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Cell Biovolume and Surface Area in Phytoplankton of Mediterranean Transitional Water Ecosystems: Methodological Aspects
Cell Biovolume and Surface Area in Phytoplankton of Mediterranean Transitional Water Ecosystems: Methodological Aspects
Cell Biovolume and Surface Area in Phytoplankton of Mediterranean Transitional Water Ecosystems: Methodological Aspects
RESEARCH ARTICLE
Abstract
1 - A series of experimental researches highlighted the role of morphometric parameters of phytoplankton
guilds as descriptors of the ecological status of transitional water ecosystems (TWs).
2 - However, at present, standardized or common methodologies for their use do not exist. In this work,
we develop a procedure for the determination of biovolume and surface area in phytoplankton guilds
of Mediterranean TWs.
3 - Phytoplankton biovolume and surface area are included among the most studied morphometric
descriptors. They can be estimated by associating the algae with similar geometric forms and
determining the volume of these by measuring the linear dimensions required for its calculation
under the light microscope.
4 - Here, a set of geometric models is suggested for calculating the cell biovolumes and surface area
of 235 phytoplankton genera, deriving from the analysis of 869 phytoplankton species, found in
transitional water ecosystems of the Mediterranean Ecoregion. The equations were designed to
minimize the effort of microscopic measurements.
5 - The similarities and differences between the geometric models here proposed and previously
published are discussed.
Keywords: morphometric descriptors, cell biovolume, surface area, phytoplankton, transitional water ecosystems.
Introduction
The Water Framework Directive (2000/60/EC)
sees phytoplankton as one of the biological
elements for evaluating the ecological status
of transitional aquatic ecosystems. However,
this regulation provides only general
indications on the relative descriptors to
be included in the monitoring programs.
Descriptors are measurable variables of a
TWB 7 (2013), n. 2
Cell biovolume and surface area in phytoplankton of Mediterranean transitional water ecosystems
101
TWB 7 (2013), n. 2
102
TWB 7 (2013), n. 2
Cell biovolume and surface area in phytoplankton of Mediterranean transitional water ecosystems
103
TWB 7 (2013), n. 2
104
TWB 7 (2013), n. 2
Cell biovolume and surface area in phytoplankton of Mediterranean transitional water ecosystems
Bacillariophyceae
Dinophyceae
Chlorophyceae
Prymnesiophyceae
Cyanophyceae
Euglenophyceae
Cryptophyceae
Chrysophyceae
Prasinophyceae
Dictyochophyceae
Xanthophyceae
Zygnematophyceae
other classes
47.9%
26.4%
7.0%
4.7%
4.7%
2.5%
1.7%
1.6%
1.2%
0.9%
0.5%
0.5%
0.5%
Figure 1. Relative contribution of the main phytoplankton classes in transitional water ecosystems in the
Mediterranean Ecoregion (data from a number of published lists of phytoplankton in TWs).
105
TWB 7 (2013), n. 2
106
TWB 7 (2013), n. 2
Cell biovolume and surface area in phytoplankton of Mediterranean transitional water ecosystems
Figure 2. Examples of phytoplankton cell measurements using image analysis system Lucia LtdNikon
Instruments SpA.
107
TWB 7 (2013), n. 2
Coefficent
ofofvariation
of biovolume
biovolume
Coefficent
variation of
(standard
errorasas%%ofof
mean)
(standard error
mean)
C.closterium
Cylindrotheca closterium
P.minimum
Prorocentrum minimum
P.micans
Prorocentrum micans
Navicula transitans
Navicula transitans
Chaetoceros whigamii
C.whigamii
100
90
80
70
60
50
40
30
20
10
0
0
10
20
30
40
50
Number
cell measured
measured
Number
ofofcells
Figure 3. Microscopical measurements of linear dimensions. In the graph is reported the coefficient of
variation in relation to the number of cells measured.
Figure 4. Coefficient of variation of average biovolume of random subset of phytoplankton guilds with
increasing numerical abundance of cells measured.
108
TWB 7 (2013), n. 2
Cell biovolume and surface area in phytoplankton of Mediterranean transitional water ecosystems
400
1000
800
700
600
500
400
300
200
150
100
50
200
25
600
Figure 5. Range of phytoplankton average biovolume (m 3 ) with the increasing of the number of cell
measured. The bars represented the minimum and maximum, the box represented the 25 and 75 percentile
and the average biovolume values.
109
TWB 7 (2013), n. 2
References
Banse K, Mosher S 1980. Adult body mass and
annual production/biomass relationships in field
populations. Ecology Monograph 50: 355379.
Basset A, Sangiorgio F, Pinna M 2004. Monitoring
with benthic macroinvertebrates: advantages
and disadvantages of body size descriptors.
110
TWB 7 (2013), n. 2
Cell biovolume and surface area in phytoplankton of Mediterranean transitional water ecosystems
111
TWB 7 (2013), n. 2
112
TWB 7 (2013), n. 2
Cell biovolume and surface area in phytoplankton of Mediterranean transitional water ecosystems
113
TWB 7 (2013), n. 2
Appendix 1 - Schedule for geometric shapes showing: mathematical equation, number and types
of linear dimensions to measure, genera to which the shape is applied, the difference with respect
to other sets of geometric shapes published and notes for species deviation.
Geometric shape and formulas
1) Sphere
V = /6 d3
A = d2
2) Prolate spheroid
V = /6 d2 h
A=
h2
2
h d
sin 1
h2 d 2
h
The shape is also used in other sets of geometric forms, in particular the set proposed by Hillebrand et al., 1999, except for the
genus Pediastrum. For this genus, Hillebrand proposed an elliptic prism applied to the whole colony. Previously Edler (1979) had
proposed a cylindrical form, also applied to the whole colony. We propose a prolate spheroid form applied to single cells. In this
way, we overcome the problems related to the estimate of the third dimension required in both previous formulas; the width of
each cell can be approximated to its thickness and to the thickness of the whole colony. This also resolves the problems of volume
overestimation of the colony, which both previous sets had put at roughly twice the actual volume.
d
h
3) Cylinder
V = /4 d2 h
A = d (d/2 +h)
This model is easy to apply and is generally calculated automatically by most image analysis software.
114
TWB 7 (2013), n. 2
Cell biovolume and surface area in phytoplankton of Mediterranean transitional water ecosystems
Appendix 1 - Continued.
sin1
2
2
2h2
(1) Ellipsoid -10%, (2) A. sphenoides: prolate spheroid
4 h 2 a c
4) Ellipsoid
V = /6 a c h
For some genera, there are some differences with respect to the geometric forms used in other sets. In particular, for Cymbella and
Amphora the more recent papers have proposed a cymbelloid form, but some linear dimensions are very difficult to measure and are
not practicable in routine analysis (two measures are in the transapical section of the cell). We argue that less bias results from using
the more simple form of ellipsoid, although it can still overestimate biovolume by 35%.
5) Cone
V = /12 d2 z
A=
d d
l
2 2
d
Number and type of linear dimension to measure 2: d =
diameter; h = height ; for area l = side of cone
d1
l
6) Truncated cone
V= (
z
d2
12
A=
2
1
2
2
d 22 d12 2 ld 2 d1
a
b
ZYG: Cosmarium
h d d1 d2 d ) 2
This form was used just for one genus and was applied following the
most similar geometric form in agreement with the most recent sets.
Some species can be considered as prolate spheroid
BAC: Asterionella, Bacillaria, Cymatopleura, Hantzschia, Pinnularia,
Rhabdonema, Synedra(1), Tabellaria (2), Thalassionema,
Thalassiothrix CHL: Tetraedon
Notes:
(1) Species with elliptic valves should be calculated as prism on elliptic
base
(2) Transapical axis (width) is measured as the mean of the minimum
and the maximum
115
TWB 7 (2013), n. 2
Appendix 1 - Continued.
A=
a b a b c
a b
c
4
a
c
Notes:
(1) Sigmoid or rhombic species can be calculated as prisms
on a parallelogram base, elliptic species as elliptic prisms,
and linear species as boxes. (2) Species with linear valves
should be calculated as box
This form was introduced for the first time in Hillebrands paper. However, the genus Nitzschia includes species of different
form. In this case, the most similar geometric forms should be used .
10) Cube
V = a3
A = 6a2
Notes:
(1) C.quadrata = sphere x 4;
This model was applied to the same genera proposed in other sets of geometric forms.
116
TWB 7 (2013), n. 2
Cell biovolume and surface area in phytoplankton of Mediterranean transitional water ecosystems
Appendix 1 - Continued.
1
l mh
2
A l m 3 h l )
Eunotia, Phaeodactylum
A = /4 (ab + ac + bc) + ac
a
b
b1
V= [(b 1 +b b 1 b) a]/3
117
TWB 7 (2013), n. 2
Appendix 1 - Continued.
Geometric shape and formulas
It is a combined solid of rotation, very easy to apply. This follows Suns set of geometric forms. For Group DIN (2), this form
was applied to the Protoperdinium. The Protoperidium genus (2) is highly variable in shape and for some species different
geometric models can be required.
ZYG: Staurastrum
V = /6 h (d 1
A=
2 +d
d2+ d2
2)
d 22 d12 2l d 2 d1
d2
d1
BAC: Cylindrotheca
h2
d2
h1
d1
d 1=d 2
118
TWB 7 (2013), n. 2
Cell biovolume and surface area in phytoplankton of Mediterranean transitional water ecosystems
Appendix 1 - Continued.
Geometric shape and formulas
DIN: Katodinium,
EUG: Eutreptia,
Eutreptiella
A=
d l 2h
2
d h
l
A=
d2
d l d
z
d
This form was used for phytoflagellate and Dinophyceae genera. It was applied in accordance with other sets of geometric forms
analyzed in this work and was also used for some Dinophyceae species that differ from the usual shape of their genera. It is a
combined solid of rotation, very easy to apply.
c
z
Number and type of linear dimensions to measure
4: a = length; c = width; h = height; z = height of cone
a
h
This shape was applied in accordance with Hillenbrand et al 1999, but only to the Euglena genus.
The euglenoid algae are variable in shape and cross section. Most euglena species are not round, but flattened in cross section.
Therefore, the obtuse pole is calculated as a half ellipsoid and the acute pole as a cone with an elliptic base. Edler,1979 and
Sicko-Goad at al., 1977 propose a similar shape with a cylinder instead of a cone. Finally, Sun and Liu (2003) proposed the
cylinder+half sphere+cone, but we do not agree with this geometric shape because it often does not coincide with the real shape of
the cell.
119
TWB 7 (2013), n. 2
Appendix 1 - Continued.
a2
b2
A = 2a1 b1 2c a1 b1 2 a 2 b2 a2 b2 c
a1 b1
b1
c
BAC: Climacosphaenia
It was in accordance with Hillebrand et
al.s (Hillebrand et al., 1999) set of
geometric shapes
BAC: Corethron,
120
TWB 7 (2013), n. 2
Cell biovolume and surface area in phytoplankton of Mediterranean transitional water ecosystems
Appendix 2 - List of genera showing the counting units (C.U.) for the application of geometric
models. The table is sorted according to taxonomic group and lists the genera alphabetically. The
list was based on the analysis of 869 species and was drawn up on the basis of the floristic list
published in Caroppo and Cardellicchio 1995,; Caroppo, 2000, Gilabert 2001 Facca et al., 2002,
Perez et al., 2002, Nuccio et al., 2003, Vadrucci et al., 2004; Hendwood, pers comm. and for data
collected during the TW Reference NET project. Shape code are referred to Appendix 1.
genus
shape
Bacillariophyceae
Achnanthes
Actinoptychus
Amphiprora
Amphora
Asterionella
Asterolampra
Asteromphalus
Asterionellopsis
Bacillaria
Bacteriastrum
Bellarochea
Berkeleya
Biddulphia
Campylodiscus
Cerataulina
Chaetoceros
Climacosphenia
Cocconeis
Corethron
Coscinodiscus
Coscinosira
Cyclotella
Cylindrotheca
Cymatopleura
Cymbella
Dactylosolen
Detonula
Diatoma
Dimerogramma
Diploneis
Ditylum
Ellerbeckia
Epithemia
Eucampia
Eunotia
Fragilaria
Fragilariopsis
Gomphonema
Grammatophora
Hantzschia
Guinardia
Gyrosigma
Haslea
Hemiaulus
Hyalodiscus
Lauderia
8
3
8
3
7
3
3
11
7
4
11
8
8
8
3
8
20
3
21
3
3
3
16
7
4
3
3
8
8
8
11
3
12
8
12
8
8
13
8
7
3
9
8
3
1
3
C.U.
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
genus
shape
C.U.
Leptocylindrus
Licmophora
Lioloma
Lyrella
Mastogloia
Melosira
Navicula
Nitzschia
Paralia
Phaeodactylum
Pinnularia
Planktoniella
Pleurosigma
Porosira
Proboscia
Psammodictyon
Pseudonitzschia
Pseudosolenia
Rhabdonema
Rhaphoneis
Rhizosolenia
Skeletonema
Stauroneis
Stephanodiscus
Stictocyclus
Striatella
Surirella
Synedra
Tabellaria
Thalassionema
Thalassiosira
Thalassiothrix
Toxarium
Trachyneis
Triceratium
Tropidoneis
3
13
3
8
8
3
8
9
3
12
7
4
9
3
3
9
9
9
7
9
3
3
8
3
3
8
4
7
7
7
3
7
3
8
11
8
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
colony
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
121
TWB 7 (2013), n. 2
Appendix 2 - Continued.
genus
Chlorophyceae
Actinastrum
Ankistrodesmus
Carteria
Chlamydomonas
Chlorogonium
Coelastrum
Crucigenia
Crucigeniella
Desmodesmus
Gonium
Kirchneriella
Micractinium
Monoraphidium
Pediastrum
Planktonema
Scenedesmus
Schroederia
Tetraedon
Tetrapedia
Tetrastrum
Treubaria
Chrysophyceae
Calycomonas
Chrysococcus
Dinobryon
Mallonomas
Monochrysis
Ochromonas
Paulinella
Spumella
Cryptophyceae
Leucocryptos
Chroomonas
Cryptomonas
Hillea
Plagioselmis
Rhinomonas
Rhodomonas
Dinophyceae
Akashiwo
Alexandrium
Amphidinium
Amphisolenia
Blastodinium
Ceratium
shape
C.U.
14
14
1
1
14
1
10
2
2
2
14
1
14
8
3
2
14
7
1
1
5
single cell
single cell
single cell
single cell
single cell
single cell or colony
single cell
single cell
5
2
2
2
2
18
5
18
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
18
18
2
2
18
18
18
single cell
single cell
single cell
single cell
single cell
single cell
single cell
4
4
4
3
4
22
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
colony
single cell
single cell
single cell
single cell
single cell
single cell
single cell
genus
shape
C.U.
Cochlodinium
Dinophysis
Diplopelta
Diplopsalis
Glenodinium
Gonyaulax
Gymnodinium
Gyrodinium
Goniodoma
Heterocapsa
Heterodinium
Katodinium
Lingulodinium
Mesoporos
Minuscula
Nematodinium
Oblea
Oxytoxum
Oxyphysis
Oxyrrhis
Peridinium
Phalacroma
Pheopolykrikos
Podolampas
Polykrikos
Pronoctiluca
Prorocentrum
Protoceratium
Protoperidinium
Ptychodiscus
Pyrocystis
Pyrophacus
Scrippsiella
Torodinium
Warnowia
Dictyochophyceae
Apedinella
Dictyocha
Parapedinella
Pseudopedinella
2
4
4
18
3
14
4
4
1
14
14
18
4
8
18
4
1
2
14
2
4
4
4
4
4
18
4
1
14
4
2
4
4
2
2
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
2
1
1
6
single cell
single cell
single cell
single cell
122
TWB 7 (2013), n. 2
Cell biovolume and surface area in phytoplankton of Mediterranean transitional water ecosystems
Appendix 2 - Continued.
genus
Cyanophyceae
Anabaenopsis
Anabaena
Aphanizomenon
Coelosphaerium
Chroococcus
Coccoid cyanobacteria
Gomphosphaeria
Gloeocapsa
Filamentous cyanobacteria
Lyngbya
Merismopedia
Microcystis
Nodularia
Nostoc
Oscillatoria
Phormidium
Snowella
Spirulina
Synechococcus
Trochiscia
Woronichinia
Euglenophyceae
Astasia
Euglena
Eutreptia
Eutreptiella
Lepocinclis
Phacus
Strombomonas
Trachelomonas
Prasinophyceae
Aulacomonas
Halosphaera
Mamiella
Micromonas
Pachysphaera
Pterosperma
Pyramimonas
Tetraselmis
shape
C.U.
3
3
3
2
1
1
1
1
3
3
10
1
3
3
3
3
2
N of 100 m filaments
N of 100 m filaments
N of 100 m filaments
colony
single cell
single cell
single cell
single or colony
N of 100 m filaments
N of 100 m filaments
single cell
part of colony
N of 100 m filaments
single
N of 100 m filaments
N of 100 m filaments
colony
3
1
1
1
N of 100 m filaments
single cell
single cell
colony
4
19
17
17
14
8
4
4
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
2
1
2
2
2
1
5
2
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
genus
shape
C.U.
Prymnesiophyceae
Acanthoica
Anoplosolenia
Calciopappus
Calciosolenia
Chrysochromulina
Coccolithus
Coronosphaera
Emiliania
Gephyrocapsa
Halopappus
Lohmannosphaera
Ophiaster
Phaeocystis
Pontosphaera
Prymnesium
Rhabdosphaera
Syracosphaera
2
14
5
4
2
1
1
1
1
2
1
2
19
1
18
1
1
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
single cell
3
2
3
single cell
single cell
single cell
1
1
single cell
single cell
8
3
3
15
single cell
single cell
single cell
single cell
3
1
1
1
single cell
single cell
single cell
single cell
Trebouxiophyceae
Lagerheimia
Oocystis
Stichococcus
Xanthophyceae
Meringosphaera
Rhizochloris
Zygnematophyceae
Closterium
Cosmarium
Mougeotia
Staurastrum
others classes
Bicosta
Ebria
Hermesinum
Pseudobodo
123