HELGOLANDER MEERESUNTERSUCHUNGEN
Helgol~inder Meeresunters. 34,355-369 (1981)
Structure and significance of cruciate flagellar root
systems in green algae: Female gametes of Bryopsis
lyngbyei (Bryopsidales)
M. M e l k o n i a n
Botanisches Institut, Westf~lische Wilhelms-Universit5t;
Schlol~garten 3, D-4400 Mfinster, Federal Republic of Germany
ABSTRACT: The ultrastructure of the flagetlar apparatus in the biflagellate female g a m e t e s of the
green alga Bryopsis lyngbyei has b e e n studied in detail. In the flagellum and basal body,
microtubule septations occur in some of the B-tubules. The transition region of the flagellum is
extremely long (260-290 nm), exhibits a stellate pattern in cross section but lacks the transverse
diaphragm. The two basal bodies form an angle of 180 ° and overlap at their proximal ends. They are
connected by a compound non-striated capping plate. Terminal caps associated with the c a p p i n g
plate partially close the proximal end of each basal body. A cruciate flagellar root system with three
different types of microtubular roots is present, i. e. the flagellar apparatus does not show 180 °
rotational symmetry. One root type contains 2 microtubules which are connected to an elaborate
cylindrical structure, presumably a mating structure. The opposite root exhibits 3 microtubules over
its entire length and is not associated with a cylindrical structure. In their proximal parts both roots
are linked to an underlying crescent body. The other two microtubular roots are probably identical
and consist of 4 (or 5) microtubules w h i c h show configurational changes. These two identical roots
insert into the capping plate and link to the inner side (i. e. the side adjacent to the other basal body)
of each basal body, w h e r e a s the other two roots attach to the outer sides of each basal body. System I
striated fibres are probably associated with each of the four roots, while system II fibres have not
b e e n observed. The flagellar apparatus of female g a m e t e s of B. 1yngbyei shows many u n i q u e
features but in some aspects resembles that of ulvatean algae. Functional and p h y l o g e n e t i c aspects
of cruciate flagellar root systems in g r e e n algae are discussed,
INTRODUCTION
In t h e p a s t f e w y e a r s t h e d e t a i l e d u l t r a s t r u c t u r e of t h e f l a g e l l a r a p p a r a t u s h a s b e e n
i n c r e a s i n g l y u s e d as a p h y l o g e n e t i c i n d i c a t o r in the g r e e n a l g a e ( s u m m a r i e s by M o e s trup, 1978; M e l k o n i a n , 1980a). T h e s e s t u d i e s h a v e c o m p l e m e n t e d a n d e x t e n d e d e a r l i e r
investigations on mitotic and cytokinetic mechanisms in green algae (summarized by
P i c k e t t - H e a p s , 1975; S t e w a r t & M a t t o x , 1975). T h e p o s t u l a t i o n of a t h i r d c l a s s of g r e e n
algae (besides the Chlorophyceae and Charophyceae sensu Stewart & Mattox), the
Ulvaphyceae sensu Stewart & Mattox, was mainly based on cytokinetic properties and
p r e s e n c e of s c a l e s o n m o t i l e c e l l s of s o m e s p e c i e s ( S t e w a r t & M a t t o x , 1978). In t h e p a s t
t w o y e a r s it h a s a l s o b e e n s h o w n t h a t t h e f l a g e l l a r a p p a r a t u s of t h e U l v a p h y c e a e s e n s u
© Biologische Anstalt Helgoland
0174-3597/81/0034/0355/$ 02.00
356
M. M e l k o n i a n
Stewart & Mattox c o n t a i n s some special structures c o n f i n e d to motile cells of this g r e e n
algal group (Melkonian, 1979; S l u i m a n et al., 1980; M e l k o n i a n , 1980b; Hoops et al.,
1981). O n e result of these studies has b e e n the f i n d i n g that fibrous structures associated
with b a s a l b o d i e s e x h i b i t a great diversity a n d are p o t e n t i a l l y more useful as p h y l o g e n e tic m a r k e r s c o m p a r e d to the rather u n i f o r m m i c r o t u b u l a r flagellar root system (see also
d i s c u s s i o n i n M e l k o n i a n , 1980a). The u n i f o r m i t y of the m i c r o t u b u l a r flagellar root
system i n various not closely r e l a t e d groups of g r e e n a l g a e has b e e n i n t e r p r e t e d as
reflecting a g e n e r a l i m p o r t a n t f u n c t i o n of these systems a n d some f u n c t i o n a l e x p l a n a tions h a v e b e e n offered ( M e l k o n i a n , 1980b).
O n l y few ultrastructural studies have b e e n u n d e r t a k e n on motile cells of siphonal e a n g r e e n a l g a e (Crawley, 1966; Burr & West, 1970; Woodcock & Miller, 1973; Moestrup
& Hoffman, 1975; Hori, 1977; Hori & Enomoto, 1978; Roberts, 1980; Roberts et al., 1980;
Roberts et al., 1981, i n prep.) a n d these have not yet led to self-explanatory results. It has
b e e n n o t e d that c o n n e c t i n g fibres i n Bryopsis are very similar to those found in Ulva a n d
Enteromorpha ( M e l k o n i a n , 1980a). From a study on the s t e p h a n o k o n t zoospores a n d the
a n i s o g a m e t e s of Derbesia tenuissima, Roberts et al. (1980; 1981 in prep.) also c o n c l u d e d
that the flagellar a p p a r a t u s of the r e p r o d u c t i v e cells of Derbesia r e s e m b l e d that of
u l v a l e a n a l g a e a n d therefore they t e n t a t i v e l y i n c l u d e d the s i p h o n a l e a n g r e e n algae in
the U l v a p h y c e a e s e n s u Stewart & Mattox. It is h o w e v e r clear that too few species of
s i p h o n a l e a n g r e e n a l g a e h a v e b e e n i n v e s t i g a t e d i n detail to m a k e such a proposal
s e n s i b l e at this time.
The p r e s e n t study o n the flagetlar a p p a r a t u s of female g a m e t e s of Brgopsis was
partially u n d e r t a k e n to a d d to our k n o w l e d g e a b o u t the flagellar a p p a r a t u s i n siphonal e a n g r e e n a l g a e a n d partly b e c a u s e Bryopsis offers a n excellent opportunity to study the
effect of e x t r e m e a n i s o g a m y o n the d e t a i l e d structure of the flagellar apparatus. The
m a l e g a m e t e s h a v e p r e v i o u s l y b e e n s t u d i e d i n some detail (Hori, 1977; M e l k o n i a n ,
1980a).
This study is the third c o n t r i b u t i o n of a series (see also M e l k o n i a n , 1978, 1979),
w h i c h aims to e v a l u a t e structural v a r i a b i l i t y a n d f u n c t i o n a l i m p l i c a t i o n s of cruciate
flagellar root systems i n g r e e n algae.
MATERIAL AND M E T H O D S
C u l t u r e s of f e m a l e a n d m a l e strains of Bryopsis lyngbyei H o r n e m a n n (Bryopsidales)
were k i n d l y p r o v i d e d by Dr. Dr. h. c. P. K o r n m a n n (Helgoland). The a l g a e were cultured
i n a n E r d - S c h r e i b e r solution, c o n t a i n i n g 0.2 g N a N O a, 0.02 g Na2HPO 4 x 10 H20 a n d
50 m l soil extract per 1 1 of filtered seawater. T h e g e n e r a l culture c o n d i t i o n were: 14/10 h
l i g h t / d a r k cycle, 10 °C a n d a p p r o x i m a t e l y 2500 Lux light intensity. Plastic petri dishes
were u s e d as culture vessels. G a m e t e discharge in the female g a m e t a n g i a u s u a l l y occurs
shortly after the b e g i n n i n g of the light period. After discharge, female g a m e t e s a c c u m u lated n e a r the light source at the meniscus, w h e r e they could be collected as a
c o n c e n t r a t e d s u s p e n s i o n with a micropipette. The cells were fixed for electron microscopy as p r e v i o u s l y d e s c r i b e d ( M e l k o n i a n , 1979). Fixed cells were w a s h e d with ErdS c h r e i b e r - s o l u t i o n (4 °C) a n d further processed b y s t a n d a r d methods (Melkonian, 1975).
Sections were cut with a d i a m o n d knife a n d o b s e r v e d i n a S i e m e n s Elmiskop IA.
Flagellar root systems
357
RESULTS
The biflagellate female
gametes
of
Bryopsis 1ynffbyei
measure
around
9 ~m in
l e n g t h a n d 5 ~ m i n w i d t h a n d a r e p e a r - s h a p e d . T h e g e n e r a l u l t r a s t r u c t u r e of t h e f e m a l e
g a m e t e is v e r y s i m i l a r to t h a t of f e m a l e g a m e t e s of
Bryopsis hypnoides
(Burr & West,
1970) a n d n e e d n o t b e d e s c r i b e d i n d e t a i l . A l o n g i t u d i n a l s e c t i o n of a f e m a l e g a m e t e
cp
rI
"" "'
,,/f'~:
rll
~
eds
Fig. 1. S c h e m a t i c 3 - d i m e n s i o n a l reconstruction of the flagetlar a p p a r a t u s of f e m a l e g a m e t e s of
Abbreviations: cp = c a p p i n g plate c o m p o s e d of two n o n - s t r i a t e d c o n n e c t i n g
fibres; tc = t e r m i n a l cap, partially closing t h e p r o x i m a l e n d of e a c h b a s a l body; r I = t w o - s t r a n d e d
microtubular root; tit = f o u r - s t r a n d e d m i c r o t u b u l a r root ( s o m e t i m e s with 5 microtubules); cs =
cylindrical structure associated with the t w o - s t r a n d e d m i c r o t u b u l a r root; cb = crescent b o d y w i t h
striations; e d s = electron d e n s e s h e e t s u n d e r l y i n g e a c h root m i c r o t u b u l e of all four m i c r o t u b u l a r
roots; sP1 = specialized area of the p l a s m a l e m m a n e a r the cylindrical structure (probably a m a t i n g
structure)
Bryopsis lyngbyei.
358
M. M e l k o n i a n
r e v e a l i n g d i s t r i b u t i o n of m a j o r c e l l o r g a n e l l e s m i g h t t h e r e f o r e b e s u f f i c i e n t for t h e
p r e s e n t p u r p o s e (Fig. 2).
A d i a g r a m m a t i c p r e s e n t a t i o n of t h e f l a g e l l a r a p p a r a t u s is s h o w n in Fig. 1 a n d m a y
b e u s e d as a r e f e r e n c e to t h e f o l l o w i n g d e s c r i p t i o n a n d s u b s e q u e n t m i c r o g r a p h s .
C r o s s s e c t i o n s of t h e f l a g e l l a r a x o n e m e i n t h e shaft r e g i o n r e v e a i in a d d i t i o n to t h e
u s u a l 9 ÷ 2 p a t t e r n of m i c r o t u b u l e s u s u a l l y o n e b i p a r t i t i o n in a B - t u b u l e of o n e o u t e r
d o u b l e t p e r s e c t i o n (Fig. 8). T h e t r a n s i t i o n r e g i o n of t h e f l a g e l l u m is u n u s u a l l y l o n g
( 2 6 0 - 2 9 0 nm) a n d in cross s e c t i o n e x h i b i t s t h e u s u a l s t e l l a t e p a t t e r n f o u n d in m o s t g r e e n
a l g a e (Figs 6, 7). In l o n g i t u d i n a l s e c t i o n s h o w e v e r t h e t r a n s i t i o n a l r e g i o n l a c k s t h e
t r a n s v e r s e d i a p h r a g m (Fig. 6). E a c h b a s a l b o d y is a b o u t 3 6 0 - 4 0 0 n m t o n g a n d in cross
s e c t i o n s a c a r t w h e e l p a t t e r n is o b s e r v e d i n t h e p r o x i m a l r e g i o n of e a c h b a s a l b o d y . T h e
t w o b a s a l b o d i e s a r e p a r a l l e l to o n e a n o t h e r ( a n g l e b e t w e e n t h e m : 180 °) a n d o v e r l a p at
t h e i r p r o x i m a l e n d s b y a r a t h e r c o n s t a n t d i s t a n c e of 170 n m (Fig. 3).
B a s a l b o d i e s a r e i n t e r c o n n e c t e d b y t w o p r i n c i p a l n o n - s t r i a t e d c o n n e c t i n g fibres
f o r m i n g a c a p p i n g p l a t e (Fig. 4), E a c h c o n n e c t i n g f i b r e a r i s e s f r o m a d i f f e r e n t b a s a l b o d y
but both touch each other midway between the basal bodies and thereby provide the
c o n n e c t i o n b e t w e e n t h e t w o b a s a l b o d i e s . In m o s t s e c t i o n s h o w e v e r t h e s e t w o fibres
a p p e a r as a s i n g l e c a p p i n g p l a t e (e. g. Fig. 4). T h e c o n n e c t i n g f i b r e s a t t a c h at t h e distal
e n d s of t h e r e s p e c t i v e b a s a l b o d i e s a n d in cross s e c t i o n s t h r o u g h this a r e a t h e fibre has a
c u r v e d a p p e a r a n c e a n d is l i n k e d to 4 d i f f e r e n t t r i p l e t s of t h e b a s a l b o d y (Fig. 23). In
a d d i t i o n e a c h c o n n e c t i n g f i b r e h a s o n e d i s c r e t e a t t a c h m e n t site to t h e p l a s m a l e m m a at
t h e m o s t a p i c a l e n d of t h e c e l l (Figs 9, 17, 22). T h e c o m p o u n d c a p p i n g p l a t e t h e r e f o r e
a r c h e s a n t e r i o r l y to f o r m a b r i d g e w i t h a h o r i z o n t a l m i d - r e g i o n b e t w e e n t h e t w o
a n c h o r i n g p r o c e s s e s (e. g. Fig. 4). T h e l a t e r a l r e g i o n s of t h e c a p p i n g p l a t e e x t e n d
p o s t e r i o l a t e r a t t y . W h e r e a s t h e l e n g t h of t h e i n d i v i d u a l c o n n e c t i n g f i b r e s r o u g h l y corresp o n d s to t h e o v e r a l l l e n g t h of t h e b a s a l b o d y (ca. 400 nm) t h e w i d t h of t h e c a p p i n g p l a t e
v a r i e s b e t w e e n 200 a n d 250 nm. In t h e o v e r l a p r e g i o n b o t h b a s a l b o d i e s are l i n k e d
t o g e t h e r b y e l e c t r o n d e n s e m a t e r i a l w i t h i n c o n s p i c u o u s s t r i a t i o n s (Fig. 3).
T h e p r o x i m a l e n d of e a c h b a s a l b o d y is p a r t i a l l y c l o s e d b y a t e r m i n a l cap, i. e. an
Fig. 2. Longitudinal section through a female gamete revealing major organelle distribution. The
chloroplast is posteriorly located, contains numerous starch grains, a pyrenoid and a very large
eyespot (arrows). X 8500
Fig. 3. Transverse section through the tip of the female gamete, b = basal bodies; 2 = two-stranded
microtubular root; 3 = three-stranded microtubular root; 4 = four- (or five-)stranded microtubular
roots; open arrow = cylindrical structure in cross section associated with the two-stranded
microtubular root. Notice basal body overlap, x 60 000
Fig. 4. Vertical longitudinal section through the flagellar apparatus, b = basal body; the capping
plate arches up towards the plasmalemma to which it links (arrow). × 52 500
Fig. 5. Oblique section through the flagellar apparatus, m = mitochondrial profile; 3 = threestranded microtubular root (the four lines visible in this root type reflect oblique sections through
the electron dense sheets that underly the root microtubules; these sheets may bifurcate [see e. g.
Fig. 18]; open arrow = oblique section through the cylindrical structure, x 45 000
Fig. 6. Longitudinal section through transition region and basal body. The length of the transition
region is indicated by two open arrows, x 67 500
Fig. 7. Cross section through the transition region revealing stellate pattern. × 82 500
Fig. 8. Cross section through the free part of a flagellum. Two opposite outer doublet exhibit
microtubule septations in their B-tubules (arrows). x 82 500
C~
Q
360
M. M e l k o n i a n
e l e c t r o n d e n s e p l a t e (Fig. 24). T h e t e r m i n a l c a p is 1 5 - 2 6 n m t h i c k a n d is d i r e c t l y l i n k e d
to t h e c a p p i n g p l a t e of t h e r e s p e c t i v e b a s a l b o d y .
F o u r m i c r o t u b u l a r roots m a k e u p t h e f l a g e l l a r root s y s t e m {Figs 3, 5). E a c h b a s a l
b o d y is a s s o c i a t e d w i t h t w o of t h e f o u r roots. T r a n s v e r s e s e c t i o n s t h r o u g h t h e a p i c a l part
of a f e m a l e g a m e t e s h o w t h a t t h e f o u r roots a r e not a r r a n g e d in a t y p i c a l c r u c i a t e p a t t e r n ,
o p p o s i t e roots a r e c o n s i d e r a b l y d i s p l a c e d a g a i n s t e a c h o t h e r a n d a p p e a r to t a k e n o n s y m m e t r i c a l p a t h s i n s i d e t h e c e l l (Figs 3, 5). F u r t h e r m o r e at l e a s t t w o o p p o s i t e roots a r e
s t r u c t u r a l l y n o t i d e n t i c a l (Figs 3, 5). O n e root is a s s o c i a t e d w i t h a n e l e c t r o n d e n s e ring,
t h r e e - d i m e n s i o n a l l y a c y l i n d e r ( d e s c r i b e d in d e t a i l b e l o w ) , w h e r e a s t h e o p p o s i t e root is
n o t a s s o c i a t e d w i t h s u c h a s t r u c t u r e a n d a p p e a r s to c o n t a i n m o r e m i c r o t u b u l e s t h a n t h e
o t h e r root (Figs 3, 5). T h e f l a g e l l a r root s y s t e m d o e s not, t h e r e f o r e , s h o w 180 ° r o t a t i o n a l
symmetry.
T h e t w o s t r u c t u r a l l y d i f f e r e n t o p p o s i t e roots a t t a c h to t h e o u t e r s i d e s of t h e r e s p e c t i v e b a s a l b o d i e s (Figs 3, 5, 9, 10, 22, 23).
C r o s s - s e c t i o n s t h r o u g h t h e root a s s o c i a t e d w i t h t h e c y l i n d r i c a l s t r u c t u r e (Figs 9-16)
i n d i c a t e t h a t this root t y p e c o n s i s t s of t w o m i c r o t u b u l e s a l o n g its l e n g t h . T h e root
m i c r o t u b u l e s a r e n o t d i r e c t l y c o n n e c t e d to b a s a l b o d y t r i p l e t s b u t t h r o u g h e l e c t r o n d e n s e
m a t e r i a l (Figs 9, 11, 22, 23). In t h e p r o x i m a l p a r t s of t h e root m i c r o t u b u l e s , e l e c t r o n d e n s e
m a t e r i a l o v e r - a n d u n d e r l i e s t h e root m i c r o t u b u l e s {Figs 9-11). T h e u n d e r l y i n g m a t e r i a l
f o r m s a c h a r a c t e r i s t i c c r e s c e n t b o d y . In cross s e c t i o n s t h r o u g h t h e root t h e c r e s c e n t b o d y
is 2 0 0 - 2 5 0 n m w i d e (Figs 9 - 1 1 ) ; t h e c r e s c e n t s h a p e , h o w e v e r , is o n l y r e v e a l e d w h e n t h e
root is s e c t i o n e d in v e r t i c a l , l o n g i t u d i n a l d i r e c t i o n (e. g. Fig. 17). T h e c u r v a t u r e of t h e
c r e s c e n t b o d y is t o w a r d s t h e c e l l i n t e r i o r (Fig. 17). S e v e r a l s t r i a t i o n s in t h e c r e s c e n t b o d y
a r e c l e a r l y d i s c e r n i b l e in s u c h l o n g i t u d i n a l s e c t i o n s (Fig. 17). A s i m i l a r c r e s c e n t b o d y
w i t h s l i g h t l y s m a l l e r d i m e n s i o n s is a s s o c i a t e d w i t h t h e o p p o s i t e root (Fig. 22). T h e t w o s t r a n d e d root is l i n k e d to t h e c r e s c e n t b o d y b y t w o e l e c t r o n d e n s e sheets, w h i c h r u n t h e
w h o l e l e n g t h of this m i c r o t u b u l a r root (Figs 9-16). O n e of t h e s e s h e e t s o f t e n b i f u r c a t e s
(Figs 11, 13, 15). T h e e l e c t r o n d e n s e m a t e r i a l a b o v e t h e t w o root m i c r o t u b u l e s a t t a c h e s to
t h e c a p p i n g p l a t e (Fig. 9) a n d a l s o to t h e c y l i n d r i c a l s t r u c t u r e (Figs 9-11).
T h e r o o t o p p o s i t e t h e t w o - s t r a n d e d r o o t i n cross s e c t i o n s a l w a y s consists of 3
m i c r o t u b u l e s (Figs 18-21). I d e n t i f i c a t i o n of this root in cross s e c t i o n s is e a s y b e c a u s e it is
Figs 9-16. Cross sections through the two-stranded microtubular root and associated cylindrical
structure from proximal {Fig. 9) to distal parts {Fig. I6)~ All micrographs x 75 000
Fig. 9. Cross section through two-stranded microtubular root near its origin, b = basal body; cp =
capping plate; cb = electron dense crescent body; open arrow = cylindrical structure in cross
section associated with the two-stranded root by electron dense material
Fig. 10-12. Serial sections through the two-stranded microtubular root. The plasmalemma, overlying the cylindrical structure, exhibits a semicircular bulge (Fig. 12). In this area the crescent body
terminates. The root tubules are intimately associated with the cylindrical structure
Figs 13, 14. In the posterior region of the cylindrical structure, an area of the plasmalemma, adjacent
to the plasmalemma overlying the cylindrical structure, appears to be thickened (arrows in Figs
13, 14)
Figs 15, 16. Cross section through distal parts of the two-stranded microtubular root. The cylindrical
structure has terminated. In Fig. 15 electron dense material is still associated with the root
microtubules. Electron dense sheets underlying individual root microtubules often show bifurcations (arrows). In Fig. 16. The root microtubules are only accompanied by the electron dense sheets
o
~J
362
M. M e l k o n i a n
proximally associated with a crescent body (the other two roots lack a crescent body), but
l a c k s t h e c y l i n d r i c a l s t r u c t u r e (Fig 1 8 - 2 1 ) . O v e r l y i n g e l e c t r o n d e n s e m a t e r i a l c o n n e c t s
t h i s r o o t to t h e b a s a l b o d y (Fig. 19). E a c h m i c r o t u b u l e of t h e t h r e e - s t r a n d e d r o o t l i n k s to
t h e c r e s c e n t b o d y t h r o u g h a n e l e c t r o n d e n s e s h e e t (Fig. 18). T h e s e s h e e t s r u n t h e w h o l e
l e n g t h of t h e t h r e e - s t r a n d e d r o o t a n d s o m e t i m e s t h e y a l s o b i f u r c a t e ( F i g s 1 8 - 2 1 ) .
The other two opposite microtubular roots approach the basal bodies in their overlap
r e g i o n a p p r o x i m a t e l y a t r i g h t a n g l e s to t h e l o n g i t u d i n a l p l a n e of t h e t w o b a s a l b o d i e s
( s e e a l s o Fig. 1). T h e y r u n v e r y c l o s e to t h e p r o x i m a l e n d of o n e b a s a l b o d y (Fig. 24) a n d
a p p e a r t o t e r m i n a t e a t t h e c o n n e c t i n g f i b r e of t h e o t h e r b a s a l b o d y (Fig. 4), i n a n a r e a
w h e r e b o t h c o n n e c t i n g f i b r e s t o u c h e a c h o t h e r . In t h e m o s t p r o x i m a l c r o s s s e c t i o n s , t h e s e
r o o t s e x h i b i t t y p i c a l r o o t t u b u l e c o n f i g u r a t i o n s a n d a 3 + 1 or 4 + 1 - p a t t e r n is p r e s e n t
( F i g s 2 4 - 2 6 ) . T h e t u b u l e of t h e s e c o n d r o w ( t u b u l e 1, t e r m i n o l o g y a c c o r d i n g to M e l k o n i a n , 1978) is d i r e c t l y c o n n e c t e d to t h e t e r m i n a l c a p of o n e b a s a l b o d y (Fig 24), t h e o t h e r
t h r e e or f o u r m i c r o t u b u l e s l i n k to t h e c a p p i n g p l a t e (Fig. 24), In m o r e d i s t a l c r o s s s e c t i o n s
t h r o u g h t h e s e roots, r o o t t u b u l e 1 c h a n g e s p o s i t i o n r e l a t i v e to t h e o t h e r m i c r o t u b u l e s a n d
j o i n s t h e u p p e r r o w ( F i g s 2 5 - 2 7 ) . In t h i s r o o t t y p e , e a c h m i c r o t u b u l e is a g a i n a s s o c i a t e d
w i t h a n e l e c t r o n d e n s e s h e e t of s i m i l a r s t r u c t u r e a s i n t h e o t h e r t w o r o o t s (Figs 2 4 - 2 7 ) ;
o v e r l y i n g e l e c t r o n d e n s e m a t e r i a l is a l s o p r e s e n t b u t o n l y i n t h e m o r e d i s t a l p a r t s of t h e s e
r o o t s ( F i g s 26, 27). A c r e s c e n t b o d y is a b s e n t a n d o p p o s i t e r o o t s a p p e a r to b e i d e n t i c a l i n
s u b s t r u c t u r e . A f e w s e c o n d a r y c y t o s k e l e t a t m i c r o t u b u l e s a r e s o m e t i m e s s e e n to o r i g i n a t e
n e a r t h e r o o t m i c r o t u b u l e s (Fig. 25).
T h e c y l i n d r i c a l s t r u c t u r e w h i c h is a s s o c i a t e d w i t h t h e t w o - s t r a n d e d m i c r o t u b u l a r
r o o t is t h e m o s t c o n s p i c u o u s f e a t u r e of t h e f l a g e l l a r a p p a r a t u s of t h e f e m a l e g a m e t e s
(Figs. 9 - 1 6 , Fig. 17). F i g u r e s 9 - 1 4 r e p r e s e n t c r o s s s e c t i o n s t h r o u g h t h e c y l i n d r i c a l s t r u c t u r e f r o m its p r o x i m a l (Fig. 9) to its d i s t a l (Fig. 14) e n d . I n l o n g i t u d i n a l s e c t i o n , t h e
c y l i n d r i c a l s t r u c t u r e m e a s u r e s a r o u n d 1.4 ~ m i n l e n g t h (Fig. 17). T h e d i a m e t e r of t h e
e l e c t r o n d e n s e r i n g is v a r i a b l e , d e p e n d i n g u p o n w h i c h p a r t of t h e s t r u c t u r e is s e c t i o n e d .
T h e c e n t r a l r e g i o n h a s a m a x i m u m d i a m e t e r of 160 n m (Fig. 12). T h e r i n g is n o t
c o n t i n u o u s , b u t i n a n a r e a of 60 n m w i d t h , a m o r p h o u s e l e c t r o n d e n s e m a t e r i a l p e n e t r a t e s
i n t o t h e r i n g i n t e r i o r (Figs 1 1 - 1 3 ) . E l e c t r o n d e n s e m a t e r i a l o v e r l y i n g t h e t w o root
Fig. 17. Longitudinal section t h r o u g h the cylindrical structure (limits of the cylindrical structure are
g i v e n by the o p e n arrows), b = basal bodies; cb = two crescent bodies of opposite roots; cp =
c a p p i n g plate~ m = mitochondrial profile; 2 = longitudinal section t h r o u g h two-stranded microtubular root. x 67 500
Figs 18-21. Cross sections t h r o u g h the t h r e e - s t r a n d e d root (the root opposite the two-stranded root)
from proximal (Fig. 18) to distal (Fig. 21) regions. All micrographs × 75 000
Fig. 18. Cross section t h r o u g h proximal parts of the t h r e e - s t r a n d e d root. Electron d e n s e sheets link
i n d i v i d u a l root tubules to the u n d e r l y i n g crescent body (cb). b = basal body~ notice bifurcation of
one of the electron d e n s e sheets. A l t h o u g h t h e r e is some fuzzy material overlying the root
microtubules (Figs 19, 20) t h e r e is no cylindrical structure present in this root type.
Fig. 22. Cross section t h r o u g h the central region of the flagellar a p p a r a t u s b = basal bodies in their
overlap region; 3 = oblique section of t h r e e - s t r a n d e d microtubular root; open arrow = oblique
section t h r o u g h cylindrical struture. Only one of the two opposite roots is associated with a
cylindrical structure, but b o t h are associated with a crescent body (cb), × 60 000
Fig. 23. Cross section t h r o u g h a basal body. Long arrow = capping plate in cross section apparently
l i n k e d to 4 microtubule triplets of the basal body; open arrow = cylindrical structure in oblique
section, x 75 000
r~
11
364
M. Melkonian
microtubules link these tubules to the amorphous material (Figs 11-14). The cylindrical
s t r u c t u r e c a u s e s t h e p l a s m a l e m m a o v e r l y i n g t h e s t r u c t u r e to b u l g e c o n s i d e r a b l y o u t w a r d s s o t h a t a s e m i c y l i n d r i c a t p r o t r u s i o n of t h e c e l l s u r f a c e is f o r m e d {Figs 12, 13).
T o w a r d s t h e p l a s m a l e m m a , t h e e l e c t r o n d e n s e r i n g is s u r r o u n d e d b y g r a n u l a r m a t e r i a l
( F i g s 9 - 1 4 ) . A d j a c e n t to t h e s e m i c y l i n d r i c a l p l a s m a l e m m a a r e a i n s o m e c r o s s s e c t i o n s ,
p a r t of t h e p l a s m a l e m m a f a c i n g a w a y f r o m t h e f l a g e l l a r a p p a r a t u s a p p e a r e d to b e
t h i c k e n e d ( F i g s 13, 14, a r r o w s ) . T h i s p l a s m a l e m m a r e g i o n i n c r o s s s e c t i o n s m e a s u r e s
0 . 3 - 0 . 4 g m . T h i s a r e a is c o n f i n e d to t h e p o s t e r i o r r e g i o n of t h e s e m i c i r c u l a r b u l g e .
The electron dense material, overlying the root microtubules in some horizontal
l o n g i t u d i n a l s e c t i o n s , a p p e a r e d to b e s t r i a t e d , it w a s h o w e v e r n o t p o s s i b l e to d e t e r m i n e
t h e s t r i a t i o n p a t t e r n n o r to d e c i d e if 4 o r o n l y t w o s t r i a t e d r o o t s a r e p r e s e n t . S y s t e m II
f i b r e s ( t e r m i n o l o g y a c c o r d i n g to M e l k o n i a n , 1980a) w e r e n o t f o u n d i n t h e f l a g e l l a r
a p p a r a t u s of f e m a l e g a m e t e s .
°27
Figs 24-27. Cross section t h r o u g h root type II (four- or five-stranded root) from more proximal
{Fig. 24} to more distal (Fig. 27) regions. All micrographs × 75 000
Fig, 24. Cross sections t h r o u g h 5-stranded root in the region of basal body overlap, b = basal
bodies; long arrow = c a p p i n g plate; small arrow = root t u b u l e 1 links to the terminal cap
Fig. 25. More distal cross section t h r o u g h 5-stranded root, Two secondary cytoplasmic microtubules
(arrows) a p p a r e n t l y originate at this root. In this region no electron d e n s e material overlies the root
microtubules
Figs 26, 27. Gradual c h a n g e in root t u b u l e configuration, root tubule 1 (root tubule in the second
row} forms one row with the other four microtubules. Notice electron dense sheets associated with
individual microtubules
F l a g e l l a r root systems
365
DISCUSSION
Functional considerations
The f l a g e l l a r a p p a r a t u s of f e m a l e g a m e t e s of Bryopsis lyngbyei shows m a n y u n u s u a l
features that h a v e not y e t b e e n f o u n d in o t h e r g r e e n a l g a e . O n e of t h e m o r e c o n s p i c u o u s
structures in the f l a g e l l a r a p p a r a t u s is t h e c y l i n d r i c a l structure a s s o c i a t e d w i t h t h e twos t r a n d e d m i c r o t u b u l a r root. This structure is not p r e s e n t in the f l a g e l l a r a p p a r a t u s of
m a l e g a m e t e s in Bryopsis lyngbyei ( M e l k o n i a n , 1980a a n d u n p u b l i s h e d observations) or
Bryopsis maxima (Hori, 1977). T h e structure is s i n g u l a r a n d c o n n e c t e d to a s p e c i a l i z e d
r e g i o n of the p l a s m a l e m m a n e a r the f l a g e l l a r bases. It is s u g g e s t e d that it r e p r e s e n t s a
m a t i n g structure, an o r g a n e l l e a s s o c i a t e d w i t h the p l a s m a l e m m a of g a m e t e s a n d
e n g a g e d in the g a m e t i c fusion process ( G o o d e n o u g h & Weiss, 1975, M e l k o n i a n , 1980b).
T h e m a t i n g structures of Chlamydomonas reinhardii ( G o o d e n o u g h & Weiss, 1975) a n d
Ulva lactuca ( M e l k o n i a n , 1980b), w h i c h h a v e b e e n s t u d i e d in detail, are also a s s o c i a t e d
w i t h a t w o - s t r a n d e d m i c r o t u b u l a r root. Unfortunately, direct proof that t h e c y l i n d r i c a l
structure acts as a m a t i n g structure d u r i n g g a m e t i c fusion in Bryopsis is lacking, b e c a u s e
g a m e t i c fusion in our cultures w a s a rare e v e n t a n d sufficient q u a n t i t i e s of g a m e t e pairs
w e r e not a v a i l a b l e . T h e a b s e n c e of a d e f i n e d m a t i n g structure in m a l e g a m e t e s s u g g e s t s
that g a m e t i c fusion m i g h t occur in a way, w h i c h is different to the w e l l k n o w n
m e c h a n i s m s of Chlamydomonas reinhardii ( r e v i e w by G o o d e n o u g h , 1977) or Ulva
lactuca ( M e l k o n i a n , 1980b). In this c o n t e x t it is i n t e r e s t i n g to n o t i c e that in the o o g a m o u s
g a m e t i c fusion of Prasiola stipitata ( F r i e d m a n n & Manton, 1960) t h e m a l e g a m e t e s m a k e
contact w i t h t h e e g g s by o n e of t h e i r f l a g e l l a . M a l e g a m e t e s of Bryopsis Iyngbyei e x h i b i t
an u n u s u a l s w i m m i n g b e h a v i o u r in w h i c h o n e of the two f l a g e l l a is h e l d in an anterior
position, w h e r e a s the other f l a g e l l u m is t r a i l i n g ( u n p u b l i s h e d observations). It m i g h t
w e l l be that g a m e t i c fusion in Bryopsis is i n i t i a t e d in a s i m i l a r w a y as in Prasiola.
The association of a t w o - s t r a n d e d m i c r o t u b u l a r root w i t h a m a t i n g structure a n d our
u n p u b l i s h e d o b s e r v a t i o n s that a 4- or 5 - s t r a n d e d m i c r o t u b u l a r root is often s e e n n e a r the
e y e s p o t of t h e f e m a l e g a m e t e s of Bryopsis lyngbyei, further a d d to the i d e a that
m i c r o t u b u l a r f l a g e l l a r roots m i g h t b e e s s e n t i a l for d e t e r m i n i n g t h e e x a c t position of b o t h
the m a t i n g structure a n d t h e e y e s p o t in m o t i l e cells of m a n y g r e e n a l g a e (see also
M e l k o n i a n , 1981).
An i n t r i g u i n g o b s e r v a t i o n with r e s p e c t to the cruciate m i c r o t u b u l a r root system in
f e m a l e g a m e t e s of Bryopsis lyngbyei is that a strict 180 ° r o t a t i o n a l s y m m e t r y (as d e f i n e d
by Floyd et al., 1980) is absent. It has p r e v i o u s l y b e e n a s s u m e d that the m i c r o t u b u l a r
roots in cruciate f l a g e l l a r root systems, as w e l l as t h e b a s a l b o d y - a s s o c i a t e d fibrous
structures, e x h i b i t 180 ° r o t a t i o n a l s y m m e t r y (Floyd et aL, 1980). In g r e e n a l g a e a 180 °
rotational s y m m e t r y of the f l a g e l l a r a p p a r a t u s w a s not f o u n d in f e m a l e g a m e t e s of Ulva
lactuca, in w h i c h fibrous roots are u n e q u a l l y d i s t r i b u t e d on o p p o s i t e s i d e s of the f l a g e l l a r
a p p a r a t u s (Melkonian, 1980b). T h e s i n g l e e y e s p o t a n d m a t i n g structure i m p o s e a typical
left-right s y m m e t r y onto most g r e e n a l g a l cells. In g a m e t e s of Ulva lactuca it has b e e n
s h o w n that the e y e s p o t and m a t i n g structure are l o c a t e d on t h e s a m e side of the cell if
one uses the p l a n e of b e a t of the two f l a g e l l a to s e p a r a t e a cell into two h a l v e s ( R o b e n e k
& M e l k o n i a n , 1981). T h e p r e s e n t observation, that o n e pair of o p p o s i t e m i c r o t u b u l a r
roots contains different m i c r o t u b u l e n u m b e r s in t h e i n d i v i d u a l roots, a p p e a r s to b e the
366
M. M e l k o n i a n
first report i n the g r e e n algae. If one a s s u m e s that m i c r o t u b u l e n u m b e r s in a root are
f u n c t i o n a l l y i m p o r t a n t (at least with respect to the t w o - s t r a n d e d root only one exception
has b e e n noted, see Moestrup, 1978), a n i d e n t i c a l r e p l i c a t i o n of this root b e c o m e s a
necessity. V a r i a t i o n of root t u b u l e n u m b e r s i n opposite roots could t h e n m e a n that either
one or both of these roots are n o n - f u n c t i o n a l i n the original sense. E v i d e n c e for such a n
a s s u m p t i o n also comes from a recent o b s e r v a t i o n that i n zoospores of a species l a c k i n g
a n eyespot, root t u b u l e n u m b e r s in the respective root type are more v a r i a b l e in opposite
roots t h a n i n r e l a t e d species with zoospores e x h i b i t i n g a n eyespot (Melkonian, in
preparation).
Phylogenetic considerations
Before the d e t a i l e d structure of the flagellar a p p a r a t u s i n female g a m e t e s of Bryopsis
Iyngbyei is c o m p a r e d with that of other g r e e n algae, it is essential to compare it with the
flagellar a p p a r a t u s of the m a l e gametes. Bryopsis g a m e t e s show a n extreme case of
a n i s o g a m y a n d the differences f o u n d i n the flagellar a p p a r a t u s b e t w e e n female a n d
m a l e g a m e t e s m i g h t be a reflection of this a n i s o g a m y . It has a l r e a d y b e e n noted that
m a l e g a m e t e s lack the cylindrical structure, p r e s u m a b l y a m a t i n g structure (see above).
System II fibres are p r e s e n t in the m a l e g a m e t e s of Bryopsis 1yngbyei a n d Bryopsis
m a x / m a ( M e l k o n i a n 1980a; Hori, 1977} associated with a m i c r o t u b u l a r root c o n t a i n i n g 4
m i c r o t u b u l e s (unpubl. observations}. No system If fibres have b e e n found i n the flagellar
a p p a r a t u s of f e m a l e gametes. T h e y m i g h t b e f u n c t i o n a l i n the p e c u l i a r s w i m m i n g
b e h a v i o u r of the m a l e g a m e t e s (see above). O n the other hand, the crescent body
u n d e r l y i n g two m i c r o t u b u l a r roots i n the female g a m e t e is not p r e s e n t i n the m a l e
gamete. Differences in the d e t a i l e d structure of the m i c r o t u b u l a r roots m i g h t exist, b u t no
d e t a i l e d i n f o r m a t i o n on m i c r o t u b u l a r roots i n the m a l e g a m e t e s exists. In s u m m a r y both
g a m e t e types a p p e a r to have the following features of their flagellar apparatus in
c o m m o n : (a) a c o m p o u n d n o n - s t r i a t e d c a p p i n g plate, (b) t e r m i n a l caps, (c) m i c r o t u b u l e
s e p t a t i o n s i n B - t u b u l e s of outer doublets, (d) a n u n u s u a l l y long transition region with
a b s e n c e of a transverse d i a p h r a g m , (e) b a s a l b o d y overlap, (f) a cruciate m i c r o t u b u l a r
root system i n w h i c h the t w o - s t r a n d e d roots l i n k to the side of each b a s a l body that is not
a d j a c e n t to the other b a s a l body.
Recently Roberts et al. (1981, i n prep.) h a v e c o m p a r a t i v e l y s t u d i e d the flagellar
a p p a r a t u s of the a n i s o g a m e t e s of Derbesia tenuissima. The flagellar apparatus of the
m a l e g a m e t e s is n e a r l y i d e n t i c a l to that of Bryopsis m a l e gametes. The female g a m e t e s
of Derbesia tenuissima, however, h a v e some p e c u l i a r i t i e s in the structure of their
flagellar apparatus. The m i c r o t u b u l a r root system is cruciate with a 5-3-5-3-pattern a n d
therefore differs from that d e s c r i b e d i n this study. T h e path of the roots a n d the w a y in
w h i c h they o r i g i n a t e at the b a s a l bodies suggests that the 5 - s t r a n d e d roots correspond to
the 2- a n d 3 - s t r a n d e d roots of f e m a l e g a m e t e s of Bryopsis lyngbyei, w h e r e a s the 3s t r a n d e d roots correspond to the 4-(5-)stranded roots of m a l e a n d female gametes of
Bryopsis lyngbyei. A n e x p l a n a t i o n for this h e t e r o g e n e i t y of the m i c r o t u b u l a r root
systems of female g a m e t e s b e t w e e n Bryopsis a n d Derbesia is difficult to give. It should
h o w e v e r be n o t e d that a n eyespot is l a c k i n g i n female g a m e t e s of Derbesia a n d the
c y l i n d r i c a l structure also a p p e a r s to be a b s e n t i n female g a m e t e s of Derbesia. It is a g a i n
s u g g e s t i v e that a m i c r o t u b u l a r root system that is not associated with a n eyespot or
m a t i n g structure w o u l d b e m o d i f i e d d u r i n g evolution. In addition, Roberts et al. (in
F l a g e l l a r root systems
367
preparation) found a system II fibre i n the female g a m e t e s of Derbesia tenuissima, b u t
orientation of the fibre a n d association with the 5 - s t r a n d e d root, seems to i n d i c a t e that
the fibre is not h o m o l o g o u s to the system II fibre f o u n d i n the m a l e g a m e t e s of both
g e n e r a a n d m a y b e more r e l a t e d to the crescent b o d y of female g a m e t e s of Bryopsis
1yngbyei. Otherwise the structure of the flagellar a p p a r a t u s of a n i s o g a m e t e s of Derbesia
tenuissima agrees well with the g e n e r a l characteristics g i v e n a b o v e (a)-(f) for the
flagellar a p p a r a t u s of a n i s o g a m e t e s of Bryopsis.
If one compares the flagellar a p p a r a t u s of Bryopsis g a m e t e s with that of other g r e e n
algae it is obvious that the system is most similar to that of some g r e e n a l g a e that have
recently b e e n classified as a separate class, the U l v a p h y c e a e s e n s u Stewart & Mattox
(Stewart & Mattox, 1978; S l u i m a n et al., 1980). T h e features that the flagellar a p p a r a t u s
of Bryopsis g a m e t e s share with that of the U l v a p h y c e a e s e n s u Stewart & Mattox i n c l u d e
the c a p p i n g plate, t e r m i n a l caps, m i c r o t u b u l e septations, basal b o d y overlap, positional
relation of root types to basal bodies a n d i n part t r a n s i t i o n r e g i o n ultrastructure. The
U l v a p h y c e a e are a d d i t i o n a l l y characterized b y the p r e s e n c e of system I a n d system II
fibrous roots (Melkonian, 1980a; SMAC or rhizoplasts i n the t e r m i n o l o g y of Floyd et al.,
1980). System I fibres have not b e e n c o n c l u s i v e l y d e m o n s t r a t e d in female g a m e t e s of
Bryopsis, b u t the o v e r l y i n g electron d e n s e m a t e r i a l associated w i t h all four roots m i g h t
well be striated in horizontal l o n g i t u d i n a l section. A system I fibre associated with a twostranded root was found in zoospores of Derbesia tenuissima (Roberts et al., 1980).
System II fibres a p p e a r to be restricted to m a l e g a m e t e s of s i p h o n a l e a n g r e e n a l g a e
(Hori, 1977; M e l k o n i a n 1980a; Roberts et al., 1981, i n prep.). It is therefore not clear
w h e t h e r homology exists b e t w e e n the system II fibres of m a l e g a m e t e s of s i p h o n a l e a n
g r e e n a l g a e a n d the p r o m i n e n t system II fibres of the U l v a p h y c e a e s e n s u Stewart &
Mattox.
The C h l o r o p h y c e a e s e n s u Stewart & Mattox (Stewart & Mattox, 1975) have a
differently constructed flagellar apparatus, i n c l u d i n g p r e s e n c e of distal a n d proximal
striated c o n n e c t i n g fibres, a b s e n c e of t e r m i n a l caps a n d m i c r o t u b u l e septations a n d
basal body overlap, lack of system II fibres (at least in the a d v a n c e d m e m b e r s of this
group) a n d a different ultrastructure of the t r a n s i t i o n region. The positional r e l a t i o n of
root types to the respective basal b o d i e s is also different i n the C h l o r o p h y c e a e s e n s u
Stewart & Mattox a n d since this character has p r e v i o u s l y not b e e n u s e d for p h y l o g e n e t i c
considerations, some r e m a r k s should b e made. In the U l v a p h y c e a e s e n s u Stewart &
Mattox, in Bryopsis, Derbesia a n d some other g r e e n a l g a e related to each other (Microthamnion, Friedmannia, Trebouxia, Pleurastrum, the C h r o o l e p i d a c e a e ; M e l k o n i a n , in
preparation) basal body overlap results i n the formation of two different sides of each
basal body i n a basal body pair: a n outer side, facing a w a y from the other b a s a l body,
and a n i n n e r side to w h i c h the other b a s a l b o d y i n a p r o x i m a l r e g i o n attaches, tt is f o u n d
that in these groups of g r e e n a l g a e t w o - s t r a n d e d m i c r o t u b u t a r roots a p p r o a c h a n d l i n k to
the outer side of each b a s a l body, w h e r e a s m u l t i - m e m b e r e d roots (e. g. 4- or 5 - s t r a n d e d
roots) approach a r e g i o n b e t w e e n both b a s a l b o d i e s a n d l i n k to the i n n e r side of each
basal body. In the C h l o r o p h y c e a e s e n s u Stewart & Mattox, the opposite situation occurs.
Although there is no b a s a l body overlap in the Chlorophyceae, the two basal bodies of a
pair are displaced a g a i n s t each other b y a d i s t a n c e of a b a s a l b o d y d i a m e t e r or slightly
less. T w o - s t r a n d e d m i c r o t u b u l a r roots a p p r o a c h the area b e t w e e n the b a s a l bodies,
opposite roots forming a straight line (e. g. M e l k o n i a n , 1978). M u l t i - m e m b e r e d roots
368
M. M e l k o n i a n
a p p r o a c h e a c h b a s a l b o d y a t a n a n g l e of a r o u n d 45°; o p p o s i t e r o o t s a r e c o n s i d e r a b l y
d i s p l a c e d a g a i n s t e a c h o t h e r ( M e l k o n i a n , 1978).
In s u m m a r y , t h i s s t u d y h a s s h o w n t h a t t h e f l a g e l l a r a p p a r a t u s of f e m a l e g a m e t e s of
B r y o p s i s l y n g b y e i is u n i q u e a n d c o n t a i n s s t r u c t u r e s n o t p r e v i o u s l y s e e n i n o t h e r g r e e n
a l g a e , b u t i n s o m e g e n e r a l a s p e c t s r e s e m b l e s t h e f l a g e l l a r a p p a r a t u s of t h e U l v a p h y c e a e
s e n s u S t e w a r t & M a t t o x . M o r e s i p h o n a l e n g r e e n a l g a e s h o u l d b e s t u d i e d w i t h r e s p e c t to
t h e d e t a i l e d s t r u c t u r e of t h e f l a g e l l a r a p p a r a t u s i n b i f l a g e l l a t e c e l l s , b e f o r e f i n a l c o n c l u s i o n s a b o u t t h e p h y l o g e n e t i c a f f i n i t i e s of t h i s g r e e n a l g a l g r o u p c a n b e m a d e .
A c k n o w l e d g e m e n t s . I would especially like to thank Dr. Dr. h. c. P. Kornmann (Helgoland) for
d o n a t i n g the cultures of Bryopsis lyngbyei and his continuous interest in this study. Mrs. I.
Wachholz, Mrs. E. M a n s h a r d and Mrs. B. Surek have h e l p e d during various stages of the work.
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