Proc. 15th INA Conf., Bohol, Philippines
J. Nannoplankton Res. 36 (2), 2016, pp.139–147 © 2016 International Nannoplankton Association
ISSN 1210-8049 Printed by The Sheridan Press, USA

139

A semi-automatic two-dimensional image system forstudying the skeletal design of the silicoflagellate genus
Corbisema (Dictyochales, Dictyochophyceae)
Hideto Tsutsui* and Richard W. Jordan
Department of Earth and Environmental Sciences, Faculty of Science, Yamagata University, 1-4-12 Kojirakawa-machi, Yamagata 990-8560,
Japan; *blacksand@mail.goo.ne.jp
Manuscript received 6th November, 2015; revised manuscript accepted 25th July, 2016.

Abstract Variations in skeletal morphology can provide valuable information on silicoflagellate classificationand evolution, as well as the ontogenetic process of skeleton formation; however, measuring hundreds of individual
skeletons can be time-consuming, and there are few, if any, existing morphometric programs that can be used to study
the triangular skeletons of Corbisema. Thus, a morphometric program previously designed to measure Stephanocha(= Distephanus) spp. was modified to investigate Corbisema spp., using C. apiculata from Mors, Denmark (early
Eocene) as a test case. Although the obtained sample set contains specimens with and without pikes, as well as
differences in the pike-strut distance, some basic statements can be made: (1) in general, the basal ring is an isosceles
triangle with two equal sides and one slightly shorter side, and (2) radial spine length and basal side length both
exhibit a unimodal distribution. The distance between the pike and strut ranges from 0 to 5µm, with a mean distance/skeleton of 0–1µm representing 77% of the total specimens, and that of >2µm representing 3%. This may be related
to their double skeleton configuration, with specimens lacking pikes or with large distances between pike and strut
possibly associated with a ‘corner-to-corner’ configuration, while those with pikes close to the struts forming ‘Starof-David’ configurations. The taxonomic implications of this result are discussed in relation to the recent literature.

Keywords computer program, Corbisema apiculata, Eocene, morphometry, Mors
1. Introduction
1.1 Morphometric studies for plankton
and microfossils
In the distant past, measurements of plankton or microfossils were done manually using the microscope graticule or from printed images – a time-consuming process
which often deterred the measurement of large numbersof individuals. Later, morphometric procedures like eigen­shape analysis (Lohmann, 1983) and automated ­image
capture (Young et al., 1996) became useful tools for the
microscopist. More recently, over the last decade or so,a number of sophisticated automatic recognition systems
like ­SYRACO (Dollfus & Beaufort, 1999; Beaufort &Dollfus, 2004), COGNIS (Bollmann et al., 2004) and­
AMOR (Knappertsbusch & Schneider, 2009) have beendeveloped which can analyze specimens on microscopeslides, speeding up measurements and improving accuracy and reproducibility.
Morphometric techniques using foraminifers, radiolarians, coccolithophores, diatoms, dinoflagellate cysts andpollen have already been discussed in numerous papers(e.g., Pappas & Stoermer, 1995; Cortese & Bjørklund,1997; Lindbladh et al., 2002; Cortese & Gersonde, 2007;
Mertens et al., 2009; Veselá et al., 2009; Aurahs et al.,2011; Cubillos et al., 2012; Schneider, et al., 2012; Siveret al., 2013; Rasband, 1997–2013). However, some disadvantages still exist, e.g., system, hardware or softwaredevelopment cost and re-measurement labor.

Some workers have used mathematical models torepresent silicoflagellate skeletal designs, noting that
more complex designs (e.g., hexagons and heptagons)
inhabit higher latitudes and thus cooler temperatures(McCartney & Loper, 1989; 1992). Most Corbisema spp.
have a triangular outline, but some species like C. apiculata have curved basal sides that could be viewed as approximating a circle, which minimizes the apical area
(McCartney & Loper, 1992). Some silicoflagellate morphometric studies, for example, noted differences in cellvolume using a three-dimensional approach (McCartney,
1988; McCartney & Loper, 1989; 1992), while others
demonstrated morphologic diversity and a relationshipbetween radial spine length and environmental conditions based on the skeletal form as a two-dimensional,
computerized wire-frame model (Tsutsui et al., 2009;
Tsutsui & Takahashi, 2009). These latter studies useda landmark-based geometric morphometrics program(Tsutsui, 2000) to calculate element lengths and angles
at designated landmarks (i.e., with coordinates) present
on all of the selected specimens.
1.2 Silicoflagellate taxonomy
Silicoflagellates are unicellular marine phytoplankton
belonging to the Class Dictyochophyceae (see recent review by Jordan et al., submitted) and form a monophyleticclade within the class according to recent molecular phylogeny data (Chang et al., 2012). However, there is still

140

Tsutsui and Jordan

controversy about how many extant genera and species exist; many biologists believe that all extant species belong
to only one genus (Dictyocha; e.g., Moestrup & Thomsen,
1990; Chang, 2015), whilst most micropaleontologists
think that the evolutionary trends among fossil taxa support the need for three extant silicoflagellate genera (Dictyocha, Stephanocha and Octactis; e.g., McCartney et al.,2014a; 2014b – the illegitimate genus Distephanus wasrecently replaced by the new genus Stephanocha; Jordan& McCartney, 2015). The classification of fossil species ismore complicated, since generic concepts tend to be basedon small morphological differences, and the full range ofskeletal plasticity (including aberrants) within a single
taxon is difficult to document. In general, Cenozoic ­genera
can be separated by shape; two-sided (­Naviculopsis),triangular (Corbisema), quadrangular (Dictyocha and­Distephanopsis), hexagonal (Stephanocha), octagonal(Octactis) or polygonal (Bachmannocena and C­ aryocha);however, within the same genus the number of basal sides
and basal spines can, albeit more rarely, be higher orlower than generally found – thus, five-sided Dictyocha orseven-­sided Stephanocha skeletons may be encountered.
When skeleton-bearing silicoflagellates undergo cell
division a second, sometimes thinner skeleton (daughter skeleton) is laid down opposite the original skeleton(mother skeleton) (Marshall, 1934), or the mother skeleton is discarded and two identical skeletons are produced(Chang, 2015). Normally, these skeletons are identical,
not mirror-images of each other (cf. Boney, 1976), so thatthe pikes of the paired skeletons interlock on opposite
sides of the strut, or in taxa where pikes are absent, organic
material holds the two skeletons together (McCartney
et al., 2014b). Modern silicoflagellate double skeletonsalign with basal rings and basal spines closely abutting,in the so-called ‘corner-to-corner’ configuration, while
many Late Cretaceous and Paleogene silicoflagellates
employed a different strategy, with one skeleton rotated
180° in a ‘Star-of-David’ configuration (e.g., McCartney
et al., 2010; Dumitrica, 2014). So far, all known examplesof ‘Star-of-David’ configurations involve skeletons withpikes (e.g., Corbisema and Dictyocha – see McCartney
et al., 2014a, 2014b). Pike positions on single skeletons
can be used to determine which configuration a paired
skeleton would have adopted, with pikes close to the strut
associated with the ‘Star-of-David’ configuration, andpikes offset from the strut associated with the ‘corner-­tocorner’ configuration.
1.3 Aims
In this paper we use a modified version of a semi-­automatedmorphometric program developed for six-sided Stephanocha skeletons to calculate element lengths and angles ofthree-sided Corbisema skeletons, using C. apiculata in asample from Mors, Denmark as a test case. It is hoped that
this modification will allow us in the future to solve taxonomic problems in Corbisema.

2. Materials and methods
2.1 Geological setting
The Fur Formation on Mors, Denmark (Figure 1) has
been studied by scientists for hundreds of years, mainly
because of the wealth of marine fossils that havebeen found there (e.g., Nielsen, 1959; Collins et al.,2005), but also for the laminated diatomites, which
are well-preserved due to the anoxic conditions thatprevailed during their deposition (Pedersen, 1981).
These diatomite layers, intercalated with ash deposits(­Pedersen & Surlyk, 1983), are thought to be early Eocene in age, based on various microfossil groups (Perch-­Nielsen, 1976; Homann, 1991; Fenner, 1994; Jørgensen
et al., 2005) and volcanic ash radiometric ages of 54.52+/– 0.05Ma and 54.04 +/– 0.14Ma (Chambers et al.,2003). Sedimentological studies also suggested an upper Paleocene-Eocene age for the Fur Formation, withthe depositional paleoenvironment interpreted as being
marine with some brackish influence well below the
wave base (Heilmann-Clausen et al., 1985). The present study adopted an early Eocene depositional age of
the Fur Formation as suggested previously by Collins
et al. (2005) and Jørgensen et al. (2005).

8.15
57.15°N

15°W

8.30

8.45

9°E

0°

15°E

North Sea

55°N
50
57

45
ThistedBredning

N

Mors

56.45

Limfjorden

56.30

Denmark

Figure 1: Map of sample location. The Corbisema specimens usedin this study were in an outcrop sample collected from Mors Island,
Denmark. The sample accession number is E1758 and the sample
is curated in the Friedrich Hustedt Diatom Study Center at the AlfredWegener-­Institute, Helmholtz-Zentrum für Polar- und Meeresforschung(AWI), Bremerhaven, Germany. The map was produced using the mapping software of ­GEOMAR, Collaborative Research Center (SFB) 574.
http://www.sfb574.geomar.de/gmt-maps/html

A semi-automatic two-dimensional image system for… Corbisema (Dictyochales, Dictyochophyceae)

2.2 Silicoflagellate analysis
Silicoflagellates, especially Corbisema, are frequent in
the Fur Formation, so they make ideal subjects for morphometric analysis. Measurements were carried out on
the light microscope because this was the method chosen
for previous silicoflagellate morphometric studies andobservations can be made quickly. The preparation of
permanent mounts follows the method outlined in Shiono& Jordan (1996), with Mountmedia® (Wako Pure Chemical Industries, Ltd.) as the mounting medium (refractive
index = >1.50). Specimens were observed using x10,
x20 or x40 objective lenses on an Olympus® BH2 andphotographed with an AR-D300C digital camera (ARMS
System, Co., LTD) or on a BX40 with an NY-1S relay
lens (Microscope Network Co., Ltd) and EOS Kiss® X6i
camera (Canon, Inc.).
2.3 Morphometric sample selection
The morphometric program requires that the main skeletal parts are not broken; however, the radial spine tip ofCorbisema, in particular, is very fragile. Before carryingout measurements, the following four conditions must be
satisfied: (1) specimens must be photographed in apicalor abapical view; (2) specimens must not be tilted, so allradial spine tips or all basal sides should be in the same
focal plane; (3) specimens should be ‘normal’, without theloss or addition of some skeletal parts or obvious deformities (e.g., Loeblich et al., 1968; Malinverno, 2010); and(4) specimens must not be partially obscured by debris,making measurement of some skeletal parts impossible.
The common forms of C. apiculata found in this study are
shown in Figure 2a–c, with the parameters to be measured
shown in Figure 2d.
The morphometric results and statistics should be supported by a large number of specimens. Fortunately, theMors sample satisfied the following conditions: (1) goodpreservation; (2) abundant silicoflagellates, especially ofCorbisema; and (3) presence of both ‘Star-of-David’ and‘corner-to-corner’ skeletal configurations of Corbisema,as already described by McCartney et al. (2014a; 2014b;
2015b).
2.4 Measuring program and procedure
The measuring program language and source code run on
the freeware interpreter 99BASIC Interpreter Version 1.19
(Iida, 2002), which itself runs on Microsoft® Windows®.
The merit of this program is that the main source code
can be easily debugged, and the system can be downsized
relatively quickly. The primary algorithms, data architectures and program routines are based on the morphometric system of Stephanocha speculum (Tsutsui, 2000). The
measuring system of this study requires the following two
manual input steps: (1) the 8-bit bitmap load on this program, and (2) skeletal end-points, junctions and scale bar
length. The basic concept of this morphometric system isbased on Corbisema specimens with linear basal sides;

(a)

(b)

(c)

(d)

141

S1

radialspine
strut(=lateral rod) B1 basal
side
R1
P3
P1 pike
R3
A
B3
B2
P2 R2
S3

S2

20 µm
Figure 2: Two skeletal forms of Corbisema apiculata and measurement
strategy: (a) pike-bearing form in apical view; (b) pike-bearing formin tilted lateral view. The arrows point to pikes; (c) pike-less form; and(d) idealized skeleton showing parameters to be measured

however, in reality, those with bulging basal sides canalso be measured by applying the Bézier curve system.
The Bézier curve is useful for drawing a smooth curve on
computer graphics, because it can be expressed with one
control point. The Bézier curve calculation method usedin our program is based on Forrest (1972). An exampleof this calculation can be seen in Figure 3, with computer
screen shots after loading the microphotograph bitmap(Figure 3a) and of the subsequent measurement result­after bitmap loading (Figure 3b).
The calculation time of length, pike distance and angles is instantaneous. The main advantages of this morphometric system are threefold: (1) Re-measurement
work is very easy because this program can recalculate
the measurement data from the coordinate data, (2) the
measurement program can quickly process large amountsof photographic data, and (3) the labor time needed formeasuring is reduced.
Another advantage of this program is that it can automatically identify skeletal parts, which can be numbered
in a clockwise direction; for instance, in the case of Corbisema, the three spines are numbered sequentially, withspines at twelve o’clock (= 1), four o’clock (=2), and eighto’clock (=3). Other skeletal parts (struts, pikes etc) are
numbered in the same way.
The actual morphometric steps are as follows: First,
the photomicrograph is converted from an image file(such as JPEG or TIFF) to an 8-bit bitmap image, whichis reduced in size to 800 × 800 pixels. The program thencreates two data files: (1) the X/Y coordinate data on

565 346

0.1, 0.1 A NO.1-161 SHORT-SP PIKE-ON L3

0.1, 0.1 A NO.1-166 SHORT-SP PIKE-ON L3

MORLTM 1-161 1,1

MORLTM 1-166 1,1

ID area381 369 245 279 388 206 231 271 285 380 258

542 568 345 399 579 296 326 426 416
Coordinate area

697 564 113

434 394 129
398 295 100

561 454 104
775 592 74

492 442 95
295 70451 69272 10432 100.1, 0.1 A NO.1-160 SHORT-SP PIKE-ON L3
MORLTM 1-160 1,1

247 272 141

0.1, 0.1 A NO.1-154 PIKE-ON SHORT-SP L3
MORLTM 1-159 1,1

358 432 415

320 308 430 368 540 318
430 518508 537 318
670 538 160
558 407 113
598 60
740
40
410
10
274

Basal corner 3 (X and Y)
Basal corner 2 (X and Y)
Basal corner 1 (X and Y)
Spine 3 (X and Y)
Spine 2 (X and Y)
Spine 1 (X and Y)
Scale bar length (10µm)
Scale bar end (X and Y)
Scale bar start (X and Y)

85

87

Basal-strut junction 1 (X and Y)
Microscope machine type (L3: Olympus BX40)
Lens type (A: x10 and x5 zoom lens)Comments
Observation code and pike on-off(1: Normal, 1: Pike-on)
Microscope stage position (X and Y)
Slide ID and serial number
Sample logistics (MOR: Mors) and Microscope type
(LTM: Light microscope)

Table 1: Coordinate data file structure

206 274402 264
380 385 246 286 403 205 242 287 314
470 397 114
406 290 105
529 430 60

Basal-strut junction 2 (X and Y)

the computer screen is saved as ‘silicoad.ctl’ (Table 1),and (2) the morphometric data output as ‘silicolw.txt’(Table 2). Two data files of fixed length are outputted in
ASCII code. The left portion of Table 1 shows the Sample ID which consists of sample origin, microscope type,slide number, serial photograph number, objective lens,
location of sample on the slide and comments. The X/Ycoordinate data in Table 1 are listed in clockwise order
after the sample ID: radial spines numbered 1 to 3, basal
ring corner, strut position, pike position, apex point andcurved basal side. The morphometric data in Table 2
consists of the X/Y coordinate of the scale bar, scale bar
length, skeletal element length, angles and curved basalside length. A zero means the value is outside the limits ofmeasuring. These files are kept in plain ASCII Character–­
Separated Values (CSV) format, which is fully compatible with other software (e.g., SAS® by SAS Institute Inc.,
SPSS® by IBM SPSS Statistics® or Microsoft® Excel®).
Consequently, the rewrite and overwrite of file contents
can be easily carried out. The statistical data (kurtosis,

286 45

Basal-strut junction 3 (X and Y)

Figure 3: Computer screenshots of coordinate input situation and result
output: (a) Screen shot after bitmap loading, and (b) after the completed
procedure. The cross line in 3a is the target for manual input. In 3b thevertical output on the left is the coordinate data, with length, angle,
curved length and scale length shown on the right222 10

Apical junction (X and Y)(b)

222 142

Basal pike 1 (X and Y)(a)(omit)

Tsutsui and Jordan(omit)

142

Re-measuring

START

2nd START

122

Yes

Load
bmp fileLoadsilicoad.ctl
1'

Input
coordinates

Calculation Angle and length
angle, length calculate different
combination(Linear mode: spine tips, basal
corners, struts and apical junction)

Morphometric result area

118

Input
coordinates

Display
result(Scale bar and length)

Add curve?

No

Save result?

Yes

Yes

Input
Bezier Curve(Basal ring only)Savesilicoad.ctl

No

Save result?

No

Yes

Save
silicolw.txt

Save anothersilicolw.txt

END

2nd END

1

Figure 4: Morphometric program flow chart. The measurement data canbe outputted from the program, and re-measurement is possible by reusing the coordinate data fileskewness, S.D. etc) was computed with SAS® Release
9.4 TS1M2 (SAS Institute, Inc., licensed by Yamagata
University). A general flow chart showing all steps in themorphometric analysis is shown in Figure 4.

7
ID area

125
114

1
0
49 55 52 14 15 14 25 28 26 25 28 26 1

157 162 153 65 59 54 65 59 54 86 87 90 118

3
2
57 53 57 18 18 14 28 27 29 31 28 28 3
10 5

Normal morphometry

143

Start
No
morphometry?

40.1, 0.1 A NO.1-166 SHORT-SP PIKE-ON L3 106 10 3
MORLTM 1-166 1,1

9
7

13 6
0.1, 0.1 A NO.1-161 SHORT-SP PIKE-ON L3 57
MORLTM 1-161 1,1

154 142 147 55 62 61 55 62 61 88 91 84 119

125 (omit)
116
145 142 148 63 54 61 63 54 61 83 92 85 117
00
12 10 20 20 21 20 20 21 0
10 6
0.1, 0.1 A NO.1-160 SHORT-SP PIKE-ON L3 119
MORLTM 1-160 1,141 42 39 962
10 10 11 10 60 62 57 20 15 19 29 32 29 34 32 28 3
0.1, 0.1 A NO.1-159 PIKE-ON SHORT-SP L3 57
MORLTM 1-159 1,1

3
2
1 Apical junction outer angle (deg)32
1 Apical junction outer angle (µm)
32
1 Basal-strut angle (deg)32
1 Basal corner angle (deg)32
1 Spine-basal angle (deg)3
2
1 Pike length (µm)
32
1 Basal corner-pike junction (µm)
32
1 Basal ring corner-strut junction (µm)
321 Strut length (µm)
32
1 Basal side length, linear mode (µm)
32
1 Spine length
Scale length (µm)
Scale bar length (dots)
Microscope machine type (L3: Olympus BX40)
Lens type (A: x10 and x5 zoom lens)Comments
Observation code and pike on-off (1: Normal, 1:
Pike-on)Microscope stage position (X and Y)
Slide ID and serial number
Sample logistics (MOR: Mors) and Microscope type
(LTM: Light microscope)

Table 2: Measurement data file structure

152 153 148 64 55 60 64 55 60 95 96 80 100 122 136(omit)A semi-automatic two-dimensional image system for… Corbisema (Dictyochales, Dictyochophyceae)

3. Results
The morphometric program size is 51Kbytes with noprogram library or plug-ins. A total of 154 Corbisema
specimens were measured, with whorl coordinate andmeasurement data file sizes of 52Kbytes and 47Kbytes,
respectively. A summary of the morphometric (lengths
based on linear and Bézier curves) and statistical (kurtosis, skewness, standard deviation, mean and minimum/
maximum) data are given in Table 3. Since all Corbisema skeletons are similar in shape (i.e., triangular)
the data on angles between skeletal elements are notpresented here.
The results of the morphometric analysis are plottedin Figure 5. The radial spine length ranges from 3–17µmwith a mean length of ≈ 8µm, while the basal side length
ranges from 24–79µm with a mean of ≈ 51µm. The struts
range from 6–24µm with a mean length of ≈ 14µm. The
distance from basal ring corner (= one apex of the triangle)
to the strut attachment position on the basal side ranges

6
24
14.4
3
0.11
–0.24

11
36
23.8
4.7
–0.32
–0.44
(N = 181)

0
4
0.7
0.8
1.23
1.72
(N = 154)

the specimens selected randomly for the analysis represent a single taxon; however, these specimens represent a mixture of pike-less and pike-bearing forms, andshowed a significant distance between the strut and thenearest pike (Figure 5c, f ). These differences could beviewed in two different ways with respect to silicoflagellate classification.

Table 3: Primary statistical summary of measurement datafrom 11–36µm. Of all the specimens observed, 154 bore
pikes and 27 lacked pikes. The distance from the strut tothe nearest pike ranges from 0–4µm with a mean of 0.7µm.
4. Discussion
4.1 Morphometric analysis
Like most other Corbisema spp., the skeletons of C. apiculata are isosceles triangles with two equal sides (e.g.,­McCartney et al., 2014a; this study), albeit with curved
basal sides, which means their overall shape is slightly
circular. All the plots of element lengths (Figure 5a–b,d–e) exhibit a unimodal distribution, and thus there is norecognizable difference among the analyzed specimens
based on skeletal dimensions. This could suggest that
200

120

(a)

4.2 Pikes as taxonomically important
structures
Among the three modern silicoflagellate genera, two possess pikes (Stephanocha and Dictyocha) while the other(Octactis) does not. From the literature and from our own
observations, pike-bearing species never lose their pikes
and pike-less species never gain pikes, regardless of thecircumstances (cultures, aberrants, etc.). This strongly
suggests that the presence or absence of pikes is fixed in
the genetic blueprint of each species. Given this situation,
the mixture of pike-less (15%) and pike-bearing (85%)
forms in the early Eocene Mors sample could be interpreted as representing more than one taxon (see Figure 2c
and 2a–b, respectively).
Furthermore, 83% of the Mors specimens bear pikes
located close to the strut, while the remaining 17% of thespecimens have pikes more than 1µm away from the strut(Figure 5f ). Normally, pike position is a good indicatorof double skeleton configuration, with pikes close to the
250

(b)

100

200

80
100

N=543

150

60
40

50

50

20

0

250

2

4

6

0
8 10 12 14 16 18 20 22 20

Length (µm)

200

(d)

200

30

40

50

60

70

Length (µm)

(e)

80

0
90 0

1

2

3

4

150

(f)

125

6

N=154

150
100

N=543
100

50

0

3

6

9

12

15 18

Length (µm)

21 24

0
27 0

4

0
8 12 16 20 24 28 32 36 40

Distance (µm)

µm

2

25

>2

50

2

50

µm

75

100

µm

Individuals

N=543

150

0

5

Distance (µm)

µm

0

N=462

100

1-

Individuals

150

N=543

(c)

01

24
79
50.9
9.5
–0.26
–0.33

Distance from piketo strut

3
17
7.9
2.4
0.45
0.04

Distance from basal
corner to strut

StrutsMin (µm)
Max (µm)
Mean (µm)
Std dev (µm)
Skewness
KurtosisBasal sides

Tsutsui and Jordan

Radial spines

144

Category

Figure 5: Histograms showing the primary measurement summary of the (a) radial spine length, (b) basal side length, (c) the distance from pike tostrut, (d) strut length, (e) the distance from basal ring corner to strut, and (f) the pike-strut distance assigned to size categories

A semi-automatic two-dimensional image system for… Corbisema (Dictyochales, Dictyochophyceae)

145

two-­dimensional light micrographs of Corbisema apiculata
and is useful for supplementing morphologic descriptions
and for explaining pike position distances more easily.
2. The early Eocene sample from the Fur Formation ofMors, Denmark, was chosen because (1) silicoflagellates
are well preserved, (2) Corbisema apiculata is abundant,and (3) the sample has already yielded double skeletons inboth configurations.
3. The double skeleton configurations of Corbisema,‘Star-of-David’ and ‘corner-to-corner’, are a direct resultof the pike positions on the skeleton pairs. The ‘Starof-­David’ configuration always requires the presence of
pikes, which are positioned close to the base of the strut,
whilst the ‘corner-to-corner’ configuration may be held
4.3 Presence or absence of pikes­together by organic material or pikes positioned away
Silicoflagellates are currently classified using their overall from the strut. In Corbisema apiculata we encounteredskeletal shape (number of basal sides) and the presence/­ pike-less and pike-bearing forms, with the pikes of theabsence of apical ring structures. In the fossil record latter forms positioned either near (i.e., within 0–1µm) or
some genera (Corbisema, Dictyocha) contain both pike- away (>1µm distance) from the strut. This suggests thatless and pike-bearing species. At present, no genus has C. apiculata could produce double skeletons of either conbeen erected solely on the basis of pike position and the figuration, or that C. apiculata is a species complex conpresence/­absence of pikes, and doing so would radically taining more than one taxon.
­alter the current classification scheme. Furthermore, inthis study some individual skeletons of Corbisema apicu- Acknowledgements
lata had variable pike-strut distances, which in a few cases Emeritus Professor Niichi Nishiwaki (Nara University)
were different by as much as 2–3µm, suggesting that the and Emeritus Professor Shiro Nishida (Nara University ofpike-strut distance may not be as significant as discussed Education) provided useful comments and discussions onabove. In this study we averaged the pike-strut distance fossil silicoflagellates and statistics. We are deeply gratefulto reduce the effects of one ‘aberrant’ distance on a sin- to Mr. Yamamoto, veteran technical engineer of Japan Margle skeleton (Figure 5f). This procedure still left about 3% itime Self-Defense Forces, who checked the program tests,of total specimens with a pike-strut distance of 2–5µm, database architectures, and the program for debugging.
which clearly separates them from the majority of spec- We would also like to thank Friedel Hinz of the Friedrichimens which had average pike-strut distances of < 1µm. Hustedt Diatom Study Centre, Alfred Wegener Institute
Whether this is significant enough to justify separation at (AWI) for providing the Mors sample used in this study.
the species/genus level may only be answered following The comments and suggestions of Elisa Malinverno andfurther morphometric studies.
an anonymous reviewer helped to improve the manu­script.
strut associated with the ‘Star-of-David’ configurationand those some distance away from the strut associatedwith the ‘corner-to-corner’ configuration. All modern
silicoflagellates use the ‘corner-to-corner’ configuration,
regardless of whether they possess pikes (Stephanocha
and Dictyocha) or not (Octactis), and never switch tothe ‘Star-of-David’ configuration, suggesting that this is
genetically-controlled. In recent papers McCartney et al.
(2014a, 2014b) proposed that different configurations
represented different lineages, which would, if accepted,
necessitate the formation of new genera; however, theconfiguration of the generitype, C. geometrica, is presently unknown.

4.4 Future studies
Our modified morphometric program showed that theC. apiculata specimens had a unimodal distribution withrespect to basal side and spine lengths, but also showedthat there were differences in the pike-strut distances.
This story is further complicated because C. apiculata andC. triacantha are difficult to distinguish and in the literature their identification is often confused (e.g., see­Loeblich et al., 1968, pl.23, figs 1, 10 and 11, all described
as Dictyocha triacantha or D. triacantha var. apiculata
from the Recent Pacific Ocean). In a second paper we willaddress this problem using the morphometric program­described herein.
5. Summary
1. The main program routines, input methods and database code structures of Tsutsui (2000) were modified inorder to create the 51Kbyte Corbisema morphometricprogram. The morphometric data was calculated from theReferences

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