab, invader of the Pacific west coast. Photo credit: Jim Carlton

Previous data  Monitored the distribution of M. galloprovincialis along the west coast  Reference: Braby & Somero, 2005, Marine Biology 7.Santa Cruz 8. Moss Landing – North 9. Moss Landing – South 10. Moss Landing ML 11. Monterey

Previous data  Monitored the distribution of M. galloprovincialis along the west coast  MLML = the site we will be sampling as well  Reference: Braby & Somero, 2005, Marine Biology 7.Santa Cruz 8. Moss Landing – North 9. Moss Landing – South 10. Moss Landing ML 11. Monterey

Previous data  Monitored the distribution of M. galloprovincialis along the west coast  MLML = the site we will be sampling as well  Found 2 potential refuges - PA & MLML  Reference: Braby & Somero, 2005, Marine Biology 7.Santa Cruz 8. Moss Landing – North 9. Moss Landing – South 10. Moss Landing ML 11. Monterey

What will we learn from our data? 1.Is our hypothesis supported? As Mytilus galloprovincialis spreads northward it hybridizes with AND displaces Mytilus trossulus As Mytilus galloprovincialis spreads northward it hybridizes with AND displaces Mytilus trossulus 2.Is MLML a refuge?

Previous results MLML: Displacement appears to be occurring MLML: Displacement appears to be occurringvs. PA: This site appears to be a stable refuge PA: This site appears to be a stable refuge

Sampling procedure Previous evidence shows that the smaller mussel population contains more natives Previous evidence shows that the smaller mussel population contains more natives But for an accurate comparison to previous data we should sample in the same random way But for an accurate comparison to previous data we should sample in the same random way Reference: Braby & Somero, 2005, Marine Biology Reference: Braby & Somero, 2005, Marine Biology

Possible conclusions Displacement will continue at the MLML site Displacement will continue at the MLML site Stable refuge will persist at the PA site Stable refuge will persist at the PA site

Quick Quiz The data collected in this workshop will contribute to answering which of the following questions? a. Is MLML a refuge for M. trossulus? b. Is M. trossulus going extinct? c. Is M. galloprovincialis continuing a successful northward invasion? d. Is the M. galloprovincialis invasion threatening M. californianus populations?

Quick Quiz The data collected in this workshop will contribute to answering which of the following questions? a. Is MLML a refuge for M. trossulus? b. Is M. trossulus going extinct? c. Is M. galloprovincialis continuing a successful northward invasion? d. Is the M. galloprovincialis invasion threatening M. californianus populations?

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