2. 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

3. 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

4. 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

5. 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 http://www.animaweb.org/i mages/carte_logistique.gif

6. 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” http://massbay.mit.edu/exoticspecies/ballast/

7. 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

8. 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 http://www.penncov eshellfish.com/Pen nCoveMussels.htm

9. 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

10. 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

11. 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. www.answers.comwww.answers.com search phylogenetic tree

12. 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 1 2 1 2

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

14. Native

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

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

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

18. 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

19. 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 http://walrus.wr.usgs.gov/infobank/gazette/html/navigation/ncal.html

20. 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

21. 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?

22. 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

23. 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

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

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

26. 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

27. 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

28. 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

29. 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 http://www.goes.noaa.gov/GIFS/WCIR.JPG

30. 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

31. 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

32. 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

33. 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 http://www.envirohealthtech.com/images/DNA.jpg

34. 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” http://www.nearingzero.net/wordplay.html

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

36. 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

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

38. 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

39. 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 http://www.bioeng.auckland.ac.nz/images/ database/bioinformatics/mitochondria.gif

40. 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

41. 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

42. 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

43. 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 ??

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

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

46. Biotech Flowchart

47. Bioinformatics Flowchart

48. 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

49. 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

50. 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

51. 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

52. 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?

53. 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

54. 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

55. 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

56. 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?

57. 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?

58. END