Studies on sexual compatibility between HELGOLANDER MEERESUNTERSUCHUNGEN
Helgol6nder Meeresunters. 42, 469-476 (1988)
Studies on sexual compatibility b e t w e e n Ectocarpus
siliculosus (Phaeophyceae) from Chile and the
Mediterranean Sea*
D i e t e r G. Miiller
Fakult~t ffir Biologie der Universit~t; D-7750 Konstanz; Federal Repubh'c of Germany
ABSTRACT: Clonal isolates of the brown alga Ectocarpus sih'culosus (Ectocarpales} originating from
Naples (Mediterranean Sea} and southern Chile were compared in laboratory culture studies. The
two isolates showed distinctly different morphological characters, but very similar details of life
history and sexual reproduction. Gametes are sexually compatible; hybrid zygotes are formed and
sporophytes develop, which are fertile on the basis of mitotic spores. However, unilocular sporangia
were abortive, indicating segregational sterility caused by chromosomal mismatch during meiosis.
Although the biological species concept in a strict sense does not apply, and appreciable morphological variability exists in this cosmopolitan taxon, local populations are considered as representatives
of the same species.
INTRODUCTION
Ectocarpus siliculosus is a b r a n c h e d filamentous b r o w n alga. It has a cosmopolitan
distribution in cold to w a r m temperate waters. The reproduction a n d life history of this
species have b e e n studied over m a n y decades since the first reports on fertilization by
Berthold (1881). The sexual hfe history involves two generations, which are c o n n e c t e d by
fertilization a n d meiosis: dioecious haploid gametophytes alternate with a diploid
sporophyte of slightly different morphology. Sex determination is genotypic. The motile
gametes are morphologically identical, but physiologically different, showing either male
or female characters. Meiosis occurs in unilocular sporangia on diploid sporophytes.
Several accessory reproductive pathways have b e e n described. Unfertilized gametes
develop into haploid sporophytes, which show two different types of reproduction:
Plurilocular mito-sporangia perpetuate these genetically u n i s e x u a l sporophytes, while
zoids from unilocular sporangia can either re-establish the g a m e t o p h y t e phase, or
develop into n e w u n i s e x u a l sporophytes (Miiller, 1967):
The taxonomic treatment of the g e n u s Ectocarpus has varied greatly i n the past.
Newton (1931) hsted 52 taxa for the British flora. More recently, several g e n e r a such as
Feldmannia, Giffordia a n d Kuckuckia were introduced in order to separate a n u m b e r of
taxa. Russell (1966), e m p h a s i z i n g the great morphological variability, r e d u c e d the g e n u s
Ectocarpus of the British coast to two species, E. fasciculatus a n d 13. siliculosus. The very
few e x p e r i m e n t a l data available support separation of these two species. They do not
hybridize although m u t u a l attraction of gametes does occur fM~iIler & G a s s m a n n , 1980).
" Dedicated to Dr. Dr. h. c. Peter Kornmann on the occasion of his eightieth birthday.
9 Biologische Anstalt Helgoland, Hamburg
470
Dieter G. Mfiller
In a l a b o r a t o r y study, sexual compatibility a m o n g cultures of F.. siliculosus clones
from various g e o g r a p h i c a l areas w e r e c o m p a r e d (Mfiller, 1979). E x c e p t for two locahties
in North A m e r i c a with partial sexual incompatibility, zygote formation w a s o b s e r v e d
b e t w e e n all isolates. Therefore, all cultures showing p l a s m o g a m y w e r e c o n s i d e r e d to b e
m e m b e r s of the species E. siliculosus. Differences in b r a n c h i n g p a t t e r n s a n d g a m e t a n g i u m m o r p h o l o g y w e r e attributed to intraspecific variability.
In 1985, isolates from southern Chile s h o w e d zygote formation with clones from the
M e d i t e r r a n e a n Sea. With these cultures, crossing e x p e r i m e n t s w e r e e x p a n d e d to include
observations on the viability of zygotes a n d hybrid sporophytes, as well as the developm e n t of unilocular sporangia, which are the site of meiosis in b r o w n algae.
MATERIALS AND M E T H O D S
Origin of cultures
G a m e t o p h y t e s of M e d i t e r r a n e a n Ectocarpus siliculosus (DiUw.) Lyngb. w e r e coll e c t e d in the vicinity of the Stazione Zoologica at Naples, Italy, in 1959 a n d 1965 (Mfiller,
1967; Fig. 1). A s p o r o p h y t e of E. siliculosus from Chile a p p e a r e d in a r a w culture of
Year
Italy:
Chile:
Napol i
Puyug~ual)i
Nap-D
spor'oph.
female
2n
Nap-R
Cfl- I
male
femal e
spovoph.
Cli-8
2n
real e
G
1959
I
S
!
G
1965
G
f!
s
I
G
J
I
I
1985
I
o_--AeG
G
G
X
1986
G
I
I
I
I
I
I
I
|
!
I
G
G
S
/l
G s
~
G
/
S
/;
G
a/
\c.
Fig. 1. Ectocarpus sfliculosus from Naples, Italy and Puyuguapi, Chile. Pedigreee of gametophytes
used for the crossing experiments. G: Gametophyte; S: Zygotic or parthenogenetic sporophytes.
Solid hnes: Reproduction by zoids; broken lines: Propagation by fragmentation of filaments
Sexual compatibility in E. siliculosus from Chile and the Mediterranean
471
Adenocystis utricularis (Bory) Skottsb., which was collected in J a n u a r y 1985 at Puerto
P u y u g u a p i , XI Region (72036 , W; 44024 , S). Spores from unilocular s p o r a n g i a d e v e l o p e d
into m a l e a n d female gametophytes.
Maintenance
of c u l t u r e s
G a m e t o p h y t e s of the Italian isolates w e r e p r o p a g a t e d b y cutting filaments into fewcelled fragments, w h i c h r e g e n e r a t e d to n e w g a m e t o p h y t e s . A n a d d i t i o n a l pair of
g a m e t o p h y t e s was restored from a h e t e r o z y g o u s s p o r o p h y t e of the s a m e isolates (Fig~ 1).
The C h i l e a n clones could not b e Successfully p r o p a g a t e d by fragmentation, b e c a u s e
g a m e t o p h y t e filament fragments s h o w e d limited or no growth. Therefore, successive
crops of g a m e t o p h y t e s were g e n e r a t e d from unilocular s p o r a n g i a of u n i s e x u a l p a r t h e n o sporophytes (Fig. 1). Culture m e d i u m w a s natural North Sea w a t e r ( G e r m a n Bight,
salinity 28 %0, e n r i c h e d according to Provasoh (Start, 1978). Cultures w e r e k e p t at 12 +
1 ~ u n d e r illumination with d a y l i g h t - t y p e white fluorescent light at a p h o t o n fiuence rate
of 12 ~t 9 m 2 9 s t for 14 h p e r day. Culture m e d i u m was c h a n g e d at w e e k l y intervals.
Crossing experiments
Small, ram-size fragments of g a m e t o p h y t e s of opposite sex with m a t u r e m a g e t a n g i a
w e r e m o u n t e d b e t w e e n a microscope slide a n d cover-glass, using a 0,5 m m thick plastic
ring or vaseIine as spacer. In these mounts g a m e t e fusions could b e o b s e r v e d directly
u n d e r the microscope. Zygotes w e r e easily identified b y their size a n d d o u b l e o r g a n e l l e
contant. In o r d e r to follow their d e v e l o p m e n t , individual zygotes w e r e m a r k e d on the
outside of the cover-glass with a small ink-mark. T h e n the cover-glass was r e m o v e d a n d
p l a c e d u p s i d e - d o w n on the bottom of a plastic petri dish filled with culture m e d i u m . It
w a s thus possible to follow the d e v e l o p m e n t of individual zygotes until the g e r m h n g s
w e r e of sufficient size to be p i p e t t e d a n d isolated into a s e p a r a t e petri dish.
RESULTS
Development
a n d life h i s t o r y
T h e life history of Ectocarpus siticulosus from Naples, Italy has b e e n s t u d i e d a n d
d e s c r i b e d in detail (Mfiller, 1967). The isolates from Chile s h o w e d essentially the s a m e
characteristics:
(1) G a m e t o p h y t e s (Figs 2-3 a n d 6-8) are morphologically different from zygotic a n d
p a r t h e n o - s p o r o p h y t e s (Figs 10-11 a n d 12-14).
(2) Unfertihzed g a m e t e s d e v e l o p to genetically unisexual p a r t h e n o - s p o r o p h y t e s
(Figs 4 - 5 a n d 9), which can r e g e n e r a t e g a m e t o p h y t e s of the original sex t h r o u g h spores
from unilocular sporangia.
(3) Zygotes d e v e l o p to diploid h e t e r o z y g o u s sporophytes, the m e i o s p o r e s of w h i c h
d e v e l o p to e q u a l n u m b e r s of male a n d female g a m e t o p h y t e s .
(4) Zygotic a n d p a r t h e n o - s p o r o p h y t e s form plurilocular s p o r a n g i a as accessory
r e p r o d u c t i v e organs. Differences b e t w e e n isolates from Italy a n d Chile concern m o r p h o l ogy a n d growth characteristics of g a m e t o p h y t e s a n d sporophytes. G a m e t a n g i a of the
472
Dieter G. Mfiller
7
Figs 2-9. Ectocarpus siliculosus. Gametophytes and parthenosporophytes
Figs 2-3. Female gametophyte from Naples. 2 Habit (scale bar 0.5 mm); 3 Gametangia (scale bar 100
~m)
Figs 4-5. Female partheno-sporophyte from Naples. 4 Habit (scale bar 0.5 mm). Unilocular sporangium (scale bar 50 ~m)
Figs 6-8. Female gametophyte from Chile. 6 Habit (scale bar 0.5 mm). 7 Gametangia (scale bar 100
~m); 8 Gametangium (scale bar 50 ~m) Fig. 9. Male parthenosporophyte (scale bar 0.5 mm)
Sexual compatibility in E. siliculosus from Chile a n d the M e d i t e r r a n e a n
473
Chilean g a m e t o p h y t e s are more irregular in s h a p e a n d often a p p r e s s e d to the s u p p o r t i n g
filament (Figs 7-8). G a m e t o p h y t e s of the M e d i t e r r a n e a n isolates can b e p r o p a g a t e d
indefinitely b y r e g e n e r a t i o n of v e g e t a t i v e fragments, while this was found to b e impossib l e with the C h i l e a n strains. In contrast, m a t u r e sporophytes of the M e d i t e r r a n e a n clones
cease g r o w t h a n d e x h a u s t t h e m s e l v e s in s p o r a n g i a formation, while s p o r o p h y t e s of the
Chilean cultures can b e m a i n t a i n e d as filamentous mats over long p e r i o d s of time.
Crossing experiments
C o m b i n a t i o n of female g a m e t e s from N a p l e s with male g a m e t e s from Chile as well
as the reciprocal crossing s h o w e d the typical p a t t e r n of "isogamy" in b r o w n algae: the
female g a m e t e s settle d o w n on the s u b s t r a t u m a n d incorporate their flagella. T h e n m a l e
g a m e t e s are attracted a n d attach with the tip of their front flagellum to the female
gamete. Eventually one male gamete, including both of its flagella fuses with the female
gamete. The zygotes resulting from crossings of N a p l e s female • Chile m a l e d e v e l o p e d
into plants with typical sporophyte h a b i t (Fig. 15). At the a g e of a b o u t 4 w e e k s n u m e r o u s
unflocular s p o r a n g i u m initials a p p e a r e d , which did not increase in size a n d s h o w e d no
further d e v e l o p m e n t (Figs 18-20), in contrast to normal s p o r a n g i a on h a p l o i d (Fig. 5) or
diploid sporophytes (Fig. 14). Careful s c r e e n i n g of all h y b r i d plants did not r e v e a l n o r m a l
unilocular sporangia, but functional plurilocular m i t o - s p o r a n g i a on older p l a n t s (Figs
16-17). Their zoids r e e s t a b l i s h e d n e w sporophytes with the s a m e p h e n o m e n o n of
abortive unilocular s p o r a n g i u m initials. H y b r i d s from reciprocal crosses (Chile f e m a l e x
N a p l e s male) d e v e l o p e d more slowly, b u t e v e n t u a l l y s h o w e d the s a m e characteristics.
DISCUSSION
The w o r k reported a b o v e d e a l s with sexual compatibility of g e o g r a p h i c a l l y s e p a r a t e d populations. If properly done, it should include a r e a s o n a b l e n u m b e r of individuals,
r e p r e s e n t i n g the w h o l e genetic r a n g e of the populations studied. H o w e v e r , the l a r g e
a m o u n t of laboratory culture w o r k r e q u i r e d p r e c l u d e s this a p p r o a c h in a p r e l i m i n a r y
study. Moreover, Ectocarpus sfliculosus from Chile was not obviously p r e s e n t in its
n a t u r a l habitat at the time of collection, but a p p e a r e d in culture as a single e p i p h y t i c
s p e c i m e n on fragments of Adenocystis. A systematic study of 176 i n d i v i d u a l p l a n t s t a k e n
from various natural habitats at N a p l e s r e v e a l e d 94 female a n d 82 m a l e functional
gametophytes. In addition, 5 plants with g a m e t o p h y t e habit w e r e found the zoids of
which were a p p a r e n t l y non-sexual. (Mfiller, 1976). Thus, it can b e a s s u m e d with r e a s o n a b l e certainty, that a n y p a i r of sexually c o m p a t i b l e g a m e t o p h y t e s from a specific locality
can b e t a k e n as r e p r e s e n t a t i v e of the local p o p u l a t i o n for its r e p r o d u c t i v e characteristics.
E. siliculosus from t h e continental coast of southern Chile is sexually c o m p a t i b l e with
plants from the M e d i t e r r a n e a n Sea. This observation confirms a n d e x p a n d s earlier
findings: G a m e t e s of E. siliculosus from both sides of the North Atlantic, the M e d i t e r r a n e a n Sea a n d Australia w e r e found to interact sexually and to form h y b r i d z y g o t e s
(Mfiller, 1979). Since the p r e s e n t study a d d s to this pattern isolates from t h e Pacific coast
of South America. it seems possible that sexual compatibility of g a m e t e s a p p l i e s to E.
siliculosus on a w o r l d - w i d e scale. The partial sexual incompatibility f o u n d in two
populations of the North A m e r i c a n Atlantic coast (Mfiller, 1979) has b e e n i n t e r p r e t e d as
474
D i e t e r G. M/iUer
Figs 10-20. Ectocarpus siliculosus. Sporophytes originating from zygotes
Figs 10-11. Isolate from Naples. 10 Habit (scale bar 0.5 mm); 11 Unilocular sporangia (scale bar 100
~m)
Figs 12-14. Isolate from Chile. 12 Habit [scale bar 0.5 mm); Figs 13 (scale bar 100 ~tm) and 14 (scale
bar 50 ~m): unilocular sporangia
Figs 15-20. Sporophytic plants derived from hybrid zygotes Naples female • Chile male. 15 Habit
(scale bar 0.5 re.m); 16-17 Mature (16) and empty (17) plurflocular sporangia (scale bars 100 ~tm);
I8-20 Multiple initials of abortive un/locular sporangia (scale bars: Fig. 18 100 ~tm, Figs 19-20 50 ~tm)
Sexual compatibility in E. siliculosus from Chile and the Mediterranean
475
first steps towards speciation through sexual isolation. The results reported here show
clearly, that plasmogamy does not necessarily imply interbreeding between geographically separated populations. Mthough hybrid sporophytes were found to be viable
and to reproduce by mitosphores, meiosporangium initials do not continue development,
and no meiospores were formed. Since unilocular sporangium initials on diploid
sporophytes are the site of meiosis in brown algae, this finding is not unexpected for
hybrids between parents which differ enough to cause severe problems in chromosome
pairing (segregational sterihty; Dobzhansky et al., I977). Thus, the results of this study
are explained most plausibly in the following way: E. siliculosus from the Mediterranean
Sea and the Pacific coast of Chile are similar enough genetically to permit plasmogamy
and the development of viable and fe~.ile hybrids on the basis of mitosis. On the other
hand, plants from these two locations are different enough to exclude functional chromosome pairing in meiosis, prohibiting gene exchange via sexual reproduction. Even
though hybrids are formed, the two populations cannot interbreed sexually, and thus do
not fully meet the biological species definition. Nevertheless, for practical reasons it
seems permissible to use plasmogamy as a useful indicator to justify the inclusion of all
sexual populations studied so far in the species E. si~culosus. This concept seems
acceptable, since the closely related species E. faciculatus does not show plasmogamy
with E. siBculosus (Mfiller & Gassmann, 1980) and thus is fully separated.
It should be possible to evaluate experimentally the above interpretation for the
observed hybrid sterihty. Fertility in such hybrids can often be restored by polyploidization. Like in many domestic higher plants doubhng of the hybrid's chromosome set to an
allotetraploid state can be expecte d to reestablish successful meiotic chromosome pairing. Although spontaneous tetraploid individuals have been observed in cultures of E.
siBculosus (Mfiller, 1967}, high chromosome numbers corresponding to this level are not
normally observed in brown algae,
As indicated above, E. siliculosus populations on many, and perhaps all cold- and
warm-temperate coasts of the globe are genetically close enough to perform zygote
formation. This raises the question about the age of the species in geological terms, its
origin and its distribution. E. siliculosus is an opportunistic species, easily growing on
substrates such as ship hulls or drifting material. Theoretically, it cannot be excluded that
it has spread over the world with historic and modern ship traffic. This possibility,
however, seems unlikely for several reasons:
(1) Bolton (1983) used the cultures of the compatibility study (Mfiiler, 1979) to
examine their ecophysiological characteristics. He showed that local populations of E.
sillculosus are genetically adapted to the temperature conditions of their habitats and
therefore represent well-defined ecotypes. Furthermore, local populations show genetically fixed distinct morphological differences (Mfiller, 1979}.
(2) Comparing the geographically most distant representatives from the North
Atlantic, Pacific Chile and Australia, the morphological differentiation is quite substantial
and the geographic distribution indicates that E. sillculosus does not cross the tropical
belt by natural means under present climatic conditions.
(3) Recent findings on reproductive physiology of brown algae suggest that some
characters are extremely conservative in evolution. All 26 species studied from 3 families
in the order Laminariales (Laminafiaceae, Alariaceae and Lessoniaceae) were found to
use the sexual pheromone lamoxirene (Maier & Mfiller, 1986). In addition, physiological
476
D i e t e r G. Mfiller
a n d s t r u c t u r a l d e t a i l s of s p e r m r e l e a s e a n d c h e m o t a x i s a r e a l m o s t i n d i s t i n g u i s h a b l e
w i t h i n t h i s g r o u p of families. T h i s m e a n s t h a t f u n d a m e n t a l d e t a i l s of r e p r o d u c t i v e
p h y s i o l o g y i n t h e L a m i n a r i a l e s h a v e s u r v i v e d u n c h a n g e d t h e d i v e r g e n c e of families,
g e n e r a a n d s p e c i e s . T h e s e a r g u m e n t s s u p p o r t t h e c o n c l u s i o n t h a t E. sillculosus is a v e r y
o l d s p e c i e s . It m a y h a v e c r o s s e d t h e e q u a t o r b e l t d u r i n g t h e c o l d p e r i o d s of t h e
P l e i s t o c e n e j o i n t l y w i t h o t h e r c o l d - w a t e r f o r m s l i k e h4acrocystis (Lfining, 1985}. Possibly,
it m a y e v e n h a v e i n h a b i t e d t h e c o a s t s of P a n g a e a , a n d s u b s e q u e n t l y f o l l o w e d t h e d r i f t i n g
c o n t i n e n t s to t h e i r p r e s e n t p o s i t i o n . T h e s e i d e a s a r e a d m i t t e d l y s p e c u l a t i v e a t t h e p r e s e n t
t i m e . M o r e e x p e r i m e n t a l e v i d e n c e w i t h p o p u l a t i o n s f r o m o t h e r p a r t s of t h e w o r l d ,
s u p p l e m e n t e d b y b i o c h e m i c a l d a t a c o u l d h e l p to e v a l u a t e t h e a g e of t h e s p e c i e s E.
siliculosus.
Acknowledgements. Thanks are due to Dr. R. Westermeier, Valdivia, for organizing a collection
expedition to southern Chile, a n d to the Deutsche Forschungsgemeinschaft for. travel funds.
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