1. Lecture 2: Common red algal families in the vicinity of Bocas del Toro: Gracilariaceae Photo: O.Camacho Hadad & Anna Fricke

2. Halymeniaceae Rhodymeniaceae Gracilariaceae

3. vegetative structure: cellular medulla family, ordinal level Gracilaria Hydropuntia flat sp. terete sp. mixed-sized cells

4. Gracilariaceae Solieriaceae

5. Solieriaceae (Solieria) loose filiform, or mixed cellular-filiform medulla

6. Halymeniaceae (Cryptonemia)

7. -cortical unit of 3 surface cells 1 1 2 2 3

8. Conjunctor cells fuse with neighboring cells, resulting in secondary pit connection formation

9. -inherently uniaxial

10. -uniaxial growth quickly disguised -axial cells: same shape as medullary cells

11. Generic distinction: -comparative post-fertilization events of female reproductive system in establishment of cystocarp: nature of fusions - complexity of male reproductive system

12. Gracilaria intermedia cystocarps

13. pericarp carposporangia medulla cystocarp floor inner gonimo- blast

14. Gracilaria: multinucleate tubular nutritive cells to roof, to base Gracilariopsis: uninucleate conjunctor cells at base Gracilariophila (parasite) Hydropuntia: multinucleate tubular nutritive cells at base of cystocarp “TAPPING BACK”

15. -origin: 3-cortical unit again -lower cell of unit = cell 1 = supporting cell cutting off carpogonial branch initial -cells 2 & 3 = nutritive cells of carpogonial branch unit 1 1 23 unfertilized female structure

16. unfertilized 2-celled carpogonial branches

17. -2-celled carpogonial branch on supporting cell -straight trichogyne reaching thallus surface -partly fusion of cortical nutritive cells to carpogonium

18. Partly fusion of neighboring cortical cells with carpogonium; vegetative nuclei remain in unfused portion

19. -diploid zygote nucleus in uninucleate carpogonium; -fused cortical cells = nutritive cells

20. -young carpospore-bearing filaments (gonimoblasts) cut off from carpogonial fusion cell

21. How lower cells of young carpospore-bearing filaments fuse back to multinucleate carpogonial fusion cell Generic character:

22. young cystocarp: Gracilariopsis -very regular, progressive growth of carpospore- bearing filaments

23. Fusion of lower carposporophyte cells to vegetative gametophytic cells Gracilariopsis

24. Gracilaria: multinucleate tubular nutritive cells to roof, to base Gracilariopsis: uninucleate conjunctor cells at base Gracilariophila (parasite) Hydropuntia: multinucleate tubular nutritive cells at base of cystocarp “TAPPING BACK”

25. Gracilaria:Gracilariopsis: Gracilariophila: Hydropuntia: male structures in shallow or deep pits super- ficial in deep pits, con- necting to vege- tative cells

26. Gurgel & Fredericq 2004 = rbcL phylogeny of the Gracilariaceae, Bayesian Tree.

27. Global Phylogeny of the Gracilariaceae Bayesian Inferred Phylogeny Gurgel & Fredericq 2004

28. Bayesian Tree A meaningful global phylogeny of the Gracilariaceae and the evolutionary patterns found in it could only be obtained after reaching a dataset composed of 35+ taxa Before that, taxa sampling played a major issue in obtaining stable and meaningful phylogenies. So, nowadays, when new taxa (i.e. distinct rbcL DNA sequences) are added to the global phylogeny they do not change the overall topology. Ready for final taxonomic conclusions.

29. What has been established so far? G. tenuistipitata G. chilensis G. vermiculophylla 94 97 85 Gp. hommersandii (outgroup) H. urvillei H. aff. changii H. changii H. crassissima H. cornea H. caudata H. secundata H. perplexa H. preissiana H. edulis H. rangiferina H. millardetii H. eucheumatoides 82 99 89 G. beckeri 83 G. acuelata G. textorii G. ornata G. bursa-pastoris G. cervicornis G. tikvahiae G. mammillaris G. canaliculata G. salicornia G. gracilis G. pacifica 97 91 67 75 Hydropuntia Gracilaria ‘New Genus’ Genera: Chilensis-type spermatangia ‘Verrucosa’-type spermatangia Rounded carposporophyte Lobed carposporophyte Textorii-type spermatangia Verrucosa-type spermatangia Sub-Genera:

30. Is high quality-agar (gel strength) correlated to phylogeny? Is there character congruence between agar gel strength (phenotype) and molecular-based phylogenies (haplotype)? Can sound phylogenies identify lineages characterized by high-quality agar? Gurgel & Fredericq 2004

31. G. vermiculophylla G. tenuistipitata G. chilensis (90% agar worldwide, Zemke-White & Ohno 1999) Proposed as a distinct genus by Gurgel & Fredericq 2004

32. Agar Properties:Yield( %, dw/dw ) Gel Strength( g. cm -2 ) 3,6 Anhydro-galactose( %, dw/dw ) Gelling Point( °C ) Boiling Point( °C ) 1 st Source of Variation: (in the Agar Properties) Strain / population ‘Tissue’ / Thallus Age Light Regime Temperature Life-cycle Reproductive status ≠ Methodologies (Whyte et al. 1984):2 nd Source of Variation: * Extraction: Standard vs. Alkali * Strength: gel shape, Ø and crosshead speed (plunger) * 3,6 AG: Resorcinol vs. IR Spectrophotometer Data in the Literature: Fred Gurgel, pers. comm

33. Searching for Phylogenetic Patterns of Native Gel Strengths Sampling for Published Data: Obtain agar parameters for species for which we have phylogenetic data (= rbcL DNA sequences). Select methodologies that reflect the algae’s most natural conditions. –Extraction: Standard (shredding  boiling  gelling  freezing  thawing  washing) –Strength method: 1.5% gels, 1.0 cm Ø plungers Minimize Method Variation Total number of Papers Surveyed= 26 Total number of Papers Selected= 09 Fred Gurgel, pers. comm

34. G. chilensis Clade Gel Strength (g. cm -2 ) Number of species with comparable agar data Gracilaria s. s. Clade Gracilariopsis Clade Fred Gurgel, pers. comm

35. Conclusions RbcL-based phylogenies reevaluated character evolution in the Gracilariaceae and inferred a new classification for Gracilaria sensu lato. The G. chilensis-clade and the genus Hydropuntia are reinforced as distinct taxa in the Gracilaria complex. Based on a literature review, data suggest that there is a strong correlation between phylogeny and agar gel strength (agar quality) between genera, and between average genetic divergence and gel strength values within each genus.

36. ordinal & family-level characters: -based on how unfertilized female system develops in relation to vegetative growth cf. Kylin system

37. Gracilaria: multinucleate tubular nutritive cells to roof Gracilariopsis: uninucleate conjunctor cells Gracilariophila (parasite) Hydropuntia: multinucleate tubular nutritive cells at base of cystocarp

38. Problem: *differentiate among stages that are are functional, versus abortive?? *no auxiliary cells!

39. Gracilaria: male structure development

45. Gracilariopsis: male structure development

46. -tetrasporangial initials are surface cortical cells -cruciate divisision of tetrasporangia all genera:

47. Gracilariaceae from the vicinity of Bocas del Toro

48. Gracilaria curtissiae -thick thallus; thinner lateral blade-like proliferations Photo: A. Fricke & O. Camacho-Hadad

50. Gracilaria intermedia

52. Photo: A. Planas & C. Fernández)

53. Gracilaria cervicornis

54. Photo: M.C. Díaz Ruiz

55. Photo: R. Fikes

56. Gracilaria domingensis

57. Photo: A. Fricke & O. Camacho-Hadad

58. Hydropuntia caudata

59. Gracilaria galetensis

60. Gracilaria hayi

61. Global Phylogeny of the Gracilariaceae Bayesian Inferred Phylogeny GTR+inv+gama 100,00 generations, 4 chains Burn in = 20,000 Gurgel & Fredericq 2004