More questions: What are the closest relatives of the Trentepohliales?

2. More questions: • What are the closest relatives of the Trentepohliales? • Is the order Trentepohliales a monophyletic group? • What evolutionary processes have occurred within the Trentepohliales?

3. Two lineages in Viridiplantae • Trentepohlialean taxa are unequivocally within the chlorophycean lineage • The order Trentepohliales is included within the ulvophycean taxa • The order Trentepohliales is a monophyletic group • Phragmoplast?

4. Relationships within the Ulvophyceae • The marine orders are the most closely related to the Trentepohliales • In all phylogenetic analyses the Trentepohliales emerged as a sister group to the clade containing the Siphonocladales/Cladophorales complex and Dasycladales, both containing representatives mainly from the marine environment!

5. Relationships within the Trentepohliaceae • What about the relationships inside of the family? • Are the genera monophyletic? • Is the species Cephaleuros virescens, with a world-wide distribution, a monophyletic taxon? • What is the validity of several morphological characters used for separation at species and genus level?

6. Relationships within the Trentepohliaceae Four main lineages in 18S rDNA: • Cephaleuros clade • Printzinalagenifera clade • Trentepohlia aurea clade (the generitype) • Trentepohlia iolithus clade • Molecular data challenge traditional classification schemes

7. Relationships among Trentepohlialean genera On the basis of our results, Cephaleurosforms a well-defined monophyletic group, representing a more advanced clade Conversely, Trentepohliais non monophyletic and the other genera included in the analyses do not represent separate lineages, suggesting the possibility that a major rearrangement at the genus level may be necessary in the future

8. Printzinawas proposed as a new genus for nine species previously belonging to Trentepohlia • The shape of the sporangia, the arrangement of the sporangiate-laterals, the extensive development of the prostrate parts of the thallus and the occurrence in shaded habitats were considered the key characters separating Printzina from Trentepohlia • The only feature that separates conclusively the two genera is the shape of the sporangia • globular to reniform in Printzina • ovoid in Trentepohlia)

9. The position of Phycopeltisis surprising Importance of the position of the ostiole in the zoosporangium as key character T. umbrina (as P. umbrina) with an ostiole opposite to the end of attachment too T. abietina from Hawaii shows the ostiole clearly opposite to the end of attachment Our results provide therefore some evidence that the position of the ostiole may be a good phylogenetic marker

10. Relationships within a species: Cephaleuros virescens • Cephaleuros virescens Kunze, the generitype of Cephaleuros, is generally considered to have a worldwide distribution in tropical and subtropical regions • Strains of C. virescens do not form a monophyletic group; South Africa and Taiwan are more closely related to C. parasiticus than to other C. virescens

11. Current analyses using the chloroplast-encoded rbcL marker confirm 18SSU results * • These data suggest that the circumscription of C. virescens should be reconsidered • Morphological characters used to separate this species form similar taxa should be reassessed • As currently circumscribed, C. virescens represents a complex of morphologically similar entities • Suriname is the type locality of C. virescens, the actual distribution of this species might be restricted to tropical South and Central America

12. Lessons learned • A subaerial habit has developed within the class Ulvophyceae • Morphological criteria traditionally used for the circumscription of genera and species of Trentepohliales are not correlated with evolutionary patterns • Common and world-wide distributed species may represent a species complex of morphologically similar entities (cryptic species) • Many morphological characters used in taxonomy are not phylogenetically relevant • Another subaerial lineage has been found from a group containing representatives mainly from the marine environment

13. Evolution of the subaerial flora

14. From textbooks to popular articles there is an appreciation that terrestrial life emerged from the seas. And there is generally no explanation that by “seas” is meant from oceans and lakes – that is, from both marine and freshwater sources

15. Several representatives of the marine green algal class Ulvophyceae have been found to be members of the terrestrial algal flora The order Trentepohliales and our newly described taxa are examples of a direct marine invasion of terrestrial environments! This is in contrast with the general idea that terrestrial floras are descendents of freshwater ancestors and not directly from marine ancestors A direct marine invasion?

16. One intriguing, even puzzling, question is: • How marine algae, from ulvophycean ancestors, “jump” into terrestrial habitats and became permanent representatives of the land flora? • A probable answer may came from independent bits of information: • Fossils • Ancient changes of sea level • Pre-adaptive phenotypic plasticity

17. Fossil subaerial microchlorophytes Records of fossils belonging to terrestrial microchlorophytes have been found as early as the late Eocene (ca. 50 mya) Reynolds and Dilcher 1984 As Cephaleuros parasiticus Re-described as an alga Later transfer to C. villosus By Thompson & Wujek 1997 Dilcher 1965 As Pelicothallus villosus Described as a fungus

18. Fossil trentepohlialean taxa have been reported from the German Eocene Trentepohlia aurea Trentepohlia diffusa Koeck 1939: Fossilie Kryptogamen aus der eocanean Barunkohle des Geiseltales. Nova Acta Leopoldina 6:333-351 Printz 1939: Vorarbeiten zu einer Monographie der Trentepohliaceen.S. Nytt Mag. Aturvidenskapene B. 80:137-210, Taf.I-XXXII

19. Trentepohlia aurea var. acutata Trentepohlia rigidula Koeck 1939: Fossilie Kryptogamen aus der eocanean Barunkohle des Geiseltales. Nova Acta Leopoldina 6:333-351 Printz 1939: Vorarbeiten zu einer Monographie der Trentepohliaceen.S. Nytt Mag. Aturvidenskapene B. 80:137-210, Taf.I-XXXII

20. Reliable fossil records established trentepohlialean floras as far as the Eocene 50 mya • By the end of this epoch: • Continents moved closer to their present position • Tropical areas shrinking • A drying period commences • In subtropical latitudes, open woodlands with ferns and shrubby plants replaced forests • Whale ancestors left the land • Trentepohlialean taxa appeared on land over 400 my after the initial colonization of land plants in the Paleozoic (480 mya) http://3dparks.wr.usgs.gov/

21. Ancient sea level changes • Just before Eocene (50 mya) the sea level was at the highest and since then it has been receding • Much of continental North America, Africa and Australia were exposed Falkowsky et al. 2004; sea level change

22. Evolution of floras such as • Diatoms • Grasses Falkowsky et al. 2004; sea level change

23. Pre-adaptive phenotypic plasticity • Intertidal marine algae live in a variable environment under great physical stress • This may lead to a selection for a “plastic morphology” were the same genotype is expressing several morphologies under different ecological conditions • Extant ulvophyceans are examples of this adaptive phenotypic plasticity:

24. Terrestrial ulvophytes express this adaptive phenotypic plasticity by their ecological ubiquity, ability to grow in several media, and their astonishing capacity to undergo morphological modifications: Printzina Trentepohlia Cephaleuros Phycopeltis

25. Information from fossils, ancient sea level changes, phenotypic plasticity and molecular data seem to indicate that: • Terrestrial ulvophytes did not “jump” from marine to terrestrial habitats: they were “left behind” after sea levels receded during the Eocene • Ancestors of terrestrial ulvophytes may had the pre-adaptive capabilities to exploit and diversify in the new habitat and become permanent members of the subaerial flora

26. Many questions still remain to be answered, for example: • Exclusively subaerial algae have a long evolutionary history compared to other green algal orders; however, their fossil evidence is relatively more recent; what is the evolutionary history of this lineage between their ancestral marine origin and their transition to land? Yakutina and Dasyclads Cambrian 600 mya Cladophora-like Precambrian 700-800 mya Trentepohliales Eocene 50 mya

27. Many questions still remain to be answered, for example: • Exclusively subaerial algae have a long evolutionary history compared to other green algal orders; however, their fossil evidence is relatively more recent; what is the evolutionary history of this lineage between their ancestral marine origin and their transition to land? • Another intriguing question is about evolutionary reversals: are there any examples of algae that originated in the continental environment and made a transition back to the sea? Trebouxiophyceae: Stichococcus marinus? Chlorophyceae: Dunaliella spp. ? Trebouxiophyceae: marine Prasiolales (Prasiola and Rosenvingiella)

28. Some conclusions • The terrestrial habitat has been colonized by several lineages and more than one green algal group • The Charophycean, Trebouxiophycean and Chlorophycean algae made attempts to colonize the Land via freshwater habitats • The marine Ulvophycean taxa also made this conquest (at least twice) • Subaerial ulvophytes conquered the land using a direct strategy • The history of the conquest of terrestrial habitats by algae is more complex than previously recognized • New molecular techniques (environmental cloning and phylogenomics) may unravel more unknown lineages and unexpected discoveries!

29. Acknowledgments Russell L. Chapman, Scripps Institution of Oceanography Mike Guiry, National University of Ireland Paul Broady, University of Canterbury, New Zealand Thomas Friedl, University of Goettingen Fred Brooks, American Samoa Community College Alison Sherwood, University of Hawai’i at Mãnoa Joe Zuccarello, Victoria University of Wellington, NZ Bruno De Reviers, Paris Herbarium Ricardo Tsukamoto, Sao Paulo, BZ Jirí Neustupa, Charles University of Prague, Czech Republic Drs. Fabio Rindi and Sarah M. Noble, graduate students Daryl W. Lam and Haj A. Allali, several undergrads Research at the Phycolab is funded by grants from NSF Systematics-DEB, NEP, MASGC, CA&S/UA and from a RAC/UA to JLB Fieldwork funds to French Guiana, South Africa, Gabon, Panama, Suriname, Europe, and Southern Mexico partially provided by the Graduate School and the Department of Biological Sciences at UA

30. The PhycoLab in the Web http://bama.ua.edu/~jlopez

31. Ongoing Research at the PhycoLab

32. Subaerial microchlorophytes Europe Southeastern USA Morocco Panama Gabon Suriname French Guiana South Africa and Australia

33. Assembling the Tree of Life Program:

34. UA – CUBA Seaweed research collaboration Lidice Clero, CIM Dr. Ana Maria Suarez Inst. Marine Sciences University of La Havana M. Sc. Ruben Cabrera Marine Archeology Yusimi Alfonso, Acuario Nacional Mayrene Guimaraes, Cayo Coco Antonio Vega, Holguin Sandra Siret, Matanzas Abdiel Jover, Santiago Angel Moreira, Cienfuegos Juan J. Lake, Camaguey Dr. Beatriz Martinez Oceanology Institute, CITMA Ivan Martin, Villaclara