This study characterized the diversity of paramylon grain morphology across photosynthetic euglenoid species using light and scanning electron microscopy. Six distinct paramylon grain categories were identified: disks/rings, ellipses/links, pyrenoid caps, plates/large rings, bobbins, and rods. Over 1,000 light microscopy images were examined from 11 representative euglenoid species. The goal was to provide a consistent terminology for describing paramylon grains and examine their phylogenetic utility for euglenoid taxonomy.

1. Characterization of paramylon morphological diversity in photosynthetic euglenoids
(Euglenales, Euglenophyta)
ANNA K. MONFILS
1
*, RICHARD E. TRIEMER
2
AND EMILY F. BELLAIRS
1
1
Department of Biology, Central Michigan University, Mt Pleasant, MI 48859, USA
2
Department of Plant Biology, Michigan State University, Lansing, MI 48824, USA
MONFILS A.K., TRIEMER R.E. AND BELLAIRS E.F. 2011. Characterization of paramylon morphological diversity in
photosynthetic euglenoids (Euglenales, Euglenophyta). Phycologia 50: 156–169. DOI: 10.2216/09-112.1
A characteristic feature of euglenoid cells is the presence of a b1–3 glucan storage product called paramylon. The
euglenoid lineage is tremendously diverse, with a great deal of variation in paramylon grain morphology. Number,
shape, location and external morphology of paramylon have been used as diagnostic features for several euglenoid
species. The goals of this study were to examine and characterize the paramylon grains from a variety of different species
in vivo and in vitro, provide a consistent, descriptive terminology that can be used to describe paramylon grain types, and
discuss the potential phylogenetic utility of paramylon grain morphology. Over 1000 light microscopy images were
examined to survey paramylon diversity across the Euglenales, and scanning electron microscopy was used to examine
paramylon in 11 representative species (Colacium vesiculosum, Cryptoglena skujae, Discoplastis spathirhyncha, Euglena
gracilis, Lepocinclis acus, L. ovum, Monomorphina pyrum, Phacus orbicularis, P. pleuronectes, Strombomonas
borystheniensis, and Trachelomonas ellipsoidalis). The various types of paramylon grains were separated into six
distinct morphological categories: disk, ellipse, pyrenoid cap, plate, bobbin or rod, with further distinction for varieties
found within each category. These categories were then applied to the diversity found in the genera and major lineages.
This study was able to determine a high level of distinction among differing paramylon grain morphologies both within
a species and among species, and variation was found in both large and small grains. Paramylon can be used at the
generic level to support major clades and generic relationships and may provide insight into the taxonomic placement of
euglenoids currently unavailable for sequencing.
KEY WORDS: Euglenales, Euglenoid, Euglenophyta, Glucan, Paramylon
INTRODUCTION
Euglenoids are an unusually diverse group of single-celled,
asexual algae. Since the first euglenoid was detected by
Antonie Van Leeuwenhoek in 1674 (presumably Euglena
viridis Ehrenberg), extensive morphological, molecular and
ecological work has been done to discover, describe and
catalogue biodiversity in this distinct lineage. As a result of
this research effort, questions regarding monophyly of
major groups in the photosynthetic euglenoids have largely
been resolved and the major genera within the order
Euglenales are now well supported and accepted as natural
groups (for a discussion of the molecular phylogenetics in
euglenoids see Triemer & Farmer 2007). Clarifications of
generic boundaries have opened additional opportunities to
explore evolutionary trends and diversity among species.
One of the characteristic features of euglenoids is the
presence of membrane bound storage granules, called
paramylon (5 paramylum), in the cytoplasm. Gottlieb
(1850) was the first to isolate the granules and determine
they were carbohydrates. Subsequent researchers charac-
terized the basic structure of the granules to determine that
paramylon is a b1–3 glucan (Kreger & Meeuse 1952; Clarke
& Stone 1960). Paramylon serves as a storage carbohydrate
or energy reserve for euglenoids and is distinct from storage
carbohydrates found in plant groups in that is has a high
level of crystallinity (Marchessault & Deslandes 1979).
Evidence from X-ray analysis of 14 species from the
Euglenales indicates that paramylon from different species
can have variable X-ray diffraction patterns with prelim-
inary (though inconclusive) evidence suggesting two poten-
tial paramylon types (Kreger & Meeuse 1952; Leedale et al.
1965). The structure and chemistry of paramylon has been
well characterized in only a single species, Euglena gracilis
Klebs (Gottlieb 1850; Clarke & Stone 1960; Booy et al.
1981; Kiss et al. 1986, 1987).
Paramylon storage granules show great diversity in
numbers and forms within euglenoids. Paramylon grains
can be small and numerous within the cell or large and few
in number. Often, combinations of large and small grains
are present. The presence of two size classes of paramylon
grains within a single cell has been referred to as
‘dimorphic’ (Milanowski et al. 2001, 2006). In addition to
granules which are free in the cytoplasm, the paramylon
may be found capping the pyrenoids (but external to the
chloroplast).
Historically, large conspicuous grains within the cell have
served as the most reliable diagnostic paramylon feature. In
general, these larger grains tend towards consistent
placement and number, and the shape of the grains depends
on the species of euglenoid in which they are found (Gojdics
1953). Number, shape and external morphology of par-
amylon grains have been widely used as diagnostic
characters among euglenoid species (Klebs 1883; Dangeard
1901; Heidt 1937; Gojdics 1953; Pringsheim 1956; Conforti* Corresponding author (monfi1ak@cmich.edu).
Phycologia (2011) Volume 50 (2), 156–169 Published 3 March 2011
156

2. 1998; Brown et al. 2003; Shin & Triemer 2004; Kosmala et
al. 2007a; Ciugulea et al. 2008).
Research relating to nutrient availability and the
conservation of paramylon shape has been mixed. Gojdics
(1953) noted that general grain shape was persistent and
consistent during nutrient deficient times. Conforti (1998)
examined paramylon morphology relative to organic
enrichment in five species of euglenoids. In enriched media
paramylon grains in four of the five species [Lepocinclis
acus (Mu¨ller) Marin & Melkonian, Euglena spirogyra
Ehrenberg, Phacus curvicauda Swirenko and Monomor-
phina pyrum (Ehrenberg) Marin & Melkonian] increased
only in the size and abundance. There were morphological
changes reported in Phacus tortus (Lemmermann) Skvort-
sov; large bobbins and/or disc-shaped paramylon grains
were both observed, depending on environmental growing
conditions. These studies suggest the need for careful
investigation into paramylon morphology to determine the
variation and potential for plasticity within a species but
overwhelmingly support the consistent nature of large grain
morphology within euglenoids.
While paramylon is often used as a key diagnostic feature
in species identification, comparison among published
descriptions is difficult because terminology for paramylon
grain morphology is not standardized. An example of the
inconsistency in literature is illustrated in references to
grains that form the paramylon centre in Euglena viridis.
Huber-Pestalozzi (1955) describes the paramylon as round-
ish or oblong (‘rundlich oder la¨nglich’). Leedale (1967) is
less descriptive and states, ‘paramylon grains small, some
scattered in the cell and others grouped around the
paramylon centre’. Pringsheim (1956) refers to the par-
amylon grains as ‘lens-shaped’, Gojdics (1953) calls them
‘ovoid to brick-shaped’ and Zakrys´ (1986) uses the term
‘rod-like’. This inconsistency makes paramylon comparison
difficult and descriptions among species unreliable.
The objectives of this study were to (1) examine
paramylon grains from representative species in vivo and
in vitro using scanning electron and light microscopy (SEM
and LM, respectively) and provide a consistent, descriptive
terminology which can be used to describe paramylon grain
types, (2) describe paramylon grain diversity across the
Euglenales and (3) examine paramylon grain diversity in a
systematic context to determine synapomorphies and
phylogenetic utility.
MATERIAL AND METHODS
Specimen examination, verification and selection
After review of paramylon diversity across the Euglenales
using over 1000 LM images, 11 species representative of
paramylon grain diversity and distinct lineages were
selected for examination using SEM [Colacium vesiculosum
Ehrenberg, Cryptoglena skujae Marin & Melkonian, Dis-
coplastis spathirhyncha (Skuja) Triemer, Euglena gracilis,
Lepocinclis acus, L. ovum (Ehrenberg) Minkevich, Mono-
morphina pyrum, Phacus orbicularis Hu¨bner, P. pleuronectes
(Mu¨ller) Dujardin, Strombomonas borystheniensis (Roll)
Popova and Trachelomonas ellipsoidalis Singh; see Table 1
Table1.Culturecollectioninformation,paramylontypesandmeasurementdataofeuglenoidspeciesexaminedusingSEM.1
Species
Paramylongraintype
Diskor
smallring
Ellipseor
smalllink
Pyrenoidcap
(depth/width)
Plateorlargering
Bobbin
Rodor
elongatedlinkFlatCurved
ColaciumvesiculosumCCAP1211-3,3.0mmCandU(1.0–2.5
mm/0.5–2.5mm)
CryptoglenaskujaeSAG1088,4.0mm4.5–13.0mm
DiscoplastisspathirhynchaSAG1224-422.0–6.5mm5.0–11.0mm2.0–3.5mm
Euglenagracilis(strainZ)SAG1224-5/25,6.0mmshallow(,1.0mm/
1.0–3.5mm)
,2.0mm
LepocinclisacusUTEX13161.0–6.0mm2–12.0mm
LepocinclisovumSAG1244-81.0–4.0mm4.0–12.0mmring7.0–13.0mm
MonomorphinapyrumACOI1295,3.0mm,2.0mm5.0–12.0mm
PhacusorbicularisASW080541.0–4.0mm2.0–3.0mm4.0–29.0mm
PhacuspleuronectesSAG1261-3b,2.0mm8.0–11.0mm
StrombomonasborystheniensisNJS10,4.0mmCandU(1.0–4.0mm/
1.8–4.0mm)
TrachelamonasellipsoidalisNJST1,2.5mmC(0.8–1.5mm/1.0–
2.5mm)
1
Dataincludesspeciesname,culturecollectionandaccessionnumberandgraintypes[CoimbraCollectionofAlgae,Coimbra,Portugal(ACOI),Algenkulture-Sammlungander
Universita¨tWien,Vienna,Austria(ASW),NewJerseyIsolateTriemerlab(NJ),SammlungvonAlgenkulturenPflanzenphysiologischesInstitutderUniversita¨tGo¨ttingen,Germany
(SAG)andCultureCenterofAlgae,Austin,TX,USA(UTEX)].Unlessotherwisestated,allmeasurementsrepresentlengthalongthebroadestaxis.
Monfils et al.: Paramylon in photosynthetic euglenoids 157

3. for culture collection information]. All cultures were grown
in modified AF-6 medium (Watanabe & Hiroki 1997),
maintained at 20–22uC with conditions of 14:10 h light:dark
and provided with approximately 30 mmol photon m22
s21
from cool white fluorescent tubes. All taxa were examined
and photo documented, and identities were confirmed with
a Zeiss Axioskop 2 Plus microscope (Carl Zeiss, Inc.
Hallbergmoos, Germany) equipped with differential inter-
ference contrast optics. Light microscopic images were
captured with an AxioCam HRC (Hallbergmoos) photo-
micrographic system.
Paramylon extraction and preparation for SEM
Cells were lysed and paramylon was extracted using 50 ml
of cultured cells in AF-6 medium combined with acetone at
a ratio of 1:2. The solution was sonicated using a Fisher
Scientific Sonic Dismembrator Model 500. The lysate was
centrifuged and supernatant removed. Insoluble material
was washed three times, vacuum filtered onto porous
membranes and desiccated.
Dried specimens were mounted onto aluminium stubs
using carbon conductive tape and sputter-coated with gold
for 180 seconds using a Denton Vacuum Desk II sputter
coater (Moorestown, NJ, USA) or an EMSCOPE SC500
sputter coater (Ashford, Kent, Great Britain). Prepared
samples were examined and images obtained using a JEOL
JSM-840A, JEOL 6300F and/or JEOL 6400V Scanning
Electron Microscope. Prepared stubs were archived in the
CMC herbarium.
RESULTS AND OBSERVATIONS
The various types of paramylon grains were separated into
six distinct morphological categories. SEM micrographs
(Figs 1–12) and representative schematic diagrams (Figs 13–
30) are presented for each paramylon type.
DISKS/RINGS (FIGS 1, 13, 14): Round compressed crystal
structure composed of wedge-shaped segments. Disks and
rings are found in many species free in the cytosol. Upon
digestion of the central region, disks give rise to small rings.
Disks or rings are defined as less than or equal to 4.0 mm at
the widest point.
ELLIPSES/LINKS (FIGS 2, 15, 16): Oblong compressed crystal
structure composed of segments of wedges and rectangular
solids. Ellipses and links are less than twice as long as wide.
In LM, profile views of ellipses can appear as rods (see rod
description below). Ellipses and links are found in many
species free in the cytosol. Upon digestion of the central
region, ellipses give rise to small links. Ellipses and small
links are less than or equal to 7 mm on the longest axis.
PYRENOID CAPS (FIGS 3–5, 17–20): Cup-shaped paramylon
grains covering but external to the pyrenoids of the
chloroplast. The internal depth of the cap varies within
and among species. In LM, caps can appear shallow and
appressed to the pyrenoid or deeply convex with a
narrowed proximal region. When examined in lateral view
using SEM, the paramylon cap can appear shallow with a
slight concave impression and curved exterior (Figs 3, 17),
or more deeply invaginated to form a C-shaped cap with a
striated exterior and flattened sides and top (Figs 4, 18) or a
U-shaped cap with striated exterior and curved surface with
or without a rim (Figs 5, 19, 20). The caps can be up to
4.0 mm deep and range in width from 0.5 to 4.0 mm.
PLATES/LARGE RINGS (FIGS 6–10, 21–26): Large shields with
variable morphology. Often found as the dominant large
grain/s in the cell; considerable variation exists among
species in location and orientation of plates within the cell.
Multiple large plate-shaped grains of variant morphology
can be found within the same cell. Plates can be discoid,
. 4 mm wide, and exhibit characteristic wedge-shaped
segments (Figs 6, 21), fully flattened with compressed coils
(Figs 7, 23), or curved and/or trough-shaped (Figs 8, 9, 24,
25). Plates can appear asymmetrical. As in other paramylon
forms, plates can undergo dissolution from the centre
generating large flat (Fig. 22) or curved (Figs 10, 26) ring-
like structures. The large size of the grains separates plates
from the smaller disks, rings, ellipses and links. Plates can
occur singly or may be multiple in number; they can be up
to 30 mm across the longest axis.
BOBBINS (FIGS 11, 27): Layered and adjoining disks found
singly in the centre of the cell. In LM the bobbin can appear
to be a series of superimposed disks in the centre of the cell.
Bobbin-shaped grains are widest at the proximal and distal
ends and narrow towards the equator. The bobbins
typically have a hollow central core. They vary in size
and can measure from 5 to 25 mm across the broadest face
of the grain. The proximal and distal ends can have
different widths.
RODS/ELONGATED LINKS (FIGS 12, 28–30): Elongated grains
greater than two times as long as wide. Rods can be single
elongated structures (up to 75 mm) or may occur as multiple
shorter grains. In LM, profile views of ellipses and disks can
appear as rods. Rods can be straight (Figs 12, 28) or bent
(Fig. 29). Rods with degraded centres can appear as
‘elongated links’ (Fig. 30). Considerable variation occurs
in rod lengths, length sizes range from 1 to 75 mm. Rods can
be differentiated into ‘short rods’, which measure # 7.0 mm
and ‘long rods’ which are . 7.0 mm.
An examination of images from LM and SEM enabled a
comprehensive overview of paramylon morphology across
the Euglenales. With the use of SEM, paramylon grain
morphology was determined for 11 species included in this
study (for collection information and species level par-
amylon descriptions see Table 1). Multiple forms of par-
amylon are found within a single species for all taxa
examined. Trends in nine genera of the Euglenales are
outlined below.
EUGLENA (FIGS 3, 31–38): A diversity of paramylon types lay
within the genus Euglena. Close examination of SEM
images and light micrographs shows that ellipses and links
and/or short rods and associated elongated links (, 7.0 mm
on longest axis) are present in nearly all species (for
examples see E. gracilis, E. viridis and E. deses Ehrenberg,
Figs 31–35). In species with stellate chloroplasts numerous
small ellipsoid paramylon grains (, 4 mm) accumulate
158 Phycologia, Vol. 50 (2), 2011

4. around a central pyrenoid region forming a ‘paramylon
centre’ (see E. viridis, Fig. 34). When ellipses or short rods
are distributed throughout the cytoplasm and the dominant
form of paramylon in the cell, they tend to be larger and on
the high end of the characterized size limit (closer to 7.0 mm
on longest axis, see E. deses, Fig. 35). In addition to ellipses
and short rods, many Euglena species have chloroplasts
with centrally located pyrenoids that are capped on both
sides with shallow cup-shaped paramylon grains (5
diplopyrenoids, see E. gracilis Figs. 3, 32, 33). Some
members of the genus have distinct paramylon types, which
can be diagnostic for the species. Euglena ehrenbergii Klebs
has long bent or strait rods (up to 75 mm; Fig. 36) and E.
convoluta Korsikov has multiple curved trough-shaped
grains often with an opening in the centre (Figs 37, 38).
TRACHELOMONAS (FIGS 4, 39–42): Grain morphology within
Trachelomonas is much more consistent than that found in
Euglena. Ellipse grains and links are the dominant
paramylon grain type and are distributed throughout the
cytoplasm and abundant in all members of the genus. When
pyrenoids are present, they are covered by paramylon caps.
In Trachelomonas, cup-shaped paramylon caps can be
found on a single side of the pyrenoid (5 haplopyrenoid;
Figs 1–12. SEM micrographs of paramylon grain types in the Euglenales. All scale bars 5 2 mm. Culture collection information available in
Table 1.
Fig. 1. Small disk paramylon grains in Colacium vesiculosum (SEM stub CMC1).
Fig. 2. Ellipsoid paramylon grain in Lepocinclis acus (SEM stub CMC5).
Fig. 3. Shallow curved pyrenoid cap in Euglena gracilis strain Z (SEM stub CMC4). Arrow indicates cap.
Fig. 4. C-shaped pyrenoid caps and ellipsoid grains found in Trachelomonas ellipsoidalis (SEM stub CMC14 A & B). Arrows indicate caps.
Fig. 5. U-shaped paramylon caps and small disk paramylon grain in Colacium vesiculosum (SEM stub CMC1). Rimless U-shaped
pyrenoid cap indicated with a C; rim of U-shaped pyrenoid cap indicated with CR.
Fig. 6. Thickened discoid paramylon plate in Phacus orbicularis (SEM stub CMC10).
Fig. 7. Flattened paramylon plate in Phacus orbicularis (SEM stub CMC10).
Fig. 8. Curved paramylon plate in Monomorphina pyrum (SEM stub CMC9).
Fig. 9. Trough-shaped paramylon plate in Cryptoglena skujae (SEM stub CMC2).
Fig. 10. Curved ring-shaped paramylon plate in Lepocinclis ovum (SEM stub CMC7).
Fig. 11. Bobbin-shaped paramylon grain in Phacus pleuronectes (SEM stub CMC12).
Fig. 12. Long rod-shaped paramylon grain in Lepocinclis acus (SEM stub CMC6).
Monfils et al.: Paramylon in photosynthetic euglenoids 159

5. see T. ellipsoidalis; Figs 4, 39, 40) or on both sides of the
pyrenoid (diplopyrenoid; see T. hispida var duplex Deflan-
dre; Fig. 41). Some Trachelomonas species lack a pyrenoid
and have no paramylon caps, (see T. volvovocinopsis
Swirenko; Fig. 42).
STROMBOMONAS (FIGS 43, 44): As in the sister taxon
Trachelomonas, ellipsoid grains and links are distributed
throughout the cytoplasm; they are abundant and
represent the dominant form of paramylon in all
members of the genus. Chloroplasts with haplopyrenoids
capped by cup-shaped paramylon grains are the norm for
most members of the genus (see Figs 43, 44 for
haplopyrenoids in S. borystheniensis). However, diplopyr-
enoid caps are present in some taxa [S. rotunda (Playfair)
Deflandre].
COLACIUM (FIGS 1, 5, 45, 46): Colacium is a small genus of 6–
10 species. Disk- and ring-shaped paramylon grains are
distributed throughout the cytoplasm in many members of
the genus. Disk- and ring-shaped grains are the dominant
paramylon type found within a cell (Figs 1, 46). Chloro-
plasts with haplopyrenoids are capped by cup-shaped
paramylon grains (see Figs 5, 45, 46 for haplopyrenoids
in C. vesiculosum).
MONOMORPHINA (FIGS 8, 47–50): Monomorphina, as recently
amended (Kosmala et al. 2007a), is a small genus with only
Figs 13–30. Schematic of paramylon grain types in the Euglenales. All scale bars 5 2 mm.
Fig. 13. Small disk-shaped paramylon grain.
Fig. 14. Small ring-shaped paramylon grain.
Fig. 15. Ellipse-shaped paramylon grain.
Fig. 16. Small link-shaped paramylon grain.
Fig. 17. Shallow curved diplopyrenoid caps.
Fig. 18. C-shaped pyrenoid cap.
Fig. 19. Rimless U-shaped pyrenoid cap.
Fig. 20. U-shaped pyrenoid cap with rim.
Fig. 21. Thickened discoid paramylon plate.
Fig. 22. Large ring-shaped paramylon plate.
Fig. 23. Flattened paramylon plate.
Fig. 24. Curved paramylon plate.
Fig. 25. Trough-shaped paramylon plate.
Fig. 26. Curved ring-shaped paramylon plate.
Fig. 27. Bobbin-shaped paramylon grain.
Fig. 28. Long straight rod-shaped paramylon grain.
Fig. 29. Long bent rod-shaped paramylon grain.
Fig. 30. Elongated link-shaped paramylon grain.
160 Phycologia, Vol. 50 (2), 2011

6. three species. The visually dominant form of paramylon
found in all three members of the genus is the large lateral
plate. In M. pyrum (Figs 8, 47, 48) and M. pseudopyrum S.
Komala, R. Milanowski, K. Brzoska, M. Pekala, J.
Kwitowski & B. Zakrys´, there are two lateral plates. The
plates are appressed to the pellicle and conform to the
contours of the cell surface giving the plate a striated and
curved appearance (Figs 8, 48). Monomorphina aenigmatica
(Drezepolski) Nudelman & Triemer bears three lateral
plates, two positioned toward the anterior and one near the
posterior of the cell (Fig. 49). This species has a pyrenoid
which is capped on one side with a cup-shaped paramylon
grain (see M. aenigmatica, Fig. 50).
Haplopyrenoids have been reported from LM and
transmitting electron microscopic images in Monomorphina
(Nudelman et al. 2005; Kosmala et al. 2007a) but are only
known for this single species, Monomorphina aenigmatica
(Fig. 50). Disk-shaped and ellipsoid and associated ring-
and link-shaped grains are abundant and present in all
members of the genus.
CRYPTOGLENA (FIGS 9, 51, 52): Two species are noted in the
genus – Cryptoglena skujae (Figs 9, 51, 52) and C. pigra
Ehrenberg. Species within this genus bear two large,
visually dominant, shield- or trough-shaped lateral plates
that are positioned between the single C-shaped chloroplast
and the pellicle (Fig. 51). The imprint of the longitudinally
oriented pellicle strips can be seen on some of the large
plates (Figs 9, 52). Ellipsoid and link-shaped grains are
abundant and distributed in the cytoplasm (Fig. 52). The
chloroplast lacks a pyrenoid in Cryptoglena; therefore,
paramylon caps are absent in the genus.
PHACUS (FIGS 6, 7, 11, 53–58): Phacus is a genus with very
distinct and characteristic paramylon types. Several grain
morphologies are found in Phacus with the usual pattern of
one to few large plates (. 4 mm) and numerous smaller disk
and/or ellipse-shaped grains. Plates dominate the cell
interior but the number and morphology can vary by
species. In P. orbicularis, (Figs 7, 53, 54) there is a single
dominant plate, while in P. curvicauda two dominant plates
are present (Fig. 55). Frequently, one or more of the plates
will undergo a partial dissolution of centres creating a
dominant large ring-like paramylon structure as shown in
P. acuminatus Stokes (Fig. 56). One of the more unusual
forms of paramylon is the bobbin-shaped grain as found in
Figs 31–38. Whole cell light micrographs and paramylon SEM micrographs of euglenoid species in the genus Euglena. In all LM images
scale bars 5 20 mm. In all SEM micrographs scale bars 5 2 mm. Culture collection information for SEM images available in Table 1.
Fig. 31. Whole cell LM image of Euglena gracilis – strain Z (SAG 1224–5/25).
Fig. 32. Whole cell LM image of Euglena gracilis – strain Z (SAG 1224–5/25). Arrows indicate diplopyrenoid paramylon caps.
Fig. 33. SEM micrograph of paramylon extracted from Euglena gracilis – strain Z (SEM stub CMC4). Ellipse indicated with an E;
pyrenoid cap indicated with a PC.
Fig. 34. Whole cell LM image of Euglena viridis (IAM E-II, IAM culture collection, Japan). Arrow indicates paramylon centre.
Fig. 35. Whole cell LM image of Euglena deses collected at the Marsh Road Silo Pond, Okemos, MI, 42u43946.500N, 84u24942.380W, 30
April 2003. Multiple short rods present are indicated with arrows.
Fig. 36. Whole cell LM image of Euglena ehrenbergii collected at Haslett High School Baseball Pond, Haslett, MI, 42u44929.990N,
84u24917.240W, 30 June 2005. Arrow indicates long bent rod of paramylon.
Fig. 37. Whole cell LM image of Euglena convoluta collected at Baker Woodlot Pond, East Lansing, MI, 42u43901.430N, 84u28937.510W,
15 April 2005. Curved trough-shaped paramylon grains indicated with arrows.
Fig. 38. Partial cell LM image of curved trough-shaped paramylon grain in Euglena convoluta collected at Baker Woodlot Pond, East
Lansing, MI, 42u43901.430N, 84u28937.510W, 15 April 2005. Arrow indicates curved trough-shaped paramylon grain.
Monfils et al.: Paramylon in photosynthetic euglenoids 161

7. P. pleuronectes (Figs 11, 57, 58). In LM the face view of the
bobbin appears as a ring (Fig. 57). Since the bobbin has
considerable depth, the top and bottom of the bobbin can
be interpreted as superimposed plates when viewing
different focal planes but the SEM image clearly shows
the bobbin-like nature of the grain (Figs 11, 58). Chloro-
plasts lack pyrenoids in Phacus; paramylon caps are absent
in the genus.
LEPOCINCLIS (FIGS 2, 10, 12, 59–66): Lepocinclis is the sister
taxon to Phacus. Like Phacus, Lepocinclis has very distinct
paramylon. Several grain morphologies are found in
Lepocinclis with the usual pattern of one to several large
grains (. 4 mm) and numerous disk- or ellipse-shaped
smaller grains. Short rods and associated links (# 7 mm)
have also been noted. Multiple short and/or long rod-
shaped paramylon grains are dominant in larger species with
an elongate body such as L. acus (Figs 12, 59, 60) and L.
helicoideus (Bernard) Lemmerman (Fig. 61). Two elongated
links are present in many species such as L. oxyuris
Schmarda (Fig. 62) and L. spirogyroides Marin & Melko-
nian (Fig. 63). The elongated links are usually positioned
one anterior and one posterior to the nucleus. Plates are
present in many of the ovoid to fusiform Lepocinclis species
(Figs 64–66). These are positioned equatorially and can be
curved to follow the contour of the cell body. Commonly the
central region of the plate has dissolved away, giving rise to
a characteristic curved oval or ring as shown in L. ovum
(Figs 10, 64–66). Chloroplasts lack pyrenoids in Lepocinclis;
paramylon caps are absent in the genus.
DISCOPLASTIS (FIGS 67–70): Two species are currently rec-
ognized in the genus: D. spathirhyncha (Figs 67–69) and D.
adunca (Schiller) Triemer (Fig. 70). Both species have
numerous paramylon grains distributed throughout the
cytoplasm. Ellipses, links and plates are noted in the
cytoplasm. Short rods can also be present.
DISCUSSION
A comprehensive LM and SEM review of paramylon grain
diversity across the Euglenales enabled quantification of
morphological variation within the order and established a
Figs 39–46. Whole cell LM micrographs and paramylon SEM micrographs of euglenoid species in the genera Trachelomonas,
Strombomonas and Colacium. In all LM images scale bars 5 20 mm. In all SEM micrographs scale bars 5 2 mm. Culture collection
information for SEM images available in Table 1.
Fig. 39. Whole cell LM image of T. ellipsoidalis (NJ ST1). Lorica not present in cells grown in culture. Arrow indicates haplopyrenoid
paramylon cap.
Fig. 40. SEM micrograph of paramylon extracted from T. ellipsoidalis (SEM stub CMC14 A & B). Ellipse indicated with an E and
haplopyrenoid paramylon cap indicated with a PC.
Fig. 41. Whole cell LM image of T. hispida var duplex collected at North Large Meadows Pond, Okemos, MI, 42u41908.610N,
84u26951.120W, 23 June 2009. Arrows indicate diplopyrenoid paramylon caps.
Fig. 42. Whole cell LM image of T. volvocinopsis (CCAC 0115). No pyrenoids present. Small ellipsoid paramylon grains scattered
throughout cell.
Fig. 43. Whole cell LM image of S. borystheniensis (NJ S10). Arrow indicates haplopyrenoid paramylon cap.
Fig. 44. SEM micrograph of paramylon extracted from S. borystheniensis (SEM stub CMC13). Ellipse indicated with an E and
haplopyrenoid paramylon cap indicated with a PC.
Fig. 45. Whole cell LM image of C. vesiculosum (CCAP 1211-3). Arrow indicates haplopyrenoid paramylon cap.
Fig. 46. SEM micrograph of paramylon extracted C. vesiculosum (SEM stub CMC1). Disks indicated with a D and haplopyrenoid
paramylon cap indicated with a PC.
162 Phycologia, Vol. 50 (2), 2011

8. rigorous framework for a consistent descriptive terminol-
ogy. All grains observed to date, either in the extensive
review of morphology conducted in this research or
examined and documented in previous publications, fall
into the designated six size and shape classifications
proposed and described in this article. It is important to
note, natural breaks in size and shape have been noted for
descriptive purposes. The terminology we propose, com-
bined with SEM reference images (Figs 1–12) and sche-
matic drawings (Figs 13–30), provides a defined set of
terms that will help to standardize the nomenclature.
Heidt (1937) and Gojdics (1953) suggested that rings and
links were the result of the dissolution and digestion of
intact grains. When paramylon is broken down by the cell,
digestion occurs in a regular fashion from the centre
outwards. This leads to partially digested grains with
progressively larger open central regions (Leedale et al.
1965). In this study, rings, links and elongated links were
documented only in the presence of the respective
comparably sized intact grains. This supports the ontogeny
of rings (large and small), links and elongated links as
remnants of the lytic process (Gojdics 1953; Leedale et al.
1965). Therefore, disks and rings, ellipses and links, plates
and large rings, and rods and elongated links, while useful
descriptive terms, represent manifestations of the same
grain types. Consequently, in the grain types proposed in
this manuscript, small and large rings, links and elongated
links fall under the same grain descriptions as their intact
counterparts.
All taxa in the study had more than a single paramylon
grain type. This feature of having two types of paramylon
grains within a single cell has been referred to as
‘dimorphic’ with one type being larger and few in number
and the other being smaller and more numerous (Mila-
nowski et al. 2001, 2006). Many species observed in this
study possessed paramylon grain diversity within both the
large grain category (plates, bobbins and long rods), as well
as the small grain category (disks, ellipses, short rods and
pyrenoid caps). Other species and genera had multiple
forms of paramylon but only in the small grain category.
Figs 47–52. Whole cell LM micrographs and paramylon SEM micrographs of euglenoid species in the genera Monomorphina and
Cyptoglena. In all LM images and SEM micrographs scale bars 5 20 mm, with the exception of Fig. 48 (M. pyrum) in which the scale bar 5
2mm. Culture collection information for SEM images available in Table 1.
Fig. 47. Whole cell LM image of M. pyrum (ACOI 1295). Lateral plates (two) indicated with arrows.
Fig. 48. SEM micrograph of large curved paramylon plate extracted from M. pyrum (SEM stub CMC9).
Fig. 49. Whole cell LM image of M. ellipsoidalis (SAG 1261-9-1). Lateral plates (three) indicated with arrows.
Fig. 50. Whole cell LM image of M. ellipsoidalis (SAG 1261-9-1); Two cells shown. Haplopyrenoid paramylon cap indicated with arrow.
Fig. 51. Whole cell LM image of C. skujae (SAG 1088). Lateral plates (two) indicated with arrows.
Fig. 52. SEM micrograph of paramylon extracted from C. skujae (SEM stub CMC2). Ellipse indicated with an E and curved lateral
plates indicated with a LP.
Monfils et al.: Paramylon in photosynthetic euglenoids 163

9. Therefore, given the multiple forms of paramylon found in
many of the euglenoids, and the variation found amongst
the grains surveyed, we propose the term, polymorphic, to
describe the variation possible in both large and small grain
types within a single cell.
Small grains – free paramylon
In all species studied using LM or SEM, small free
paramylon grains were present in the cytoplasm (small free
grains 5 disks # 4 mm, and ellipses and short rods # 7 mm
on longest axis). Disk and ellipse shapes are differentiated
based on observed grain segments and overall shape.
Ellipses and short rods are separated based on length to
width ratios on the broadest face of the grain. Short rods
(# 7.0 mm) are differentiated from long rods (. 7.0 mm)
because this is a natural break in the variation in rod length
found across taxa. Rings, links and short elongated links
were noted in all species in which disks, ellipses and short
rods were observed, respectively. The number of small
grains present in the cells was largely dependent on
nutrition and age of the cell and was not included as a
variable for this study (see Gojdics 1953; Kiss et al. 1986;
Conforti 1998).
Disks, ellipses and short rods are difficult to distinguish
in LM micrographs. The small size makes shape difficult to
determine, and disks and ellipses can appear as short rod-
shaped grains if oriented in profile. This is a particular
limitation of these grains found on the smaller end of the
length continuum (, 7 mm). Careful observation of living
cells can be used to observe grains for reorientation (disks
and ellipses present in profile). Caution must be observed
when using these shape distinctions in taxonomic descrip-
tions based on LM alone. With this important caveat, some
careful conclusions can be drawn from the observations of
LM and SEM in this study.
Ellipse-shaped grains are found in every genus of the
Euglenales with the exception of one, Colacium. From a
Figs 53–58. Whole cell LM micrographs and paramylon SEM micrographs of euglenoid species in the genus Phacus. In all LM images scale
bars 5 20 mm. In all SEM micrographs scale bars 5 2 mm. Culture collection information for SEM images available in Table 1.
Fig. 53. Whole cell LM image of P. orbicularis (ACOI 1996). Arrow indicates large flattened paramylon plate.
Fig. 54. SEM micrograph of large flattened paramylon plate extracted from P. orbicularis (SEM stub CMC11). Small disk- and ring-
shaped paramylon grains are visible on plate surface.
Fig. 55. Whole cell LM image of P. curvicauda collected at Marsh Road Silo Pond, Okemos, MI, 42u43946.500N, 84u24942.380W, 19 May
2005. Arrows indicate two large thickened discoid paramylon plates present in the cell.
Fig. 56. Whole cell LM image of P. acuminatus collected at Marsh Road Silo Pond, Okemos, MI, 42u43946.500N, 84u24942.380W, 19
May 2005. Paramylon plates with partial dissolution of centres indicated with arrows.
Fig. 57. Whole cell LM image of P. pleuronectes (SAG 1261-3b). Bobbin shape paramylon grain indicated with arrow.
Fig. 58. SEM micrograph of bobbin-shaped paramylon extracted from P. pleuronectes (SEM stub CMC12).
164 Phycologia, Vol. 50 (2), 2011

10. phylogenetic perspective (see Fig. 71 for a simplified
phylogenetic diagram adapted from Triemer et al. 2006),
the most parsimonious explanation of this trait indicates a
basal derivation of the ellipse grain, with a loss in Colacium.
Interesting to note, Colacium has disk-shaped grains. Disk-
shaped grains are not found in its sister taxa, Strombomonas
and Trachelomonas. Disk-shaped grains are reported in
only four genera: Colacium, Lepocinclis, Monomorphina
and Phacus. The most parsimonious explanation for this
distribution of disk-shaped grains is three separate origins:
Figs 59–66. Whole cell LM micrographs and paramylon SEM micrographs of euglenoid species in the genus Lepocinclis. In all LM images
scale bars 5 20 mm, with the exception of Fig. 61 (L. helicoideus) with a scale bar 5 200 mm. In all SEM micrographs scale bars 5 4 mm, with
the exception of Fig. 60 (L. acus) with a scale bar 5 2 mm. Culture collection information for SEM images available in Table 1.
Fig. 59. Whole cell LM image of L. acus (UTEX 1316). Arrows indicate rod-shaped paramylon grains.
Fig. 60. SEM micrograph of rod-shaped paramylon grain extracted from L. acus (SEM stub CMC6).
Fig. 61. Whole cell LM image of L. helicoideus collected at Marsh Road Silo Pond, Okemos, MI, 42u43946.500N, 84u24942.380W, 22 June
2006. Arrows indicate long rod-shaped paramylon grains present in the cell.
Fig. 62. Whole cell LM image of L. oxyuris collected at Marsh Road Silo Pond, Okemos, MI, 42u43946.500N, 84u24942.380W, 22 June
2006. Elongated link-shaped paramylon grain indicated with arrow.
Fig. 63. Whole cell LM image of L. spyrogyroides (SAG 1224-10b). Elongated link-shaped paramylon grains indicated with arrows.
Fig. 64. Whole cell LM image of L. ovum (SAG 1244-8). Curved ring-shaped paramylon plate indicated with arrows.
Fig. 65. SEM micrograph of large thickened plate-shaped paramylon extracted from L. ovum (SEM stub CMC8).
Fig. 66. SEM micrograph of curved ring-shaped paramylon grain extracted from L. ovum (SEM stub CMC7).
Figs 67–70. Whole cell LM micrographs and paramylon SEM micrographs of euglenoid species in the genus Discoplastis. In LM images
scale bars 5 20 mm. In Fig. 68 (D. spathirhyncha) SEM micrographs scale bar 5 10 mm; In Fig. 69 (D. spathirhyncha) SEM micrographs
scale bar 5 2 mm. Culture collection information for SEM images available in Table 1.
Fig. 67. Whole cell LM image of D. spathirhyncha (SAG 1224-42).
Fig. 68. SEM micrograph of paramylon grains extracted from D. spathirhyncha (SEM stub CMC3).
Fig. 69. SEM micrograph of thickened discoid plate-shaped paramylon grain extracted from D. spathirhyncha (SEM stub CMC3).
Fig. 70. Whole cell LM image of D. adunca (ASW 08095). Short rod-shaped paramylon grains indicated with arrows.
Monfils et al.: Paramylon in photosynthetic euglenoids 165

11. once in the Phacus-Lepocinclis clade, once in Monomor-
phina and once in Colacium.
In the majority of species either ellipsoid or disk-shaped
grains were present. Only two species examined in this
study, (Phacus orbicularis and M. pyrum), had both disks
and ellipses present in the same cell. However, since small
grain type is difficult to detect using LM, and SEM was
conducted on only two Phacus species and a single
Monomorphina, it is unclear as to whether this is unique
to these taxa or will be found more commonly.
This SEM study confirms the presence of short rods
(# 7 mm) in Euglena gracilis, Discoplastis spathirhyncha and
Lepocinclis acus. Additional members of Euglena (for
example see LM of E. deses, Fig. 35) have rod-shaped
paramylon grains on the upper end of the ‘short’
designation and one example, E. ehrenbergii (Fig. 36;
which may not be a true Euglena species), has very long
rods (up to 75 mm). Of the two Discoplastis species
described, D. spathirhyncha has short rods observed with
SEM and D. adunca has short rod-shaped paramylon
grains on the upper end of the short designation (6–7 mm,
Fig. 70). Both short and long rods are present in L. acus
(Figs 12, 59, 60). Lepocinclis is the genus with the most
notable presence of rods with multiple short and long rod-
shaped paramylon grains dominant in the species of the
genus with larger elongated bodies (Figs 59–63). Rods
appear to be entirely absent, either short or long, in the
ovoid to fusiform smaller species of the genus (for example
see L. ovum Fig. 64).
From a phylogenetic perspective (Fig. 71), the most
parsimonious explanation for short rod-shaped paramylon
grains appears to be a presence at the base of the Euglenales
with the presence of short rod-shaped paramylon grains in
Discoplastis. Rod-shaped grains were lost twice; once in the
Phacus clade and again in the Trachelomonas-Cryptoglena
clade in which no rod-shaped grains occur. Evidence
indicates short rod-shaped grains are always found in the
presence of long rod-shaped grains; although, the inverse is
not always true.
From a taxonomic perspective, presence of small
detached ellipse, disk or short rod-shaped grains was of
little taxonomic value. Distinguishing this type using LM is
challenging at best. Consequently, while variability exists in
type and presence of multiple types, it is not possible to
apply this distinction broadly without comprehensive SEM
across the individual genera. Instead, it is the larger
dominant grains and the attached pyrenoid-associated
grains that appear to have the most taxonomic value.
Small grains – pyrenoid-associated paramylon
One of the more unusual types of paramylon grains is that
capping the pyrenoid in several taxa. In photosynthetic
Euglenales, species may lack pyrenoids or have naked
Fig. 71. Phylogenetic diagram illustrating relationships among the nine natural genera within the order Euglenales and the major paramylon
morphological types. Phylogeny modified from rooted Bayesian phylogenetic tree based on the combined small subunit and partial large
subunit rDNA sequences (Triemer et al. 2006 ). Paramylon grains illustrated at the tips of the tree indicate the presence of paramylon
morphological types in the lineage.
166 Phycologia, Vol. 50 (2), 2011

12. pyrenoids (pyrenoids lacking paramylon caps), haplopyr-
enoids or diplopyrenoids. Haplo- and diplopyrenoids bear
paramylon caps. Developmentally, paramylon caps mature
as the pyrenoid extends from the chloroplast. Differently
aged cells may have deeper caps (for a thorough review of
loricate paramylon cap development see Brown et al. 2003).
A range in cap depths was seen in our SEM samples,
suggesting that the depth of the paramylon cap may be a
plastic character. In fact, different depths of paramylon
caps are found within the same culture. This is best
illustrated in the range of cap depths observed in the SEM
study for U- and C-shaped grains (Table 1). This also may
explain why the light images from the T. ellipsoidalis culture
(Fig. 39) appeared to have deeper caps than those observed
with SEM from paramylon isolates (Fig. 40) in which none
of the caps were observed to exceed 1.5 mm in depth. Using
SEM, the deepest paramylon caps were found in Strombo-
monas (Figs 43, 44) and Colacium (Figs 45, 46). Here, both
C-shaped grains and U-shaped grains were present, some
up to 4 mm deep with striated exterior surfaces.
Haplopyrenoids are found in Trachelomonas (Fig. 39),
Strombomonas (Fig. 43), Colacium (Fig. 45) and Mono-
morphina (Fig. 50). Paramylon caps in these instances are
deeper than those found in Euglena, reaching a depth of up
to 4.0 mm in some taxa when mature (Figs 4, 5, 40, 44, 46)
and appear C- or U-shaped when viewed laterally with LM.
The most parsimonious explanation to account for the
distribution of haplopyrenoids (and the associated caps)
among the genera is that haplopyrenoids were present in
the ancestor to the Trachelomonas-Cryptoglena and lost in
the lineage leading to Cryptoglena (see Fig. 71). The
alternative explanation would require that haplopyrenoids
were acquired twice, once in the Monomorphina lineage and
again in the Colacium/Trachelomonas/Strombomonas line-
age.
Diplopyrenoids are common in Euglena, present in
some spiny Trachelomonas species (e.g. T. hispida Fig. 41
and taxa in clade A from Ciugulea et al. 2008) and found in
a few Strombomonas species such as S. rotunda (not
shown). In Euglena diplopyrenoids, the associated param-
ylon forms shallow caps , 1.0 mm deep that, under the
LM, appear as ‘parentheses’ bracketing the pyrenoid
(Figs 32, 41). The SEM study of isolated paramylon more
accurately shows the structure of the cap in Euglena. What
appears as a curved structure appressed to the surface of
the chloroplast in LM (Figs 32, 41) is a shallow cap in the
SEM (Figs 3, 33). Based on LM, the morphology of the
paramylon caps on diplopyrenoids is relatively consistent
across the genera in which they have been found. Based on
the distribution of taxa containing diplopyrenoids with
caps across the phylogenetic tree (see Fig. 71), they may
have arisen independently twice, once in the Euglena genus
and a second time in the Trachelomonas/Strombomonas
lineages.
Large grains – plates
In determining variation in euglenoid paramylon, any
dominant orbicular paramylon structure was categorized
as a ‘plate’. Considerable variation occurs in size, number,
orientation and state of dissolution of paramylon plates.
THICKENED PLATES: When plates appear large (. 4 mm)
and carry the same characteristic shape found in small
disks, they are described here as thickened plates. These
were observed using SEM in Discoglena spathirhyncha
(Fig. 69), Lepocinclis ovum (Fig. 65) and Phacus orbicularis
(Fig. 6). Where observed in D. spathirhyncha, we did not
find small disks but did observe small ellipsoid grains. So
this thickened plate shape is quite different and much larger
than the other grains found in the D. spathirhyncha culture
(Fig. 68). In P. orbicularis thickened plates were observed
(though not the dominant paramylon type). Thickened
plates are prevalent throughout the Phacus clade and
observed in multiple species, sometimes dominant in the cell
and sometimes in multiples within a cell (see Figs 55, 56; for
a discussion of Phacus paramylon plates see Kosmala et al.
2007b). Lepocinclis ovum has many plate types including
thickened plates, flattened plates and curved rings, perhaps
all developmental variants of the same shape. Thickened
plates could have arisen twice in the Euglenales, once at the
base of the Euglenales in Discoplastis and once in the
Phacus-Lepocinclis calde. Alternatively, thickened plates
are found in the ancestor of the Euglenales and were lost
once in the Euglena- Cryptoglena clade (see Fig. 71).
FLATTENED PLATES: Flattened plates appear quite a bit
different in structure than the disk-like plates. These lack
the characteristic segmented wedges of a thickened plate.
They are often nonsymmetrical and have a flattened coiled
formation to the paramylon (see Figs 7, 54). These can be
observed readily with LM, where plates appear to lack
depth characteristic of the large thickened plates (for
comparison see Figs 53, 55, 56). The large flattened plates
can reach $ 30 mm in size, and as in P. orbicularis are
dominant in the centre of the cell when viewed using LM
(Fig. 53). Thickened plates and flattened plates were
observed in cultures of both P. orbicularis and L. ovum
using SEM. Large flattened plates observed in SEM could
be artifacts of the thickened plates after breakdown due to
age and nutrition of the cell (see review of paramylon grain
structure and breakdown in Gojdics 1953). Even if
remnants of the lytic process, large flattened plates only
occur in the Phacus-Lepocinclis clade and support the sister
taxa relationship of the two genera (Fig. 71).
LARGE RINGS: It is important to make a distinction
between large flat rings and large curved rings. Large flat
ring-like structures are present in all taxa with large
thickened plates but large curved rings are only present in
the obovoid to fusiform smaller species of Lepocinclis (see
L. ovum Figs 10, 64, 66). Large curved rings are shown with
SEM in L. ovum (Figs 10, 66). Interestingly, this curved
shape is maintained even after cell lysis. Also of note, in L.
ovum, large disks and flattened plates are observed. This
implies an early stage of an intact thickened/flattened plate
grain later degraded to the formation of the large curved
ring. The thickened/flattened plate stage has not been noted
in light images so this phase of development warrants
further review. Since the curved rings appear in the
Lepocinclis group exclusively and only in smaller obovoid
to fusiform members of the genus (as opposed to the
elongated larger species), this character state could prove to
Monfils et al.: Paramylon in photosynthetic euglenoids 167

13. be a defining characteristic of some members of Lepocinclis
and taxonomically informative.
CURVED PLATES: Curved plates occur in Cryptoglena and
Monomorphina cultures observed using SEM (Figs 47–52).
LM clearly shows plates placed parietally in the cell, unlike
the centralized plates found in Phacus. As noted in
Kosmala et al. (2007a) the depth of the plate is dependent
on age and nutrition of the cell. The plates observed in M.
pyrum in SEM were thickened and appeared with striations
similar to the thick ribbed pellicle structure of the intact
cell. The SEM images shown herein (Figs 8, 48) support the
conclusion of Kosmala et al. (2007a) that cell shape affects
the paramylon surface structure in Monomorphina. Curved
shield-like plates were found in Cryptoglena that were
smooth and elongated (Figs 9, 51, 52). As in Monomor-
phina, these plates occur between the pellicle and cytosolic
contents, and reflect the topography of the cell (in the case
of Cryptoglena a smoother exterior with striations). The
presence of parietal plates in both Cryptoglena and
Monomorphina is a synapomorphy supporting the proposed
sister taxa relationship among these genera (see Fig. 71).
As previously noted, curved trough-shaped plates appear
with missing centres in E. convoluta (Figs 37, 38). These
plates are observed with LM. Currently cultures are
unavailable for sequencing or SEM analysis. However,
paramylon morphology combined with the presence of
small discoid chloroplasts lacking pyrenoids may be an
indication that this taxon is misplaced in the genus Euglena
and may be better placed among Phacus or Lepocinclis.
This is consistent with the lack of plates in any other
members of Euglena. This is a case where paramylon
morphology can provide information about taxonomy.
Large plate morphology is highly variable, though trends
can be seen in large plate evolution as a whole. Assuming E.
convoluta is misplaced, there are a few scenarios for plate
evolution (see Fig. 71). Large plates, in any of the forms,
appear to have arisen at the base of the Euglenales, with a
loss in the Trachelomonas-Colacium clade and a loss in
Euglena. An alternative is three separate derivations of
large plates: once in Discoplastis, once in the Phacus-
Lepocinclis clade and once in the Monomorphina-Cryto-
glena clade. The second option would allow for three
separate types of plates: medium-sized disks in Discoplastis
(not the dominant paramylon form), large dominant plates
in the cell centres in the Phacus-Lepocinclis clade and
parietal plates in the Monomorphina-Crytoglena clade.
Large grains – bobbins
Bobbin-shaped paramylon grains are very distinct and only
reported here in Phacus pleuronectes (Figs 11, 57, 58). In
LM they appear like superimposed thickened plates and are
often referred to as plates in the literature (Kosmala et al.
2007b). This is the first SEM imaging of bobbins
confirming the formation of a thickened disk structure
with constriction in the central region. The examples from
this culture of P. pleuronectes show variation in the depth
and differences between the width of the two orbicular
faces. Grains can appear to have a ring-like formation with
a hollowing of the central region, however, all large grains
have the characteristic bobbin shape. The bobbin shape is a
character state present in P. pleuronectes; the only other
reported bobbin paramylon is found in P. tortus (reported
from LM in Conforti 1998). Recent work by Kosmala
(2007b) differentiating P. pleuronectes and P. orbicularis
does not reference the difference in paramylon grain
morphology among the two species. Though not always
visible with a brief examination, the bobbin is distinguish-
able using LM in P. pleuronectes. With the noted
phenotypic plasticity within these two species, bobbins
could be a key diagnostic feature when differentiating P.
pleuronectes and P. orbicularis.
Large grains – long rods
Long rods (. 7 mm on longest axis) were examined using
SEM in Lepocinclis acus. These long rods can be straight
or bent and this character appears to be plastic. SEM
images of rods show the paramylon has a coiled formation
with a centre seam (Figs 12, 60). In LM images, long rods
can be described as links with a more pronounced central
cleft, thus the term ‘elongated links’ (Figs 62, 63). Where
long rods or short rods were observed in culture, small
ellipses were also noted in SEM. Small rods appear in the
culture of L. acus as well as long rods up to 12 mm,
implying there may be differences in size based on the age
of the grain and/or the cell. The longer the rod, the more
pronounced length to width ratio. Rods can be up to 75 mm
in length.
The large pronounced elongated links and rods (exceed-
ing 7 mm) are a characteristic found in elongated larger
bodied members of Lepocinclis and when present could be a
key diagnostic feature. Euglena ehrenbergii is a notable
exception (Fig. 36). However, long rod presence could be
an indication of the incorrect generic placement of this
taxon. This species has been difficult to sequence for
phylogenetic analysis and the taxonomic placement may be
open for interpretation. Like Euglena convoluta discussed
earlier, this taxon also has small discoid chloroplasts
lacking pyrenoids, a feature not found in other Euglena
species.
In conclusion, paramylon grains are highly variable
within euglenoids. This variation can be best observed using
SEM, and insights from SEM provide useful characters for
examination using LM. Terminology has been proposed,
which can be applied consistently across the Euglenales to
help inform future projects in research utilizing paramylon
grain morphology. This study was able to determine a high
level of distinction among paramylon grain morphologies
both within a species and among species and variation was
found in both large and small grains. A new term
‘polymorphic’ is proposed to accurately describe the
variation in both large and small paramlyon grain types.
Trends in large and small grains as well as a discussion of
dominant paramylon types relative to specific cultures and
across major clades have been described in detail. Variation
in small grain morphology has the potential to be useful in
taxonomic descriptions but only with the use of SEM
because it is often difficult to determine small grain types
using LM alone. Large paramylon grain types and
presence/absence of large grains can be used at the generic
168 Phycologia, Vol. 50 (2), 2011

14. level to support major clades and generic relationships and
may provide insight into the taxonomic placement of
euglenoids currently unavailable for sequencing.
ACKNOWLEDGEMENTS
Funding was provided through the Presidents Research
Investment Funds, Faculty Incentive Team Funds, Summer
Scholars Program and Undergraduate Research and
Creative Endeavors Grant at Central Michigan University
(CMU) and the Central Michigan chapter of Sigma Xi. In
addition, the authors wish to acknowledge the financial
support provided by the National Science Foundation
PEET program (Partnership for Enhanced Expertise in
Taxonomy, grant no. DEB 4-21348). The authors would
like to thank the two anonymous reviewers for constructive
comments on the manuscript. Additional thanks to S. Juris,
A. Mueller and M. Steinhilb for insights and support; P.
Oshel in the Microscopy Facility at CMU for advising on
equipment use; Matt Bennett for assistance with cultures;
Larry Burditt and Marlene Cameron for paramylon
graphics; and E. Linton for phylogenetic diagrams.
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Received 30 December 2009; accepted 26 July 2010
Associate editor: Fabio Rindi
Monfils et al.: Paramylon in photosynthetic euglenoids 169