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Content uploaded by Lenka Štenclová
Author content
All content in this area was uploaded by Lenka Štenclová on Oct 02, 2019
Content may be subject to copyright.
MOLECULAR AND MORPHOLOGICAL DELIMITATION AND
GENERIC CLASSIFICATION OF THE FAMILY OOCYSTACEAE
(TREBOUXIOPHYCEAE, CHLOROPHYTA)
0.1
98/1.00
68/-
Lagerheimia longiseta JQ315525
100/1.00
68/0.99
97/1.00
55/0.96
Oocystidium sp. SAG 81.80
97/1.00
100/1.00
86/0.99
Quadricoccus ellipticus Hq008712
73/1.00
98//1.00
Oocystis parva Jq315649
Oocystis sp. Jq315800
98/1.00
Oocystis marssonii Af228688
75/1.00
Oocystella oogama SAG 3.96
Oocystis bispora CH 99
96/1.00
77/0.97
Crucigeniella rectangularis Ah02990
88/1.00
Elongatocystis ecballocystiformis Hq008713
Ecballocystis hubeiensis Jx0885
50/-
Makinoella tosaensis Af228691
Makinoella tosaensis SAG 28.97
99/1.00
52/-
55/-
Oocystis solitaria Af228686
79/1.00
80/1.00
Planctonema lauterbornii Af38748*
Planctonema lauterborniiSAG 68.94
Auxenochlorella protothecoides Fn29893
Oocystidium polymammilatum AN9-1
Oocystidium polymammilatum Ay195966
Ooplanctella planoconvexa Fm881777
Oocystidium sp. Hq008711
Lagerheimia marssonii CCALA 365
Lagerheimia genevensis Ay122336
Lagerheimia ciliata SAG 1194
Lagerheimia longiseta SAG 57.81
Lagerheimia subsalsa SAG 2084
Frenceia aphitricha SAG 10.81
Echinocoleum elegans Fm881776
Oocystis cf. rhomboidea W Twin SlisT-7d
Quadricoccus sp. Ay197626
Quadricoccus sp. As7-C
Schizochlamydella capsulata CCMP 245
Amphikrikos cf. nanus Af228690
Oocystis cf. parva. Ay197635
Oocystis parva SAG 82.80
Oocystis heteromucosa Af228689
Oocystis heteromucosa KR 210
Oocystis cf. marssonii SAG 2085
Tetrachlorella alternans Af228687
Oocystis sp. CAUP H 1110
Granulocystis verrucosa SAG 56.81
Siderocystopsis punctifera SAG 28.81
Willea vilhelmii CCALA 515
Ecballocystopsis dichotomus Jx0884
Eremosphaera gigas ACOI 1819
Oocystis solitaria CAUP H 1106
Neglectella peisonis SAG 37.96
Eremosphaera viridis SAG 228-1
Eremosphaera viridis SAG 39.92
Tetrastrum staurogeniiforme Jq356702
Tetrastrum staurogeniiforme Jq356703
Tetrastrum heteracanthum Jq356709
Chlorella vulgaris Fr865658
Chlorella variabilis Ab206549
Micractinium pusillum Af364101
OOCYSTACEAE
SPINY CLADE
GRANULATED
CLADE 2
GRANULATED
CLADE 1
100/1.00
100/1.00
100/1.00
100/1.00
100/1.00
100/1.00
100/1.00
100/1.00
100/1.00
100/1.00
-/0.99
OOCYSTOIDEAE
MOLECULAR AND MORPHOLOGICAL DELIMITATION AND
GENERIC CLASSIFICATION OF THE FAMILY OOCYSTACEAE
(TREBOUXIOPHYCEAE, CHLOROPHYTA)
52/-
90/1.00
Lagerheimia ciliata SAG 2083
Oocystis naegelii MP STE7
OUTGROUP - CHLORELLACEAE
OOCYSTIDIUM
CLADE
NEGLECTELLA
CLADE
MAKINELLOIDEAE
- COENOBIAL
CLADE
´EREMOSPHAEROIDEAE´
MOLECULAR AND MORPHOLOGICAL DELIMITATION AND
GENERIC CLASSIFICATION OF THE FAMILY OOCYSTACEAE
(TREBOUXIOPHYCEAE, CHLOROPHYTA)
Lenka Štenclová
Faculty of Science, University of South Bohemia, Czech Republic
lenka.stenclova@prf.jcu.cz
Introduction Objectives
Results and discussion Conclusion
10 Neglectella peisonis
SAG 37.96
Material and Methods
Acknowledgment
cells. Cells propagate by autospores and daughter cells usually stay longer time or all their life enclosed in the mother
cell wall. The cell wall possesses specific ultrastructure: multi-layered with the cellulose fibrils in each layer
perpendicular to fibrils of the adjoining layer. The cell wall can be smooth or bears warts or spines and often polar
thickness. Individual genera were distributed into three subfamilies. Subfamily Eremosphaeroideae associated genera
with large cells and numerous chloroplasts. Lagerheimioideae and Oocystoideae contained representatives with small
dimensions and a few chloroplasts while Lagerheimioideae algae were characterized by spines on their cell wall.
Establishment of the molecular phylogeny proved the morphology-based delimitation and generic conception of
Oocystaceae to be inaccurate. Three species with the atypical or irregular shape of the cell: Elakatothrix viridis,
Nephrochlamys subsolitaria, Rhombocystis complanata, and spherical spiny Trochiscia hystrix were transferred to the
class Chlorophyceae. Some outside Oocystaceae coming taxa with a similar shape of the cell as Oocystis: Amphikrikos
sp., Quadricoccus ellipticus, Schizochlamydella capsulata and mucilagenous Coenochloris planoconvexa
(Ooplanctella planoconvexa) were newly included in the family. The changes indicate that the shape of the cell of
Oocystaceae representatives is rather strict.
Coenobial strains Crucigeniella rectangularis, Makinoella tosaensis and Tetrachlorella alternans and
pseudofilamentous Ecballocystis hubeiensis, Ecballocystopsis dichotomus, newly redefined as members of the
family, indicates that the family has a wider variability of the cell arangement than previously expected. Furthermore,
some authors considered including coenobial genus Tetrastrum, as well as simply filamentous Planctonema
lauterbornii, both clustering on the basis of the Oocystacea tree.
Not many taxa of Oocystaceae were sequenced so far; however internal structure and generic classification of the
family were called into question. Current finding showed that the morphologically well-defined genus Oocystis is
polyphyletic. More data are necessary to resolve which lineage represents the right Oocystis.
Nadání Josefa, Marie a Zdeňky
Hlávkových
I thank to Jan Hanys Kaštovský, Marie Pažoutová
and Jiří Košnar for help with the study and to
Marvin Fawley, Christina Bock and Lothar Krienitz for providing thier algal strains. For financial
support, I thank to University of South Bohemia and the Foundauntion "Nadání Josefa, Marie a
Zdeňky Hlávkových”
The traditional morphological conception of the family Oocystaceae included species with
typically oval or elliptical or lemon-shaped, uncommonly spherical, rhombic, spindle-shaped,
are consulted with morphological and ultra-structural findings. The research follows the previous
studies and brings together the partial results and newly available data. Comprehensive Definition:
study containing an extensive dataset allows discussing the old definition of the family with modern
findings. The traditional conception of the family and its division into the Internal structure:
subfamilies stays unsure, because of poor taxa selection, especially of the spiny representatives of
the family. The present study supplies space in our knowledge. The requirement Generic concept :
of the revision of the polyphyletic genus Oocystis is obvious since the first studies dealing with the
molecular phylogeny of the family released. To examine additional taxa of the genus help us to
detect the natural character of the genus Oocystis. The status of several other genera is tested.
Delimitation:The main purpose of the study is a new, more precise
delimitation of the family based principally on the molecular data, which
Figure 3: Phylogenetic analyses of SSU rRNA gene sequences of members of Oocystaceae with Chlorellaceae as outgroup.
Numbers at the branches indicate bootstrap support from maximum likelihood (ML, 100 replicates) and Bayesian posterior
probabilities (BI). Support ≥50% for ML and ≥0.95 for MB is shown. ML/BI (Bold taxa = one sequence added.). Drawings
demonstrate typical morphology of the individual subfamilies and clades. Lineages with ´Oocystis´ spp. marked by an asterisk.
Figure 1: Phylogenetic analyses concatenated dataset of SSU rRNA
and rbcl sequences of members of Oocystaceae with Chlorellaceae as
outgroup. Numbers at the branches indicate bootstrap support from
maximum likelihood (ML, 100 replicates) of unpartitioned alignment and
Bayesian posterior probabilities of unpartitioned (BI) and partitioned
(PBI) alignment. Support ≥50% for ML and ≥0.95 for MB is shown
ML/BI/PBI. (Bold and underlined taxa = both sequences added, bold =
one sequence added.)
0.1
Oocystidium polymammilatum AN9-1
Oocystidium polymammilatum AN2/29-4
91/1.00
0.96
50/-
0.95
Oocystidium sp. SAG 81.80
100/1.00
1.00
Lagerheimia marssonii CCALA 365
Lagerheimia genevensis SAG 48.94
Lagerheimia ciliata SAG 1194
Lagerheimia ciliata SAG 2083
100/1.00
1.00
Lagerheimia longiseta SAG 57.81
Lagerheimia subsalsa SAG 2084
100/1.00
1.00
50/0.99
1.00
Echinocoleum elegans SAG 37.93
Oocystis cf. rhomboidea W Twin SlisT-7d
100/1.00
1.00
Oocystis cf. parva Tow 6/3 P-1ou
Oocystis parva SAG 82.80
Oocystis heteromucosa SAG 1.99
Oocystis heteromucosa KR 210
100/1.00
1.00
Oocystis cf. marssonii SAG 2085
Tetrachlorella alternans SAG B42.81
99/1.00
1.00
Oocystis sp. CAUP H 1110
99/1.00
1.00
86/1.00
1.00
86/0.99
0.98
Eremosphaera gigas ACOI 1819
100/1.00
1.00
Eremosphaera viridis SAG 228-1
Eremosphaera viridis SAG 39.92
100/1.00
1.00
100/1.00
1.00
Planctonema lauterbornii *
Planctonema lauterborniiSAG 68.94
100/1.00
1.00
76/1.00
1.00
Tetrastrum staurogeniiforme KR 1996/3
Tetrastrum staurogeniiforme SAG 45.81
99/1.00
1.00
Tetrastrum heteracanthum SAG 24.81
100/1.00
1.00
Chlorella vulgaris
Chlorella variabilis
Micractinium pusillum
Auxenochlorella protothecoides
OUTGROUP - CHLORELLACEAE
OOCYSTIDIUM
CLADE
SPINY CLADE 1
-/0.95
1.00
100/1.00
1.00
100/1.00
1.00
100/1.00
1.00
100/1.00
1.00
100/1.00
1.00
100/1.00
1.00
OOCYSTOIDEAE
Ooplanctella planoconvexa
Frenceia aphitricha SAG 10.81
Quadricoccus sp. MDL6-7
Quadricoccus sp. As7-C
Schizochlamydella capsulata CCMP 245
Amphikrikos sp. SAG 30.96 *
Oocystis naegelii MP STE7
Granulocystis verrucosa SAG 56.81
Siderocystopsis punctifera SAG 28.81
Willea vilhelmii CCALA 515
Ecballocystis hubeiensis
Makinoella tosaensis SAG 28.97
Makinoella tosaensis CCALA 961
Ecballocystopsis dichotomus
´Oocystis´solitaria CAUP H 1106
´Oocystis´solitaria SAG 83.80
Neglectella peisonis SAG 37.96
100/1.00
1.00
OOCYSTACEAE
NEGLECTELLA
CLADE
GRANULATED
CLADE 2
GRANULATED
CLADE 1
MAKINELLOIDEAE
- COENOBIAL
CLADE
´EREMOSPHAEROIDEAE´
87/1.00
0.99
94/1.00
1.00
66/0.99
64/0.97
60/1.00 99/1.00
changed due to the application of molecular
phylogenetics. Some taxa preliminary defined in the
family were excluded and one another newly redefined
as member of the family. Internal structure was divided
to three subfamilies Oocystoideae, Makinelloideae and
Eremosphaeroideae. Inside Oocystoideae occurred 4
respectively 5 morphological clades. Changes in
genera Crucigeniella, Franceia, Lagerheimia,
Neglectella, Oocystidium, Oocystis, Ooplanctella and
Willea are suggested.
The delimitation and defini-
tion of the family Oocystaceae
under the standard conditions. The strains were examined by light microscopy (Olympus BX 51
microscope with digital camera Olympus DP – 71). Observations were documented with the
software DP Controller 3.1. Chosen strains (Tetrastrum and Planctonema spp.) were investigated
using transmission electron microscopy (Jeol JEN 1010) to detect the ultrastructure of the cell wall.
Strains were processed by common methods of molecular biology (DNA isolation, PCR reaction of
SSU and rbcl gene and their sequencing) and molecular phylogenetics. The final phylogenetic
trees were made using Maximum likelihood (Phyml 3.0) and Bayesian inference (Mr.Bayes 3.2.1)
based on alignments of the SSU gene, rbcl gene and concatenated SSU + rbcl datasets.
52 single-algal strains from the public or the
private culture collections were cultivated
100/1.00
Figure 2: Phylogenetic analyses of rbcl gene sequences of members of
Oocystaceae with Chlorellaceae as outgroup. Numbers at the branches
indicate bootstrap support from maximum likelihood (ML, 100 replicates)
and Bayesian posterior probabilities (BI). Support ≥50% for ML and
≥0.95 for MB is shown. ML/BI. (Bold taxa = one sequence added.)
Drawings demonstrate typical morphology of the individual Lagerheimia
lineages.
0.1
100/1.00
96/1.00
84/1.00
95/1.00
100/1.00
82/1.00
100/1.00
95/1.00
100/1.00
100/1.00
95/1.00
84/1.00
100/1.00
62/-
100/1.00
93/1.00
94/1.00
100/1.00
97/1.00
100/1.00
Siderocystopsis sp. CCALA 396
Planctonema lauterbornii Ef113462
100/1.00
79/0.98
98/1.00
100/1.00
Lagerheimia genevensis SAG 48.94
Oocystidium sp SAG 81.80
Ecballocystis hubeiensis Jx018187
Makinoella tosaensis SAG 28.97
´Oocystis´solitaria SAG 83.80
Planctonema lauterborniiSAG 68.94
Auxenochlorella protothecoides Eu038285
Oocystidium polymammilatum AN9-1
Oocystidium polymammilatum AN2/29-4
Ooplanctella planoconvexaCAUP H5502
Lagerheimia marssonii CCALA 365
Lagerheimia hindakii SAG 11.92
Lagerheimia ciliata SAG 1194
Lagerheimia ciliata SAG 2083
Lagerheimia longiseta SAG 57.81
Lagerheimia subsalsa SAG 2084
Frenceia aphitricha SAG 10.81
Echinocoleum elegans SAG 37.93
Oocystis cf. rhomboidea W Twin SlisT-7d
Quadricoccus sp. MDL6-7
Quadricoccus sp. As7-C
Schizochlamydella capsulata CCMP 245
Amphikrikos nanusSAG 2074
Oocystis cf. parva Tow 6/3 P-1ou
Oocystis parva SAG 82.80
Oocystis heteromucosa SAG 1.99
Oocystis heteromucosa KR 210
Oocystis naegelii MP STE7
Oocystis cf. marssonii SAG 2085
Tetrachlorella alternans SAG 42.81
Oocystis sp. CAUP H 1110
Granulocystis verrucosa SAG 56.81
Siderocystopsis punctifera SAG 28.81
Willea vilhelmii CCALA 515
Ecballocystopsis dichotomus Jx018186
Eremosphaera gigas ACOI 1819
´Oocystis´solitaria CAUP H 1106
Neglectella peisonis SAG 37.96
Eremosphaera viridis SAG 228-1
Eremosphaera viridis SAG 39.92
Tetrastrum staurogeniiforme SAG 45.81
Tetrastrum staurogeniiforme KR 1996/3
Tetrastrum heteracanthum SAG 24.81
Chlorella vulgaris Ab260909
Chlorella variabilis Ab260903
Micractinium pusillum Ef113451
OOCYSTACEAE
OUTGROUP - CHLORELLACEAE
OOCYSTIDIUM
CLADE
SPINY CLADE 1
GRANULATED
CLADE 2
SPINY CLADE 2
100/1.00
Planctonema sp. Ef113463
NEGLECTELLA
CLADE
´Oocystis´ cf. nephocytioides CCALA 397
Makinoella tosaensis CCALA
Amphikrikos nanusSAG 30.96
Granulocystopsis coronata SAG 33.81
-/0.99
100/1.00
-/0.95
-/0.98
MAKINELLOIDEAE
- COENOBIAL
CLADE
GRANULATED
CLADE 1
EREMOSPHAERO
IDEAE
Delimitation: Taxa Oonephris obesa and Nephrocytium agardhianum were excluded from the family
because of their affinity with t he class Chlorophyceae. The remaining s trains constituted monophyletic
clade inside Trebouxiophyceae including coenobial Willea wilhelmii (Figs. 1-3). The situatinon on the basis of the Oocystaceae tree still stayed unresolved. Neither
molecular phylogeny nor morphology and even nor ultrastructural observation can justify the inclusion of the genera Tetrastrum and Planctonema into the family. The
Genera share some morphological traits with the family; however, some traits are different, and the relevant multi-layered ultrastructure of the cell wall was not
detected.
Definition: Changes in present study, in accord with previous findings, indicate that the definition of the family regarding the typical oval, elliptical or lemon-like shape
of the cell is rather robust, and the trait can be relevant to distinguish whether taxa could be included in the family. Conversely, coenobial and simply filamentous
members, recently reclassified to the family, show the larger diversity of the cell arrangement. It is assumed that within those groups can be more adepts to the transfer
to Oocystaceae.
Internal structure: Molecular phylogenetics suggested the different organisation of the family in comparison with morphological conception. Previously distinguished
subfamilies Lagerheimioideae and Oocystoideae made one monophyletic cluster - subfamily Oocystoideae. Monophyletic coenobial clade was labelled as subfamily
Makinelloideae. Big-celled Eremosphaera spp. and the Neglectella clade on the basis of Oocystaceae tree made the third subfamily. However its suitability is
questionable, because monophyletic or paraphyletic status was not proved. Probably because of long branch attraction. 5 well-supported clades were detected inside
subfamily Oocystoideae (Figs. 1-3). The position of rest of the strains remains unclear.
Generic conception: Since the first modern study of the Oocystaceae was released, the requirement of a comprehensive revision of the genus Oocystis was obvious.
O. solitaria clustered together with Neglectella far from the other representatives of the genus Oocystis. Inside the coenobial clade occurred O. cf. nephrocytioides as
well as previously O. ecballocystiformis (currently new genus Elongatocystis). Granulated taxa O. bispora and Oocystella oogama found their position inside
Granulated clade 1. All four taxa are suggested to be excluded from the genus Oocystis (including Oocystella). The rest of the strains including the strain representing
the type species O. naegelii possess uncertain position (Figs. 1-3). Genus Lagerheimia was proved to be paraphyletic (Fig. 2). Its representatives L. ciliata, L. subsalsa
and L. longiseta are closely related with Franceia amphitricha (spiny clade 1). Strains L. genevensis and L. hindakii formed spiny clade 2 – true Lagerheimia because
L. genevensis is a type species of the genus. Placement of the strain of L. marssonii disposes of poor support and it its status is unclear. The distribution of the spiny taxa
into several (two or three) genera is in accordance with the recent trend to establish small genera. Ooplanctella planoconvexa clusters within the Oocystidium strains.
Closed phylogenetic relationship and shared morphological characteristics, especially the wide mucilaginous envelope indicate, the clade probably one genus.
Echinocoleum elegans possesses similar mucilaginous envelope, nevertheless its relationship with the Oocystidium clade and the rest of Oocystaceae is unclear. Two
coenobial strains Crucigeniella rectangularis and Willea vilhelmii share similar morphology and close phylogenetic relationship. The genus Crucigeniella is pophyletic,
therefore its taxonomy should be re-examined.
Scale bar
in all pictures
indicates
20µm.
4 Oocystidium polymammilatum
AN2/29-4
5 Nocystella oogama
SAG 3.96
6 Oocystis naegelii
MP STE7
3 Lagerheimia gemenevensis
SAG 48.94
2 Lagerheimia marssonii
CCALA 365
1 Lagerheimia ciliata
SAG 1194
7 Granulocystis verrucosa
SAG 56.81
9 Oocystis solitaria
SAG 83.80
11 Eremosphaera viridis
SAG 228-1
8 Makinoella tosaensis
CCALA 96