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CHEMTAX & FIELD SAMPLES

CHEMTAX & FIELD SAMPLES. “Carotenoids + chlorophylls” as marker pigments: improving the phytoplankton community structure derived from HPLC pigment signatures. I: Discovery of new pigments.

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CHEMTAX & FIELD SAMPLES

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  1. CHEMTAX & FIELD SAMPLES

  2. “Carotenoids + chlorophylls” as marker pigments: improving the phytoplankton community structure derived from HPLC pigment signatures. I: Discovery of new pigments • Divinyl chl a and b in free-living prochlorophytes e.g. Prochlorococcus marinus (Chisholm et al 1992; Goericke & Repeta 1992, 1993). • Monovinyl chl c3 (in addition to divinyl chl c3; Jeffrey & Wright 1987; Fookes & Jeffrey 1989) in haptophytes as Emiliania huxleyi (Garrido & Zapata, 1998). • Two new chl c pigments in Pavlova gyrans (Garrido & Zapata 1997) first noted by Fawley (1989). • Three non-polar chl c pigments detected in E. huxleyi, Prymnesium parvum and Chrysochromulina polylepis (Chl c2-MGDG; Garrido et al. 1995; Zapata et al. 1998; Garrido et al. 2000).

  3. “Carotenoids + chlorophylls” as marker pigments: improving the phytoplankton community structure derived from HPLC pigment signatures. I: Discovery of new pigments • A new fucoxanthin pigment in E. huxleyi (Garrido & Zapata, 1998) recently characterized as 4-keto-19’hex-fuco (Egeland et al. 1999) and detected in a number of other haptophytes (Zapata et al. unpublished).

  4. “Carotenoids + chlorophylls” as marker pigments: improving the phytoplankton community structure derived from HPLC pigment signatures. II: HPLC methods & CHEMTAX • The “carotenoids + chlorophylls” approach has only been included in one CHEMTAX paper (Riegman & Kraay 2001; Kraay et al. 1992 is able to resolve Chl c1, c2 and c3). • None of the HPLC methods employed in CHEMTAX papers has the ability to resolve both polar and non-polar chlorophylls (coelution of Chl c1 + c2, Chl c3 + MVChl c3, Chl b + DV Chl b, Chl a + DVChl a, non-polar Chl c´s haven´t been described).

  5. “Carotenoids + chlorophylls” as marker pigments: improving the phytoplankton community structure derived from HPLC pigment signatures. III: CHEMTAX + new HPLC methods. • We will compare CHEMTAX reconstruction of Chl a contributed by several algal classes using synthetic pigment data sets obtained by two different approaches (i.e. different HPLC methods): • A) Carotenoid approach: Wright et al 1991,…. • B) Carotenoid + chlorophyll approach: Zapata et al 2000.

  6. “Carotenoids + chlorophylls” as marker pigments: improving the phytoplankton community structure derived from HPLC pigment signatures. III: CHEMTAX + new HPLC methods. • Synthetic pigment data sets: • 1) Phytoplankton community constituted by three haptophytes ( two Phaeocystis antarctica strains from ice and water column (Australian Antarctic Division; E. huxleyi), two diatoms (Pseudonitzschia subcurvata, Chaetoceros mitra) and prasinophytes. • 2) Surface and deep water phytoplankton community: three types of haptophytes, prochlorophytes, chlorophytes, diatoms and cyanobacteria. Pigment ratios obtained from high light and low light conditions. • 3) Several haptophytes with up to seven different chl c´s and four fucoxanthin pigments.

  7. “Carotenoids + chlorophylls” as marker pigments: improving the phytoplankton community structure derived from HPLC pigment signatures. III: CHEMTAX + new HPLC methods. • DATA SET 1 • Haptophytes: • Phaeocystis antarctica (MSIA-1); (isolated from ice); fuco dominant • Phaeocystis antarctica (RG2.2); (water column), hex-fuco dominant. • Emiliania huxleyi (CS-57); MV Chl c3,hex-fuco dominant. • Diatoms: • Pseudonitzschia subcurvata (1-D); Chl c3, Chl c2 • Chaetoceros mitra (CS-70); Chl c2, Chl c1 • Prasinophytes(Wright & van den Enden, 2000)

  8. “Carotenoids + chlorophylls” as marker pigments: improving the phytoplankton community structure derived from HPLC pigment signatures. III: CHEMTAX + new HPLC methods. • DATA SET 2 • Haptophytes: • Type I: Chrysochromulina polylepis (CCMP-286);Chl c3, Chl c2,hex-fuco dominant. • Type II: Isochrysis galbana (CS-22); Chl c2, Chl c1. • Type III: Emiliania huxleyi (CCMP 70); MV Chl c3,fuco, 4k-hex-fuco and hex-fuco. • Pelagophyceans: • Pelagomonas calceolata • Cyanobacteria: • Type I:Synechococcus (Schlüter et al. 2000). • Type II: Prochlorococcus marinus (van Lenning 2000, Ph.D. Thesis). • Chlorophytes:Chlorella (Schlüter et al. 2000).

  9. “Carotenoids + chlorophylls” as marker pigments: improving the phytoplankton community structure derived from HPLC pigment signatures. III: CHEMTAX + new HPLC methods. • DATA SET 3 • Haptophytes: • I: Pavlova lutheri (CS-23);Chl c2, Chl c1,fuco. • II: Isochrysis galbana (CS-22); Chl c2-MGDG (E.huxleyi type). • III: Pavlova gyrans (CS-608); Chl c-P.gyrans. • IV: Prymnesium parvum (CS-288);Chl c3, MV Chl c3. • V: Phaeocystis pouchetii (CS-165); but-fuco and hex-fuco. • VI: Emiliania huxleyi (CS-57); 4keto-hex-fuco. • VII: Chrysochromulina polylepis (CS-286); Chl c2-MGDG (C.polylepis-type).

  10. DATA SET 1 Pigment ratio matrix 1: carotenoids + chlorophylls Pigment ratio matrix 2: carotenoids

  11. DATA SET 2 Pigment ratio matrix 1: carotenoids + chlorophylls

  12. DATA SET 2 Pigment ratio matrix 1: carotenoids

  13. DATA SET 3

  14. Results 25 and 50 % error input ratio matrix DATA SET 1 Results obtained with 25% error inputratio matrix 0.9 0.6 Phaeocystis RG 2.2 Carots + Chls Carots + Chls 0.8 0.5 0.7 0.6 0.4 0.5 0.3 0.4 0.3 0.2 0.2 0.1 0.1 0 0 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 40 0.9 Carotenoids 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0 5 10 15 20 25 30 35 Phaeocystis MSIA MSIA1CX RG22CX 40 0.6 Carotenoids 0.5 0.4 0.3 0.2 0.1 0 0 5 10 15 20 25 30 35 40 40

  15. DATA SET 1 Results obtained with 25% error inputratio matrix 0.7 Carots+Chls 0.6 0.5 0.4 0.3 0.2 0.1 0 0 5 10 15 20 25 30 35 40 P. subcurvata PSCUCX 0.7 Carotenoids 0.6 0.5 0.4 0.3 0.2 0.1 0 0 5 10 15 20 25 30 35 40

  16. DATA SET 1 Results obtained with 25% error inputratio matrix 0.8 1 Carots + Chls Carots+Chls 0.9 0.7 0.8 0.6 0.7 0.5 0.6 0.4 0.5 0.4 0.3 0.3 0.2 0.2 0.1 0.1 0 0 0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 0.8 1 Carotenoids Carotenoids 0.9 0.7 0.8 0.6 0.7 0.5 0.6 0.5 0.4 0.4 0.3 0.3 0.2 0.2 0.1 0.1 0 0 0 5 10 15 20 25 0 5 10 15 20 25 30 35 40 Chaetoceros mitra CHETCX prasinophyte PRASICX 30 35 40 30 35 40

  17. DATA SET 1 Results obtained with 50% error inputratio matrix 0.9 0.8 Carots+Chls Carots+Chls 0.8 0.7 0.7 0.6 0.6 0.5 0.5 0.4 0.4 0.3 0.3 0.2 0.2 0.1 0.1 0 0 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 40 0.8 0.9 Carotenoids Carotenoids 0.8 0.7 0.7 0.6 0.6 0.5 0.5 0.4 0.4 0.3 0.3 0.2 0.2 0.1 0.1 0 0 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 40 Phaeocystis MSIA-1 MSIA1CX Phaeocystis RG 2.2 RG22CX 40 40

  18. DATA SET 1 Results obtained with 50% error inputratio matrix

  19. DATA SET 1 Results obtained with 50% error inputratio matrix 0.7 0.7 Chaetoceros mitra CHETCX Carots + Chls Carots + Chls 0.6 0.6 0.5 0.5 0.4 0.4 0.3 0.3 0.2 0.2 0.1 0.1 0 0 0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 30 35 40 1 0.8 Carotenoids Carotenoids 0.9 0.7 0.8 0.6 0.7 0.5 0.6 0.5 0.4 0.4 0.3 0.3 0.2 0.2 0.1 0.1 0 0 0 5 10 15 20 25 0 5 10 15 20 25 30 35 40 prasinophyte PRASICX 30 35 40

  20. OUTPUT RATIO MATRIX. DATA SET 1 25% error (Car+Chl) 50% error (Car+Chl) 25% error (Car) 50% error (Car)

  21. DATA SET 2 Single analysis (Surface plus deep samples). Results obtained with 25% error inputratio matrix 0.6 E. huxleyi Carots+Chls 0.5 0.4 0.3 0.2 0.1 0 0 5 10 15 20 25 30 35 40 0.6 Carots+Chls Chlorella 0.5 0.4 0.3 0.2 0.1 0 0 5 10 15 20 25 30 35 40 1 1.2 Carotenoids Carots+Chls Prochlorococcus 0.9 1 0.8 0.7 0.8 0.6 0.6 0.5 0.4 0.4 0.3 0.2 0.2 0.1 0 0 0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 0.6 E. hux CX Carotenoids 0.5 0.4 0.3 0.2 0.1 0 0 5 10 15 20 25 30 35 40 0.6 Carotenoids Chlor CX 0.5 0.4 0.3 0.2 0.1 0 0 5 10 15 20 25 30 35 40 Prochlo CX 30 35 40

  22. DATA SET 2 Single analysis (Surface plus deep samples). Results obtained with 25% error inputratio matrix 0.45 0.45 Carotenoids Carots+Chls 0.4 0.4 0.35 0.35 0.3 0.3 0.25 0.25 0.2 0.2 0.15 0.15 0.1 0.1 0.05 0.05 0 0 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 0.6 C.polylepis Carots+Chls 0.5 0.4 0.3 0.2 0.1 0 0 5 10 15 20 25 30 35 40 0.7 Carots+Chls 0.6 0.5 0.4 0.3 0.2 0.1 0 0 5 10 15 20 25 30 35 40 Synechococcus Synec CX 40 40 0.6 Carotenoids C.poly CX 0.5 0.4 0.3 0.2 0.1 0 0 5 10 15 20 25 30 35 40 0.6 Carotenoids Pelagomonas calceolata Pelagoc CX 0.5 0.4 0.3 0.2 0.1 0 0 5 10 15 20 25 30 35 40

  23. DATA SET 2: 25% error Single analysis (Surface plus deep samples). Results obtained with 25% error inputratio matrix 0.5 Carots + Chls 0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 0 5 10 15 20 25 OUTPUT RATIO MATRIX: Car Isochrysis galbana Igalb CX 30 35 40 0.6 Carotenoids 0.5 0.4 0.3 0.2 0.1 0 0 5 10 15 20 25 30 35 40

  24. DATA SET 2: 25% error SINGLE ANALYSIS chl c3 MV chl c3 chl c2 chl c1 But-fuco Fuco 4-k-hex-fuco Hex-fuco Neo Lut Zea Chl b ap-Ehux ap-Chry Chl a 0.276 0.184 0.385 0.000 0.002 0.393 0.425 0.477 0.000 0.000 0.000 0.000 0.173 0.000 1.000 E. huxleyi 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.039 0.152 0.054 0.103 0.000 0.000 1.000 Chlorella 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.115 0.843 0.000 0.000 1.000 Prochlorococcus 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.066 0.000 0.000 0.000 1.000 Synechococcus 0.214 0.013 0.166 0.000 0.003 0.078 0.066 1.032 0.000 0.000 0.000 0.000 0.002 0.061 1.000 C.polylepis 0.184 0.000 0.401 0.000 0.503 0.779 0.000 0.000 0.000 0.000 Pelagomonas calceolata SURFACE SAMPLES chl c3 MV chl c3 chl c2 chl c1 But-fuco Fuco 4-k-hex-fuco Hex-fuco Neo Lut Zea Chl b ap-Ehux ap-Chry Chl a E. huxleyi 0.156 0.186 0.360 0.000 0.000 0.401 0.390 0.799 0.000 0.000 0.000 0.000 0.180 0.000 1.000 Chlorella 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.036 0.167 0.084 0.160 0.000 0.000 1.000 Prochlorococcus 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.611 0.447 0.000 0.000 1.000 Synechococcus 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.443 0.000 0.000 0.000 1.000 C.polylepis 0.157 0.011 0.134 0.000 0.000 0.027 0.045 1.129 0.000 0.000 0.000 0.000 0.001 0.041 1.000 Pelagomonas calceolata 0.250 0.000 0.439 0.000 0.659 0.340 0.000 0.000 0.000 0.000 DEEP SAMPLES chl c3 MV chl c3 chl c2 chl c1 But-fuco Fuco 4-k-hex-fuco Hex-fuco Neo Lut Zea Chl b ap-Ehux ap-Chry Chl a 0.241 0.016 0.217 0.000 0.004 0.385 0.209 0.133 0.000 0.000 0.000 0.000 0.074 0.000 1.000 E. huxleyi 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.053 0.102 0.009 0.019 0.000 0.000 1.000 Chlorella 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.161 1.506 0.000 0.000 1.000 Prochlorococcus 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.511 0.000 0.000 0.000 1.000 Synechococcus 0.285 0.000 0.166 0.000 0.006 0.124 0.084 0.994 0.000 0.000 0.000 0.000 0.002 0.062 1.000 C.polylepis 0.156 0.000 0.427 0.000 0.211 1.241 0.000 0.000 0.000 0.000 Pelagomonas calceolata (Cont.) OUTPUT RATIO MATRIX: Car+Chl 0.000 0.000 0.000 0.000 1.000 0.000 0.000 0.343 0.217 0.000 0.443 0.000 0.000 0.000 0.000 0.000 0.000 0.036 0.000 1.000 Isochrysis galbana 0.000 0.000 0.000 0.000 1.000 Isochrysis galbana 0.000 0.000 0.350 0.329 0.000 0.678 0.000 0.000 0.000 0.000 0.000 0.000 0.041 0.000 1.000 0.000 0.000 0.000 0.000 1.000 0.000 0.000 0.231 0.144 0.000 0.309 0.000 0.000 0.000 0.000 0.000 0.000 0.030 0.000 1.000 Isochrysis galbana

  25. DATA SET 2 Single analysis (Surface plus deep samples). Results obtained with 50% error inputratio matrix 0.7 Carots+Chls 0.6 0.5 0.4 0.3 0.2 0.1 0 0 5 10 15 20 25 30 35 40 0.7 Carots+Chls 0.6 0.5 0.4 0.3 0.2 0.1 0 0 5 10 15 20 25 30 35 40 0.9 1.2 Carotenoids Carots+Chls 0.8 Prochlorococcus 1 0.7 0.6 0.8 0.5 0.6 0.4 0.3 0.4 0.2 0.2 0.1 0 0 0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 30 35 0.7 E. huxleyi E. hux CX Carotenoids 0.6 0.5 0.4 0.3 0.2 0.1 0 0 5 10 15 20 25 30 35 40 0.7 Carotenoids Chlorella Chlor CX 0.6 0.5 0.4 0.3 0.2 0.1 0 0 5 10 15 20 25 30 35 40 Prochlo CX 40

  26. DATA SET 2 Single analysis (Surface plus deep samples). Results obtained with 50% error inputratio matrix 0.4 0.45 Carotenoids Carots+Chls 0.4 0.35 0.35 0.3 0.3 0.25 0.25 0.2 0.2 0.15 0.15 0.1 0.1 0.05 0.05 0 0 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 40 0.9 Carots+Chls 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0 5 10 15 20 25 30 35 0.5 0.5 Carotenoids Carots+Chls 0.45 0.45 0.4 0.4 0.35 0.35 0.3 0.3 0.25 0.25 0.2 0.2 0.15 0.15 0.1 0.1 0.05 0.05 0 0 0 5 10 15 20 25 0 5 10 15 20 25 Synechococcus Synec CX 40 0.6 C.polylepis C.poly CX Carotenoids 0.5 0.4 0.3 0.2 0.1 0 40 0 5 10 15 20 25 30 35 40 P. calceolata Pelagoc CX 30 35 40 30 35 40

  27. DATA SET 2: 50% error Single analysis (Surface plus deep samples). Results obtained with 50% error inputratio matrix 0.4 Carots+Chls 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 0 5 10 15 20 25 30 35 40 OUTPUT RATIO MATRIX: Car Isochrysis galbana Isgalb CX 0.7 Carotenoids 0.6 0.5 0.4 0.3 0.2 0.1 0 0 5 10 15 20 25 30 35 40

  28. DATA SET 2: 50% error SINGLE ANALYSIS chl c3 MV chl c3 chl c2 chl c1 But-fuco Fuco 4-k-hex-fuco Hex-fuco Neo Lut Zea Chl b ap-Ehux ap-Chry Chl a 0.227 0.202 0.354 0.000 0.002 0.258 0.468 0.477 0.000 0.000 0.000 0.000 0.150 0.000 1.000 E. huxleyi 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.039 0.152 0.054 0.103 0.000 0.000 1.000 Chlorella 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.110 0.392 0.000 0.000 1.000 Prochlorococcus 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.066 0.000 0.000 0.000 1.000 Synechococcus 0.125 0.008 0.105 0.000 0.002 0.049 0.042 0.470 0.000 0.000 0.000 0.000 0.001 0.033 1.000 C.polylepis 0.185 0.000 0.270 0.000 0.271 0.579 0.000 0.000 0.000 0.000 Pelagomonas calceolata SURFACE SAMPLES chl c3 MV chl c3 chl c2 chl c1 But-fuco Fuco 4-k-hex-fuco Hex-fuco Neo Lut Zea Chl b ap-Ehux ap-Chry Chl a 0.280 0.325 0.597 0.000 0.000 0.263 0.815 0.349 0.000 0.000 0.000 0.000 0.153 0.000 1.000 E. huxleyi 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.084 0.268 0.150 0.286 0.000 0.000 1.000 Chlorella 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.612 0.380 0.000 0.000 1.000 Prochlorococcus 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.516 0.000 0.000 0.000 1.000 Synechococcus 0.212 0.047 0.327 0.000 0.000 0.063 0.097 3.665 0.000 0.000 0.000 0.000 0.002 0.108 1.000 C.polylepis 0.111 0.000 0.408 0.000 0.758 0.270 0.000 0.000 0.000 0.000 Pelagomonas calceolata DEEP SAMPLES chl c3 MV chl c3 chl c2 chl c1 But-fuco Fuco 4-k-hex-fuco Hex-fuco Neo Lut Zea Chl b ap-Ehux ap-Chry Chl a 0.177 0.015 0.215 0.000 0.004 0.363 0.197 0.126 0.000 0.000 0.000 0.000 0.078 0.000 1.000 E. huxleyi 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.009 0.040 0.004 0.009 0.000 0.000 1.000 Chlorella 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.137 1.390 0.000 0.000 1.000 Prochlorococcus 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.511 0.000 0.000 0.000 1.000 Synechococcus 0.234 0.000 0.130 0.000 0.005 0.097 0.061 0.869 0.000 0.000 0.000 0.000 0.002 0.047 1.000 C.polylepis 0.171 0.000 0.310 0.000 0.263 1.198 0.000 0.000 0.000 0.000 Pelagomonas calceolata (Cont.) OUTPUT RATIO MATRIX: Car+Chl 0.000 0.000 0.000 0.000 1.000 0.000 0.000 0.133 0.205 0.000 0.443 0.000 0.000 0.000 0.000 0.000 0.000 0.036 0.000 1.000 Isochrysis galbana 0.000 0.000 0.000 0.000 1.000 0.000 0.000 0.516 0.319 0.000 0.613 0.000 0.000 0.000 0.000 0.000 0.000 0.026 0.000 1.000 Isochrysis galbana 0.000 0.000 0.000 0.000 1.000 0.000 0.000 0.119 0.096 0.000 0.328 0.000 0.000 0.000 0.000 0.000 0.000 0.030 0.000 1.000 Isochrysis galbana

  29. DATA SET 2 Results after splitting data in two groups (using low light and high light pigment ratios for surface and deep samples): 25% error inputratio matrix. 0.6 E. huxleyi Carots+Chls 0.5 0.4 0.3 0.2 0.1 0 0 5 10 15 20 25 30 35 40 0.6 Chlorella Carots+Chls 0.5 0.4 0.3 0.2 0.1 0 0 5 10 15 20 25 30 35 40 0.8 0.9 Carots+Chls Carotenoids 0.8 0.7 0.7 0.6 0.6 0.5 0.5 0.4 0.4 0.3 0.3 0.2 0.2 0.1 0.1 0 0 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 40 0.6 E. hux CX Carotenoids 0.5 0.4 0.3 0.2 0.1 0 0 5 10 15 20 25 30 35 40 0.6 Carotenoids Chlor CX 0.5 0.4 0.3 0.2 0.1 0 0 5 10 15 20 25 30 35 40 Prochlorococcus Prochlo CX 40

  30. DATA SET 2 Results after splitting data in two groups (using low light and high light pigment ratios for surface and deep samples): 25% error inputratio matrix. 0.4 Carot+Chls 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 0 5 10 15 20 25 30 35 40 0.6 C.polylepis Carot+Chls 0.5 0.4 0.3 0.2 0.1 0 0 5 10 15 20 25 30 35 40 0.6 P. calceolata Carot+Chls 0.5 0.4 0.3 0.2 0.1 0 0 5 10 15 20 25 30 35 40 0.35 Synechococcus Synec CX Carotenoids 0.3 0.25 0.2 0.15 0.1 0.05 0 0 5 10 15 20 25 30 35 40 0.6 C.poly CX Carotenoids 0.5 0.4 0.3 0.2 0.1 0 0 5 10 15 20 25 30 35 40 0.6 Pelagoc CX Carotenoids 0.5 0.4 0.3 0.2 0.1 0 0 5 10 15 20 25 30 35 40

  31. DATA SET 2 Results after splitting data in two groups (using low light and high light pigment ratios for surface and deep samples): 25% error input ratio matrix. 0.4 Carot+Chls 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 0 5 10 15 20 25 30 35 40 0.4 Carots 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 0 5 10 15 20 25 30 35 40 Isochrysis galbana Isgalb CX

  32. DATA SET 2 Results after splitting data in two groups (using low light and high light pigment ratios for surface and deep samples): 50% error inputratio matrix. 0.4 0.45 Carotenoids Carots+Chls 0.4 0.35 0.35 0.3 0.3 0.25 0.25 0.2 0.2 0.15 0.15 0.1 0.1 0.05 0.05 0 0 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 40 0.7 Carots+Chls 0.6 0.5 0.4 0.3 0.2 0.1 0 0 5 10 15 20 25 30 35 40 0.9 0.9 Carots+Chls Carotenoids 0.8 0.8 0.7 0.7 0.6 0.6 0.5 0.5 0.4 0.4 0.3 0.3 0.2 0.2 0.1 0.1 0 0 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 E. huxleyi E. hux CX 40 0.7 Carotenoids Chlorella Chlor CX 0.6 0.5 0.4 0.3 0.2 0.1 0 0 5 10 15 20 25 30 35 40 Prochlorococcus Prochlo CX 40 40

  33. DATA SET 2 Results after splitting data in two groups (using low light and high light pigment ratios for surface and deep samples): 50% error inputratio matrix. 0.4 0.4 Carotenoids Carots+Chls 0.35 0.35 0.3 0.3 0.25 0.25 0.2 0.2 0.15 0.15 0.1 0.1 0.05 0.05 0 0 0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 30 35 40 0.6 C.polylepis Carots+Chls 0.5 0.4 0.3 0.2 0.1 0 0 5 10 15 20 25 30 35 40 0.5 0.5 Carotenoids 0.45 0.45 0.4 0.4 Carots+Chls 0.35 0.35 0.3 0.3 0.25 0.25 0.2 0.2 0.15 0.15 0.1 0.1 0.05 0.05 0 0 0 5 10 15 20 25 0 5 10 15 20 25 Synechococcus Synec CX 0.6 Carotenoids C.poly CX 0.5 0.4 0.3 0.2 0.1 0 0 5 10 15 20 25 30 35 40 P. calceolata Pelagoc CX 30 35 40 30 35 40

  34. DATA SET 2 Results after splitting data in two groups (using low light and high light pigment ratios for surface and deep samples): 50% error input ratio matrix. 0.7 Carots+Chls 0.6 0.5 0.4 0.3 0.2 0.1 0 0 5 10 15 20 25 30 35 40 Isochrysis galbana Isgalb CX 0.7 Carotenoids 0.6 0.5 0.4 0.3 0.2 0.1 0 0 5 10 15 20 25 30 35 40

  35. DATA SET 3 25% error 50% error 0.8 0.8 0.7 0.7 0.6 0.6 0.5 0.5 0.4 0.4 0.3 0.3 0.2 0.2 0.1 0.1 0 0 0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 30 35 40 0.6 0.8 I. galbana CS 22 0.7 0.5 0.6 0.4 0.5 0.3 0.4 0.3 0.2 0.2 0.1 0.1 0 0 0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 30 35 40 0.7 0.8 P. gyrans CCMP608 0.7 0.6 0.6 0.5 0.5 0.4 0.4 0.3 0.3 0.2 0.2 0.1 0.1 0 0 0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 30 35 40 P.lutheri CS 23 P. lutheri CS 23 PlutCX P.lut CX I. galbana CS 22 I.galb CX I.galb CX P. gyrans CCMP608 Pgyr CX PgyrCX

  36. DATA SET 3 25% error 50% error 0.8 0.7 P. parvum CS 288 0.7 0.6 0.6 0.5 0.5 0.4 0.4 0.3 0.3 0.2 0.2 0.1 0.1 0 0 0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 30 35 40 0.4 0.35 P. pouchetii 0.35 0.3 0.3 0.25 0.25 0.2 0.2 0.15 0.15 0.1 0.1 0.05 0.05 0 0 0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 30 35 40 0.7 0.8 E. huxleyi 0.7 0.6 0.6 0.5 0.5 0.4 0.4 0.3 0.3 0.2 0.2 0.1 0.1 0 0 0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 30 35 40 0.8 0.8 0.7 0.7 0.6 0.6 0.5 0.5 0.4 0.4 0.3 0.3 0.2 0.2 0.1 0.1 0 0 0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 30 35 40 P. parvum CS 288 PparvumCX Pparvum CX PpouchCX P. pouchetii CS 165 Ppouch CX Ehux CX E. huxleyi Ehux CX C. polylepis Cpoly CX Cpoly CX C. polylepis CS 286

  37. DATA SET 3 25% error c-pavlova Chl c3 MV c3 Chl c2 Chl c1 BFx Fucox 4keto-HFx HFx ap-EHUX ap-chry Chl a 0.000 0.000 0.000 0.040 0.033 0.000 0.068 0.000 0.000 0.000 0.000 1.000 Pavlova lutheri CS 23 0.000 0.000 0.000 0.039 0.038 0.000 0.373 0.000 0.000 0.021 0.000 1.000 I. galbana CS 22 0.032 0.000 0.000 0.018 0.130 0.000 0.417 0.000 0.000 0.000 P. gyrans CS 213 50% error c-pavlova Chl c3 MV c3 Chl c2 Chl c1 BFx Fucox 4keto-HFx HFx ap-EHUX ap-chry Chl a 0.000 0.000 0.000 0.036 0.006 0.000 0.317 0.000 0.000 0.000 0.000 1.000 Pavlova lutheri CS 23 0.000 0.000 0.000 0.025 0.017 0.000 0.040 0.000 0.000 0.007 0.000 1.000 I. galbana CS 22 0.033 0.000 0.000 0.016 0.099 0.000 0.288 0.000 0.000 0.000 P. gyrans CS 213 OUTPUT RATIO MATRIX 0.000 1.000 0.000 0.086 0.002 0.028 0.036 0.000 0.221 0.000 0.000 0.023 0.000 1.000 P. parvum CS 288 0.000 0.326 0.000 0.253 0.000 0.426 0.262 0.279 0.525 0.102 0.000 1.000 P. pouchetii CS 165 0.000 0.189 0.033 0.191 0.000 0.007 0.006 0.000 1.865 0.089 0.000 1.000 E. huxleyi CS 57 0.000 0.172 0.000 0.203 0.000 0.007 0.155 0.093 1.610 0.002 0.084 1.000 C. polylepis CS 286 0.000 1.000 0.000 0.078 0.003 0.041 0.056 0.000 0.411 0.000 0.000 0.029 0.000 1.000 P. parvum CS 288 0.000 0.209 0.000 0.118 0.000 0.061 0.166 0.133 0.322 0.067 0.000 1.000 P. pouchetii CS 165 0.000 0.466 0.047 0.173 0.000 0.007 0.006 0.000 0.668 0.065 0.000 1.000 E. huxleyi CS 57 0.000 0.224 0.000 0.328 0.000 0.009 0.186 0.126 0.574 0.003 0.031 1.000 C. polylepis CS 286

  38. CONCLUSIONS • CHEMTAX interpretation of synthetic data yielded better results when comparing the “chlorophylls+carotenoids approach” with the classic carotenoid analysis. • CHEMTAX output is highly dependent on adecuate guesses of field pigment ratios (50% error in input values led to large errors in reconstructed phytoplankton classes). • Samples collected from different depths/light intensities must be separated prior to CHEMTAX analysis. Knowledge about pigment responses with light would improve CHEMTAX results by providing suitable seed values to different light conditions.

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