Biodiversity hot spots of subaerial algae

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Subaerial Algae in Tropical Rainforests, part II
Juan M. Lopez-Bautista The University of Alabama
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Overview

• Biodiversity and systematics of subaerial algae
• Collection and study of subaerial algae
• Molecular systematics of subaerial
  microchlorophytes
• Evolution of subaerial microchlorophytes

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Molecular systematics of subaerial
microchlorophytes
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Klebsormidium Klebsormidium is one of the most
common genera of terrestrial Charophyta It
consists of few species of green algae occurring
on soil, subaerial surfaces and semi-aquatic
habitats all around the world
Pisa Italy 2005
The systematics of this genus has been entirely
based on traditional morphology. The
species-level relationships in Klebsormidium have
not been examined with molecular tools
Klebsormidium Galway Ireland 2004
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• Klebsormidium
• - One of the most widespread genera of
  terrestrial and freshwater green algae
• - Cosmopolitan distribution
• - Uniseriate filaments no differentiated
  holdfast one parietal chloroplast covering a
  half-2/3 of the cell wall, with a single pyrenoid
• 22 species currently described
• Species-level systematics based almost entirely
  on morphology
• Reproduction by biflagellate zoospores

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Principal morphological characters used for
species identification in Klebsormidium

• Width of filaments
• Length of filaments
• Cell shape
• Presence/absence of constrictions between
  adjacent cells
• Occasional presence/absence of biseriate parts
• Presence of H-shaped pieces
• Shape of the chloroplast

Long
Short
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Characters observable in culture

• Growth habit filamentous or fragmented
• Presence/absence of a superficial hydrorepellent
  layer
• Inducibility of zoospore release
• Morphology of the zoosporangial aperture
• Germination pattern of sporelings

Fragmented
Filamentous
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• CITIES SAMPLED
• Bergen (Norway)
• Stockholm (Sweden)
• Copenhagen (Denmark)
• Galway (Ireland)
• London, Plymouth and
• Manchester (England)
• Hamburg, Rostock and
• Konstanz (Germany)
• Bordeaux and Marseilles
• (France)
• Porto (Portugal)
• Pisa, Siena and Pavia
• (Italy)
• La Valletta (Malta)
• Koper (Slovenia)
• Prague (Czech Republic)

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Morphology (field)

• Very uniform morphology virtually no differences
  between strains from different cities
• Filaments mostly long, sometimes mixed with
  short fragments
• Filaments unbranched, 6-9 ?m wide
• Slight constrictions between adjacent cells
  present in many strains
• Cells regularly cylindrical
• Chloroplast with smooth margin
• Globular enlargements present in one strain
  (Pisa)
• No biseriate parts or pseudo-branches

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Morphology (culture)

• All strains growing well in both media used
• Production of superficial hydrorepellent layer
  observed in the strains from Bergen, Galway,
  Hamburg and Konstanz
• The strains from Marseilles, Pisa, La Valletta,
  Hamburg and Porto showed a marked tendency to
  fragmentation after a few weeks the strains from
  Pisa and Marseilles consisted entirely of short
  fragments (2-5 cells)
• Release of zoospores easily inducible only in
  the strain from Galway release aperture
  indistinct
• Sporelings in the Galway strain germinating with
  unipolar and bipolar pattern

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• Although the morphology of the field material
  was virtually identical to Klebsormidium
  flaccidum, preliminary experiments in culture
  have shown a very large range of morphological
  variation
• Most strains produce only 3D filaments that
  remain submerged
• Others populations produce also a superficial
  layer of parallel filaments that cover completely
  the surface of the medium,
• In other strains, the filaments get fragmented
  into many short fragments, giving the cultures
  the appearance of a green soup

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Samples originally corresponding to Klebsormidium
flaccidum are spread along the rbcL tree
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Samples originally corresponding to Klebsormidium
flaccidum are spread along the rbcL tree On the
other hand, samples corresponding to different
morphological species are grouped together
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Samples originally corresponding to Klebsormidium
flaccidum are spread along the rbcL tree On the
other hand, samples corresponding to different
morphological species are grouped
together Furthermore, specimens not possible to
ascribe to any known taxa, and from distant
locations, are found to be genetically similar!
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Conclusions

• The populations of Klebsormidium occurring in
  European cities belong to at least 4 different
  evolutionary lineages
• In the field material, these show an almost
  identical morphology and there are no
  morphological characters useful to distinguish
  them
• More differentiation observed in culture, in
  which different strains show different growth
  habits
• The taxonomy of Klebsormidium needs critical
  reconsideration none of the morphological
  characters used for identification have
  phylogenetic relevance
• The characterization of Klebsormidium flaccidum,
  type species of the genus, is a taxonomic mess
  that can be solved only sequencing the type
  specimen depending on its identity, the whole
  genus may require a radical reassessment

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Lessons learned

• Overall, these experiments and analyses
  indicated that a great deal of genetic diversity
  is hidden behind a very similar morphology
• Morphology and cytology, even in simple and
  common forms, can be misleading
• Unialgal cultures and phylogenetic analyses are
  needed to re-evaluate these so called
  cosmopolitan species

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Spongiochrysis hawaiiensis
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Dense yellow-orange coating believed to be
Trentepohlia by the local scientists
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• Interesting unicellular green alga, reproducing
  with a budding-like mechanism known only in two
  subaerial species of the class Trebouxiophyceae
• Marvania geminata Hindák 1976
• Marvania aerophytica Neustupa Sehjonová
  2003/Stichococcus ampulliformis Handa et al. 2003
  

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• However, sequences of the 18S rRNA, showed
  unequivocally that this alga is a member of the
  Cladophorales Siphonocladales lineage
  (Ulvophyceae)
• Since this group was so far known to include
  basically marine species with complex thalli,
  this was a discovery of exceptional interest,
  that shed new light on the phylogeny of this
  algal group
• Autosporulation has arisen separately at least 3
  times (twice in Trebouxiophyceae and once in
  Ulvophyceae)

Phylogenetic tree for a 18S rDNA sequence
alignment of 1732 characters, with 55
representatives of the Viridiplantae and
Spongiochrysis hawaiiensis.
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In a restricted analysis limited to 41
representatives of this group, our samples were
included in the Aegagropila-clade with high
support



Phylogenetic tree for an 18S rDNA sequence
alignment of 1595 characters, with 41
representatives of the Siphonocladales
Cladophorales complex, two outgroup taxa, and
Spongiochrysis hawaiiensis
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• These data suggest that a marine ancestor gave
  origin to Spongiochrysis
• Simplification of the thallus
• Carotenoid productionSimilar trends during the
  colonization of terrestrial habitats have been
  found in the evolution of green algae

Phylogenetic tree for an 18S rDNA sequence
alignment of 1595 characters, with 41
representatives of the Siphonocladales
Cladophorales complex, two outgroup taxa, and
Spongiochrysis hawaiiensis
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Phylogenetic Considerations
This alga represents a new subaerial lineage
from the class Ulvophyceae It is the first
known subaerial member of the Cladophorales
Siphonocladales lineage For the unicellular
habit and for the subaerial habitat this species
is well differentiated from all other members of
this lineage and can be considered the first
known successful step of these algae into
subaerial habitats New genus, new
species Spongiochrysis hawaiiensis
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Lessons learned

• The subaerial habit has developed in an algal
  group that was formerly believed to be entirely
  aquatic (the Cladophorales)
• A new subaerial lineage exists in the class
  Ulvophyceae
• It also showed that identical morphologies and
  identical mechanisms of reproduction have
  developed independently in separated green algal
  lineages
• Most importantly, great evolutionary surprises
  can be found in forest environments, if detailed
  surveys based on modern methodologies are carried
  out

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The Order Trentepohliales
The group is unusual (to say the least!) in many
respects
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Unusual features

• Exclusively terrestrial and orange-red (unlike
  the majority of green algae, which are aquatic
  and green)
• Differentiated sexual and asexual reproductive
  structures including an absolutely intriguing,
  unique zoosporangial abscission process
• Remarkable bilaterally keeled flagella and
  flagellar apparatus (i.e. basal bodies and
  associated components), and
• Evolutionarily puzzling phragmoplast-mediated
  cytokinesis found no where else in the entire
  chlorophyte lineage and analogous to but distinct
  from the phragmoplast-mediated cytokinesis of
  some green algae and the land plants in the
  charophycean lineage

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The order Trentepohliales
Five genera currently recognized
Phycopeltis
Printzina
Trentepohlia
Cephaleuros
Discoid Epiphytic Isomorphic 28 species
Prostrate Epiphytic or epilithic Isomorphic 8
species
Erect Epiphytic or epilithic Isomorphic 38
species
Discoid Obligated epiphyte Heteromorphic 15
species
Reduced Endophytic Heteromorphic 4 species
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Trentepohlia Martius 1817 The genus Trentepohlia
is the most diversified of the trentepohliacean
algae, consisting of branched heterotrichous
filaments, growing epilithic or epiphytic on the
bark of trees, or in lichenic associations at
exposed habitats forming conspicuous masses,
usually yellow to orange in color As currently
redefined, Trentepohlia shows a profused erect
system, with a scarce or absent prostrate system
sporangia are ovoid, sporangiate-laterals
solitary or grouped, borne terminally or on an
enlarged terminal head-cell of a branched
sporangiophore gametangia are terminal
only Described species 35
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Physolinum H. Printz 1921
The species type for the genus Physolinum was
originally described as Trentepohlia monile,
later the specific epithet was changed to T.
moniliformis The genus Physolinum was based on
the discovery of aplanospores as the sole
reproductive structures found in this alga, and
concluding that was markedly different from
Trentepohlia Described species 1
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• Printzina Thompson et Wujek 1992
• Printzina is remarkably similar to Trentepohlia,
  except
• the presence of globular to reniform sporangia,
  and
• an almost pseudoparenchymatic thallus with few or
  any upright filaments
• Described species 9

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Phycopeltis Millardet 1870 The filaments of
Phycopeltis grow openly or form a
pseudoparenchymatous thallus, but always
superficial upon the plant host or other surfaces
Another feature distinguishing Phycopeltis from
other superficial trentepohlialean genera is the
terminal position of the papilla-pore or ostiole
on the sporangium, which is opposite to the end
of attachment whereas, in the other genera it is
basal and adjacent to the area of
attachment Described species 25
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Cephaleuros Kunze in Fries 1829 This genus is
reported as an obligate epiphyte and
subcuticular, that may be parasitic The prostrate
portion can be open-filamentous to
pseudoparenchymatous Sporangiophores bear one or
more head cells subtending sporangiate-laterals C
ephaleuros is one of the most studied genera
among the Trentepohliales, in part for its
worldwide distribution, its obvious presence and
economic damage to their host Described species
15
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Stomatochroon Palm emend. Thompson and Wujek
1997 This alga grows as branching filaments
endophytically in the substomatal chamber and
protruding its sporangiophores through the
stomata The reduced morphology of this alga is
extraordinary, one species has become reduced to
a single massive and lobed anchoring
cell Described species 4
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Life cycles They have been described as either
an isomorphic or heteromorphic alternation of
generations
Isomorphic generations occur in taxa where the
vegetative morphology is simpler (Trentepohlia
Phycopeltis),
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Whereas a more complicated heteromorphic
alternation is said to occur in taxa with a
complex vegetative morphology (Cephaleuros
Stomatochroon) and possibly secondarily reduced
morphology
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Reproductive Cells sporangium is unique to this
order Trentepohliales displays a characteristic
abscission between the sporangium and the
suffultory cell (or stalk cell) The sporangium
and suffultory cell form together the
sporangiate-lateral, which representa an
important taxonomic character for the
circumscription of the order
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Abscission The abscission involves a central area
rich in plasmodesmata surrounded by a thickened
area or internal ring In the periphery of this
abscission septum there is a second area of
thickened wall material, the external ring the
region between the rings lacks plasmodesmata
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Flagella The flagellate cells are compressed in
a dorsiventral fashion with either two (gametes)
or four (zoospores) flagella
The overlapping configuration of the basal bodies
in Trentepohliales is CCW and it has been cited
as an evidence for an affinity with the
Ulvophyceae
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Cytokinesis
A phragmoplast-type cytokinesis has been reported
for C. parasiticus and Trentepohlia odorata
Chapman and Henk 1986
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• Ecology and Distribution
• The geographic distribution of the
  Trentepohliales is basically pantropical
• All Trentepohliales are subaerial, none having
  ever been found in aquatic habitats, freshwater
  or marine
• The presence of sporopollenin-like substances in
  the cell walls, as well as a special pattern of
  carbohydrates and alcohols, probably are
  adaptative features against desiccation in the
  subaerial habitat
• Trentepohliales are well known to form lichenic
  associations with fungi
• Trentepohlia commonly occurs upon rocks or tree
  bark
• Phycopeltis is an epiphyllous alga, growing on
  the surface of leaves
• Cephaleuros is a strict epiphyte living beneath
  the cuticle of leaves, twigs, and fruits
• Stomatochroon is found in the air chambers and
  stomata of leaves

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• Questions on the evolution of the Trentepohliales
• Where does the Trentepohliales belong in the
  various proposed classification schemes of green
  algal classes?
• Phragmoplast cytokinesis suggested an affinity
  with Charophyceae
• The CCW flagella can be cited as evidence for an
  affinity with the Ulvophyceae
• Based on biochemical and physiological features
  they have been classified as Pleurastrophyceae
• Moreover, Trentepohliales share a rare
  ultrastructural feature (presumptive mating
  structure in the gametes) with members of a
  fourth class, the Chlorophyceae

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Molecular studies in green algae
VIRIDIPLANTAE (CHLOROBIONTA, GREEN PLANTS)
CHLOROPHYTA
STREPTOPHYTA
Charophycean lineage
Chlorophycean lineage
CHLOROPHYCEAE
TREBOUXIOPHYCEAE
LAND PLANTS
CHAROPHYCEAE Chlorokybales Klebsormidiales Coleoch
aetales Zygnematales Charales
ULVOPHYCEAE
Mesostigma
PRASINOPHYCEAE
PRASINOPHYCEAE
PRASINOPHYCEAE
PRASINOPHYCEAE
ANCESTRAL GREEN FLAGELLATE