Multilocus Sequence Typing MLST HMC 2012 what is MLST? powerful population genetics technique DNA sequences of internal fragments of multiple genes identifies allelic variants to.
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Multilocus Sequence Typing MLST HMC 2012 what is MLST? powerful population genetics technique DNA sequences of internal fragments of multiple genes identifies allelic variants to characterize, subtype and classify members of bacterial populations Evolutionary Pattern Diversity Population Genetics Evolutionary Process form & function mutation morphology recombination physiology natural selection phenotype/genotype genetic drift allelic diversity migration MLST data general protocol 1) sampling and isolation of marine Vibrio on TCBS Agar plates 2) restreak and pick single colonies --> liquid cultures general protocol 3) extract DNA, perform PCR to amplify genes 4) Sanger sequencing (for and rev Primer for each gene) 5) alignment of reads, quality check, computational analysis DNA Extraction Gene Amplification Sequencing, Phylogenetic Analysis metadata local site (Hopkins and Point Lobos) metadata global site (California and New Zealand) 2 sites 2 sites metadata time (2007 - 2012) source (anemone and water) Gyrase • Two subunits encoded by gyrA and gyrB • Topoisomerase Type II: o ATP-dependent initiation of the double-strand breakage of DNA o Introduces negative supercoils in DNA o Only present in bacteria: target for antibiotics Malate dehydrogenase • NAD+-dependent oxidation of malate to oxaloacetate o Involved in several metabolic pathways (e.g. TCA cycle) • Homodimeric soluble 30-35 kDa protein RecA • Protein responsible for maintenance and • • repair of DNA DNA-dependent ATPase Several functions all related to DNA repair ompK • - 26 kDa outer membrane protein - transmembrane beta-barrel protein involved in ion transport - receptor for KVP40, a broad-hostrange vibriophage isolated from sea water Main Questions 1) Is there an observable population structure? If so, how does this relate to the ecology of the environment? (Null) Hypothesis: No structure/pattern. “Everything is everywhere.” 2) How is evolution occurring in the environment? a) Rate: Are the genes evolving at a similar rate? Hypothesis: Housekeeping genes are evolving slower than ompK. b) Process: How much recombination vs. mutation is observed in populations? Hypothesis: Significant amounts of both recombination and mutation at a fixed ratio within the population. Data Overview GENE gyrB mdh recA ompK total unique alleles sequences sequences (98%) 502 159 61 550 158 59 486 195 56 65 59 25 Rarefaction Analysis mdh 45 40 40 35 35 30 30 25 25 20 20 15 15 10 2012 10 5 NZ 300 280 260 240 220 200 180 160 140 120 110 90 80 60 40 20 0 0 2007-2011 1 20 40 60 73 90 110 120 140 160 180 200 220 240 260 280 300 320 340 360 5 1 Number of alleles (98% similarity) gyrB recA 35 30 ompK 30 25 25 20 20 15 15 10 10 5 5 0 0 1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 Number of sequences 2012 0 2 gyrB New Zealand 2012 38 1 New Zealand 0 mdh 1 12 0 11 0 34 1 2007-2011 2007-2011 8 12 New Zealand 2012 0 1 31 recA 0 12 Number of shared alleles - ‘allele’ defined at 98% sequence similarity 2 2007-2011 10 Main Questions 1) Is there an observable population structure? If so, how does this relate to the ecology of the environment? (Null) Hypothesis: No structure/pattern. “Everything is everywhere.” Concatenated housekeeping genes A homogenous and phylogenetically distant clade Hopkins Point Lobos Water Anemone Years These are different from the rest of the isolates with 100% bootstrap support. Unfortunately, we didn’t isolate any this year. 66 60 82 A ubiquitous, low diversity clade Recent diversification? 86 population structure, but no clear sorting by geography or time Individual gene trees gyrB recA mdh Derived clades gyrB Derived clades recA Derived clades mdh Opportunity to look for recombination •Divergent •Low diversity •100% bootstrap support We might expect them to share an evolutionary history MultiDimensional Scaling Finds the best way to visualize distance information on a 2 or 3 dimensional plot. In this case we used the maximum likelihood estimates for evolutionary distances between isolates. On the plot, evolutionarily distant isolates are far apart while closely related isolates are close together. The difference between multidimensional scaling and trees A • A is equally related to B, C and D. B MDS 2 D C D A MDS 1 B C The difference between multidimensional scaling and trees A D MDS 2 B • A is equally related to B, C and D. • On the tree, A is only close to B C D A MDS 1 B C The difference between multidimensional scaling and trees B C D D MDS 2 A • A is equally related to B, C and D. • On the tree, A is only close to B • This can make it hard to visualize categorical clusters A MDS 1 B C The difference between multidimensional scaling and trees B C D D MDS 2 A • A is equally related to B, C and D. • On the tree, A is only close to B • This can make it hard to visualize categorical clusters • The MDS plot solves this problem by putting closely related species close together on the page A MDS 1 B C MDS - multidimensional scaling Site Hopkins Pt. Lobos MDS - multidimensional scaling Source Water Anemone MDS - multidimensional scaling Year 2007 2008 2009 2010 2011 2012 ompK phylogenetic tree pK om K p 1b m W e o H 7 e 8 PA A4 4b 0 1 pK 20 01 0 9 H H A om 2 0 11 2 0 7 2b 2 Z 8 PA 6e N Z 8 PW N 00 1 2 01 53 2 Z 40 N Z 26 N Z 31 N Z 69 pK N Z 84 om K N Z 72 N Z 73 A6aomp A2h N Z 8 H 30 K H N 00 2 mp gy’ 6 2 0 0 o A5 ’ 2 010 2 H A8a 2 01 2 H ’2 01 0 ’2 Z 5 8 N Z1 9 N Z1 9 N Z4 2 N Z5 N Z 82 N Z 83 N Z 85 N Z 77 N Z 79 N Z 81 N 86 N Z 80 N Z 75 NZ 76 NZ 107 N Z 1 0 9 5 o m pK NZ 06 23 20 1 0 6 NZ 108 NZ 35 NZ 12 NZ 6 NZ 5 NZ 2 NZ 3 NZ 13 NZ NZ 48 NZ 22 NZ 17 NZ 20 NZ 45HA1a ompK 2008 1a ompK 2008 HW a ompK 2008 HA7 a ompK 2008 HA5 a ompK 2008 HA8 ’ HA3b ’2012 e 2010 ompK HW3 2012 HA1d 2006 237 ompK NZ 23 2012 HA7a 2012 HA1b 2011 PW3a 2011 PW3d 2007 PA1 9 ompk 2011 HW3f 2011 HA8a 2011 HA3d 2012 HW4 2012 PA4 a 2012 PA4 c 2012 HA1ae 2012 HA4a NZ 64 NZ 65 NZ 60 NZ 70 NZ 62 NZ 59 NZ NZ 58 NZ 56 201257 2011 PA5c NZ 4 HW3e NZ 4 NZ 38 NZ 37 NZ 3 2 NZ 2 1 N Z 15 NZ 11 NZ 14 N 55 N Z 68 N Z 71 N Z 10 NZZ 1002 N 94 N Z 91 N Z 89 N Z8 N Z8 7 NZZ 748 N 6 N Z6 1 N Z1 3 N Z 9 03 N Z9 6 N Z9 5 NZZ 923 N 2 Z 90 N 01 9 9 2 Z 0o 2 0 0 3 3 mp K 2 00 6 HA 20 011 6 2 257 6 o 9a 2 1 2 00 1 PW 0 o m ’2 00 9 PW 3 m pK ’2 01 9 HHA 3f c pK 7 2 0 2 20 01 10 HAA7a d o 0 o m 2 20 01 10 o om mp 7b’ om pK 20 0 1 m p K p 11 8 P P W p K K H P A K H W 1 c H A 3 7b W 2a W e ’ 3a o 3b m pK 10 Z 4 pK N Z 9 N Z 1 2 om pKpk N Z 4 4 m N Z 5 11 W1ed o om pk N Z 1 P 5 7 om N Z 8 HA 1 8 A N 0 20 008 7 HHA1 4d 2 0 0 7 HA 1 a a ’ 2 0 0 2 PA 1 3b 2 01 2 HW 7f HA 3d 2 0 1 2 A p K HW 2 01 2 P m K ’2 1 o p ’ pK 200100 om A1gj 2 0 1 2 P A 1 c o m m pK 2 01 2 P W 2 e o ’2 01 H A17 b’ 2 009 P A15 a’ pK 2 009 2 P A15 om pK 2 1 P a m ’20012 HA34d oompKK ’2 008 HA 8b mp c 2 009 HA 8c o A2 2 009 HA K P A5d c’ 2 009 omp K P W3 2 10 omp K H 8h 20 0 m p H A k 201010 oompKK HA8 4e ’2 10 mp PW d 20 10 o mpK PA1 l 20 10 o mpK HA8 b 20 0 o p K A 1 20110 ommpK P pK 20 10 o 0 o m 20 06 22 A3f’ 20 12 H 1c ’20 12 HA 2 0 39 NZ 41 NZ 16 NZ NZ 25 NZ 24 ompK NZ 27PA11eompk 2008 PA8a ompK 2008 PA5a pK 2008 245 om pk 2006 HA5 8 om 2007 PW8f 2011 PW8e 2011 PA6b 2011 PA10b 2011 2a 2011 PA 14a ompK 2009 PW 6b ompK 2009 PA1 2a ompK 2009 PA1 d 2012 PA8 ’2012 PA8i’ ’2012 PA8h’ 2012 PA62a ’2012 PA15c’ ’2012 PW2b’ 2012 PA5f 2012 PA7c 2011 HW5d 2 2 00 2 00 7 2 00 7 H 2 00 7 H A6 2 0 00 7 7 H H A A 6 9 0 7 H A 4 4 o om 2 A 2 7 2 00 HA 1 2 om mp pk 20 20 006 6 2 1 1 9 o om pk k 2 10 06 2 75 o m pk 2 0 1 o 2 7 2 o m pk ’2 01 0 m 61 o m pk ’20 01 0 o om pK ommp pK 2 1 0 m pK HA p K 20 009 0 omomppK H 2mK 20 09 HW pK K HHA2A2j 0 9 HW 1 b H A n HW 1a o A3 2i’ m d 20 1d oom pK ’ p 1 ’2 201 1 P mp K ’20012 1 H A3 k 20 12 HHA6A4f f 1 d A 2 2 2 ’20 010 20 011 HA74c’ ’ 1 1 0 om 2 H e 20 om pK HA A2f 20 0 6 2 p K P A 3 g 20 06 2 22 HA2 7a 20006 2 55 oompKa’ 6 58 m 2002006 259 omppK 200 8 H 262 omp K 20 7 H A4a omp K 20 07 H W1 1 omp K 20007 HWA1 6 oompkK 2007 HW 2 1 o mpk 2 7 P 2 4 mp 200007 P A1 5 oompkk 200 7 PA1A1 7 o mpk m 7 200 PA1 10 om pk 20077 PA211 omppk 200 PA2 3 omp k 20 7 PA2 7 ompkk 200707 PA2 98 ompk 2007 PA2 10 ompk ompk 2008 PW1 1 om pk 2008 HW1c om 2008 HW2d om pK pK 2d 2010 PW ompK ompK 2010 om PA1e 2010 ompK HA10a pK ’2010 om PA3e pK PA1f’ ’2010 om ’2010 ompK PA9a’ pK PA9f’ ’2012 PA 2012 PA8b’ 201 PA81i c 2010 ompK2 PA2 a 2009 HA4c omp K 2009 HA7e ompK 2009 PA17d ompK 2011 PW8c 2011 PA7b 2011 HW5f 2 201011 HA 1 20 0 20 PA1 3ac 20099 PA16 11 HA0 PA 1 e o m 3 f 6 ’2012c omppKK 2012 PW2a’ 2012 PA4a 2011 HA3e 2011 PW6c 2011 PW8d PW8b 2011 PW 2011 PW 6d 6b 2011 PW 2011 PA 6a 7c 2011 PA 2011 PA 7a 6a 2011 PA2e 2011 PA2 2011 HA4 d 2012 PA1ha 2012 HA3c ’2010 ompK HA1g 2010 ompK PW4g’ 2010 ompK PW4b ’2012 PA55e’ ’2012 PA55c’ ’2012 PA62c’ ’2010 ompK HA2e’ 2010 ompK PW4h 2010 ompK PW4d 2010 ompK PA9b 2010 ompK PA5e 2010 ompK PA3a f 2010 ompK PA2m 2010 ompKKHA8 HA2d 2010 omppK HA2b 2010 om PA62d’ ’2012 PA5a’ ’2012 PW4 2012 pK 14e om pK 2009 PW 14b om pK 2009 PW 13b om pK PW 2009 PW13a om kK 09 e om kK 20 PA20 2009 PA20d om kK 2009 PA20c om kK om a 2009 PA20 ompK 2009 PA16ad ompK 20099 PA12 ompKK 200 9 PA11ed omppK 1 0 20 9 PA1 2e om pK 20009 HW 2d om pK 20 9 HW 2b om pK 200 9 HW 2a om pK 200 9 HW 1e ommpK 20009 HWA9c o mpK 20 09 H 12b o mpKk 20 9 PA 3c oomp K A 200009 HW2 4 omp K 2 7 P 2b omp K 20008 PWW1a omppK 20 08 P A12ed om pK 20 08 P A12 c om pK 20 08 P A12 b ommpKK 20 08 P A121d o mp k 20 08 P A1 1b o omppk 20 08 PPA1 0b om pk 20 08 P A 1 9 b o m p K 20 008 8 PAA9a om pK 2 0 0 8 P 2 b om p K 2 0 W 3b om pK 20 0 8 H A 1 6e o m p K 20 08 H HAA6d c ommpK k 20 008 8 H A6 b o omppk 2 0 0 H A 6 5 m pk 2 008 H 2 2 o m pk 2 00 8 P W 2 1 o m p k 2 0 7 PW 2 6 o o m p k k 20 007 PWA2 2 1 ommp k 2 007 7 P A 1 8 o mp pk 2 00 7 PPA 1 3 2 o m pk 2 0 0 7 A 1 o m pk 2 00 7 P A 1 1 5 o m pk k 2 0 0 7 P A 2 3 o om p k 2 0 0 7 P W 1 2 om p 2 00 H W 1 0 om 2 07 7 H W 7 1 5 2 0 00 7 H A A 7 2 00 7 H H 2 0 0 07 2 20 2006 223 ompK 2006 224 ompK 2006 225 ompK 2006 226 ompK 2006 227 ompK 2006 228 ompK 2006 243 ompK 2006 246 2006 263 ompK 2006 266 ompK 2006 268 ompK 2007 HA1 ompK 2 ompk 2007 2007 HA2 6 ompk 2008 HA2 8 ompk 2008 HA17a om 2008 HW2e om pK 2006 PA11a ompK pK 2006 233 om 2007 267 om pK 2006 HA2 10 pK 2006 234 om ompk 2006 264 om pK 2007 265 om pK H A2 pK 20 20 07 HA2 1 om 20 08 HA 9 om pk 2 0 0 8 H W 2a o m pk NZ 06 23 2c o pK pK 200 28 6 ompm K 20 6 23 20 08 P 2 om 20 11 H W2c pK 2 0 0 9 H W 5c o mp K 20 09 P A3a 20112 PAW14 ompK 2 15 d o ’2 N 012 PA4 d mpK N Z 34 PA7i’b 2 Z 43 20008 P 0 2 8 W 2 008 PW 1c 2 008 PA 2e omp N 008 PW 1a om K 2 Z 3 PW 1b ompKpK 1d o m 2 0 09 0 om pK ’20010 PA pK N 1 om 16 2 Z 0o p d N 01 78 mpKK PAomp 20 Z 1 0 o m 1 K 2 0 05 pK PA9 3f d 20 006 9 PW ’ PA N 0 2 5c N Z 6 19 13 N Z 10 22 o c N Z 11 4 1 ommpKomp K 2 Z 66 0 pK 2 01 6 7 20 0 1 1 1 P N N Z 11 P A3 N Z 1 P A3 d N Z 9 01 A 3b a 2 Z 9 7 N 00 4 6 8 Z 9 29 H A3 d om pK 2006 271 ompK 2010 ompK PA9c 2010 ompK HW8g ’2012 PA4f’ ’2012 HA5a’ ’ ’2010 ompK PA7c PA7d 2010 ompKHA1 0d 2010 ompK K HA3c 2010 omppK PA1c 2010 om 4d 2012 PApK 11c om 2009 PA 10b ompK 2009 HA 8e ompK 2009 HA 8d ompK pK 2009 HA 8a om pK om 2009 HA HA5d 2009 HA4b ompK pK 2009 HA4a om pK 2009PA11b om ompK 2009 PA11a HA2e’ 2009 ’2012K PA1aa’’ p 7 0 om P W ’201 ’2012 2 PA7hd’ 201 2 HA55b ’20112 PA 2b 20 2 HA 8’ 20 1 2 H A 8 j ’201pK HAA5f m 0 o 12 H 7 c ’ 201 20 12 HA 7d 20 12 PA 8e 7f’ ’20 pK HW P A 1 g’ m 0 o m pK P A g 201010 o mpK PA77e ’2 10 o mpK PA 5b ’20 010 oompKK PAA5a 2 0 m p P 2e 20110 oompKK PAA9dk 2 0 1 0 o m p 1 P A9 i 20 10 20111 P PA99f 20 20 011 PA 9c 2 11 P A 9 a 20 11 PA 9h 20011 PA 9e l 2 011 PA A9 o 2 011 1 P A9 n 2 01 P A9 2 1 1 P 9m j 20 011 PAPA9 g 9 b 2 1 A 1 1 20 201 1 PPA9 8b f 1 8 20 011 HWHA 8e 2 11 1 HA 7e a 20 20111 HA 1 e W 3 20 11 P A 0c c 20011 1 P A1 8 3c 2 201 1 P HWW 1f 1 1 H W 2 0 01 11 1 H 2 0 1 2 20 2 2 01 2 01 1 2 0 1 1 HW 2 01 1 H W 1 20 011 1 HHW 1c e 2 1 A 1 2 01 1 HA 8 b 2 01 1 HA 6e d 2 01 1 HHA 6d 2 0 1 1 H A 6b 2 01 1 H A 5 e 20 011 1 H A5 5d 2 1 H A b 2 01 1 H A 5 a 2 011 1 H A 1b 20011 PWA11a 2 11 H 3 c 20 011 HWW5e e 1 1 H 5a H W N W7 7f N Z3 e NZZ 516 47 20007 HAW2comp 2 00 8 H 6 pK pK 2 008 23 2 006 8 2 om om c 2 Z 2 23 2 N 006 PW 5c ompK pK 2 008 HW 3a om 2 011 HA 14d 2 009 P W 5 d 2 009 PA1 b 2 012 PA4 i’ 2 12 PA7 20 012 pK ’ 2 Z 34 om K N Z 43 W1c omp K N 08 P W2e omp K 2 0 0 8 P A 1a o m p K 20 08 P W1b omp 2 0 0 8 P W1 d 20 08 P mp3Kf 20 30 16d oA 1 Z N 09 PA pK P A9d’ 20 10 om pK P 20 1 0 o m c ’20 78 PA 5 NZ10 ompK pK 20 1 05 c om NZ 9 PW13mpK 200 6 219 o mpK 200 6 221 o 200 104 N Z 11 0 NZ 66 NZ 7 NZ 6 PA3d 2011 PA3a 2 0 1 1 PA 3 b 2011 1 NZ 10 N Z 97 NZ 98 N Z 46 pK 2009 HA3d om NZ 29 2008 HW1b ompK 2011 PA7e 2009 HA4e ompK 2011 HA4b NZ 7 NZ 8 2008 PA2b ompK 2011 PW6e NZ 53 NZ 40 NZ 26 NZ 31 NZ 69 NZ 84 NZ 7 2 N Z 73 2008 HA 2006 3 6a ompK 0 om p K 2010 o2m ’2012 H pK HA2h A5gy’ ’201 NZ 520 HA8a’ NZ NZ 1198 NZ NZ 5429 NZ NZ 82 NZ 883 NZ 5 NZ 777 NZ 9 N Z 81 NZ 8 6 NZ 8 0 NZ 75 NZ 76 N 10 7 109 20Z NZ 06 23 1 N 0 5 om pK NZZ 1086 NZ 35 N 12 N Z6 NZZ 5 N 2 N Z3 NZZ 13 N 4 N Z2 8 NZZ 172 N 2 2 Z4 0 20008 5 20 0 8 HA ’2 01 9 P A1 5a m K 2 00 9 P A1 a o mp K 2 00 12 P 3 o mp K 2 0 12 HA 4d o p c ’2 0 8 HA 8b om 2 ’2 00 9 HA 8c PA 5d c’ 2 0 0 9 A pK P A 3 2 00 9 H m pK HW 8h 2 00 0 o m p K A 8k 2 0 1 0 o m K H A 4e 2 01 0 o mp K H W d 2 01 0 o m p K P A1 l ’2 01 0 o mp P A8 2 01 o pK H 1b 2 010 o m p K PA K 2 010 om pK mp 2 010 om 0 o 2 010 22 3f’ 2 006 HA 1c 2 012 HA ’ 2 0 12 9 2 Z3 N Z 41 N Z 16 N Z 25 pK N Z 24 om N Z 27 A11e ompkK N 08 P A8a omp 20 08 P A5a pK 20 08 P 45 om ompk 20 06 2 A5 8 20 07 H W8f 2 0 1 1 P 8e 2 0 11 P W 6 b 20 11 PA 10b 20 11 PA 2a K 20 11 PA 14a ompK 20 09 PW 6b omp K 20 9 PA1 2a omp 200 9 PA1 200 2 PA8d 201 2 PA8i’ ’ ’201 2 PA8h ’2012 PA62a 201 2 PA15c’ ’201 2 PW2b’ ’201 PA5f 2012 PA7c 2012 HW5d 2011 H W 1 a o m p d’ K 20 1d o mpK 2 11 m p ’ 2 0 01 1 P A 3 k ’20 12 H HA4f f 20112 HAA6d’ 2 0 2 H 4 c’ 20 2 11 A7e ’2 110 om012 HHA2f 0 A3 p 0 200 ompKK PA7 g 200 6 222 HA2aa 200 6 255 ompK ’ 200 6 258 ompK 20 6 259 ompK 200 06 262 ompK 2007 8 HA4a ompK 200 HW1 1 ompK 20077 HA1 6 ompk 2007 HW2 1 oompk mp k 2007 HW2 4 om 200 PA1 5 om pk 2007 7P PA1 7 om pk p A 1 10 2007 P ompkk A 2007 PA1 11 ompk 2 3 o 2 0 07 P A m pk 2 7 om 2007 PA pk 2007 PA 2 8 ompk 2007 PA2 2 9 ompk 2007 PW1110 ompk ompk 2008 HW1c om pK 2008 HW2d ompK 2008 PW2d ompK 2010 ompK PA1e 2010 ompK HA10a 2010 ompK PA3e ’2010 ompK PA1f’ ’2010 ompK PA9a’ ’2010 ompK PA9f’ ’2012 PA8b’ 2012 PA1i 2012 PA8c 2a 2010 ompK PApK 2009 HA4c om pK 2009 HA7ed om ompK 2009 PA17 c 2011 PW8 7b 201 1 P A 5f 2011 HW Protein similarity network: ompK • Pairwise BLAST of all sequences • Similarity network not based on alignment • Analysis with Cytoscape software • Used to represent similarity network between protein families Protein similarity network: no identity cut off Protein similarity network: 90% identity cut off Protein similarity network: 99% identity cut off Structure Analyzes population structure based on distances at multiple loci for each genotype provided. K=6 K=4 K = 4, Sorted by year. 2007 2008 2009 2010 2011 2012 Structure K=4 K = 4, Sorted by location. Hopkins Point Lobos Main Questions 1) Is there an observable population structure? If so, how does this relate to the ecology of the environment? (Null) Hypothesis: No structure/pattern. “Everything is everywhere.” Finding: There is population structure. Difficult to tell what factors determine it. Main Questions 1) Is there an observable population structure? If so, how does this relate to the ecology of the environment? (Null) Hypothesis: No structure/pattern. “Everything is everywhere.” 2) How is evolution occurring in the environment? a) Rate: Are the genes evolving at a similar rate? Hypothesis: Housekeeping genes are evolving slower than OmpK. b) Process: How much recombination vs. mutation is observed in populations? Hypothesis: Significant amounts of both recombination and mutation at a fixed ratio within the population. recA Do genes evolve at similar rates? recA mdh mdh gyrB gyrB What about the outliers? ompK But ompK is different… gyrB Positive vs. Negative Selection within Genes: dN/dS gyrB rec A Positive vs. Negative Selection within Genes: dN/dS md h omp K SplitsTree concatenated sequences from CA, 2007-2012 mainly HA 2008 and 2009 Phi Test for SplitsTree test for recombination null hypothesis: no recombination concatenated 2007-2012 p=0 concatenated 2012 recA 2012 gyrB 2012 mdh 2012 ompK 2012 p=0 p=0.003 p=0.19 p=0.97 p=0.6 different rate of recombination in genes (?) Structure Communities Mapped Onto SplitsTree 2012 Clonal Backbone of Vibrio Population ClonalFrame identifies and removes recombination events that disrupted clonal inheritance. Recombination vs. Mutation Mean r/m = 3.8 for concatenated housekeeping genes. Structure: Low recombination. Trees, SplitsTree: Varying levels of recombination vs. mutation depending on gene/genotype. Main Questions 1) Is there an observable population structure? If so, how does this relate to the ecology of the environment? (Null) Hypothesis: No structure/pattern. “Everything is everywhere.” Finding: There is population structure. Difficult to tell what factors determine it. 2) How is evolution occurring in the environment? a) Rate: Are the genes evolving at a similar rate? Hypothesis: Housekeeping genes are evolving slower than OmpK. Finding: Evidence was found for different rates of evolution. b) Process: How much recombination vs. mutation is observed in populations? Hypothesis: Significant amounts of both recombination and mutation at a fixed ratio within the population. Finding: Different contributions of recombination vs. mutation in various genes and genotypes. Next Year • More meta data • • • oEnvironmental oPhysiological More distant sites Seasonal sampling Data curation Thank you to… • Alfred • Chris • Paul • Kosh • Julian • Bradley • Ian • the HMC 2012 class!!