J. Phycol. 34, 126–137 (1998)
SEXUAL REPRODUCTION IN THE PENNATE DIATOMS PSEUDO-NITZSCHIA
MULTISERIES AND P. PSEUDODELICATISSIMA (BACILLARIOPHYCEAE)1
Nickolai A. Davidovich
Diatoms Biology Laboratory, Karadag Branch of the Institute of Biology of the Southern Seas, Feodosiya, 334876 Ukraine
and
Stephen S. Bates2
Fisheries and Oceans Canada, Gulf Fisheries Centre, P.O. Box 5030, Moncton, New Brunswick, Canada E1C 9B6
ABSTRACT
Clones of the domoic-acid-producing pennate diatom
Pseudo-nitzschia multiseries (Hasle) Hasle and of the
potentially toxic P. pseudodelicatissima (Hasle) Hasle
normally decrease in cell size in culture until they eventually die without undergoing sexual reproduction to regain the largest cell size. However, we induced sexual reproduction by mixing individual exponentially growing
clonal cultures of the appropriate minimal cell size under
the same conditions that are normal for vegetative growth.
We observed pairing of parent cells (gametangiogamy); production of four morphologically isogamous, nonflagellated
gametes per gametangial pair; rearrangement of the gametes and their fusion to form zygotes, revealing physiological anisogamy; enlargement of auxospores; and formation
of long initial cells. Our observations of allogamous reproduction are consistent with those reported for other dioecious pennate diatoms. Clones of P. pseudodelicatissima
from the Black Sea and from the CCMP culture collection
failed to auxosporulate when mixed together, although they
are the same species according to scanning electron microscopy. The range in apical length of P. multiseries was
broader than that reported in the literature for field samples, necessitating a modification of the species description.
Knowledge of the pattern and timing of sexual reproduction in Pseudo-nitzschia spp. may provide insights into
their bloom dynamics.
Such a fundamental biological characteristic as
sexual reproduction should not be ignored in studies of Pseudo-nitzschia because of its possible relationship to bloom dynamics and cell toxicity. As in most
other diatoms, continued vegetative division results
in a decrease in mean cell size, which must be restored to its maximal size by auxospore formation,
usually by means of sexual reproduction (Drebes
1977, Round et al. 1990, Mann 1993a). However,
there is only one paper containing observations of
sexual reproduction in P. multiseries Hasle (Hasle)
(Subba Rao et al. 1991), and it has provoked considerable debate and criticism (Fryxell et al. 1991,
Rosowski et al. 1992, Subba Rao et al. 1992, Mann
1993a, b). The main contention is that the type of
sexual reproduction reported is contrary to the normal process known for pennate diatoms. Subba Rao
et al. (1991) reported the presence of flagellated
gametes in P. multiseries cultures, an observation
more consistent with a contamination by fungal parasites (Rosowski et al. 1992). The absence of flagellated gametes is characteristic of pennate diatoms
(Geitler 1935, Mann 1993a). Furthermore, Subba
Rao et al. (1991) concluded that oogamy (production of large, nonmotile female gametes that are fertilized by small, motile, flagellated male sperm) was
the mode of reproduction. This contradicts numerous other observations of auxosporulation in pennate diatoms that show that allogamy (accomplished
by the pairing of gametangia, or ‘‘parent cells’’), not
oogamy, is the most frequent mode of their sexual
reproduction (Geitler 1969, 1973, Wiese 1969, Drebes 1977, Round et al. 1990). In some pennate diatoms, constitutional automixis or apomixis takes
place, whereas others illustrate heterogamy (MagneSimon 1960, Roshchin 1994). Only in Rhabdonema
spp. was modified oogamy described (von Stosch
1958, Geitler 1973), and this is not the typical mode
of oogamy. Therefore, the report of flagellated gametes and of oogamy in P. multiseries (Subba Rao et
al. 1991) is anomalous. The only other report of
sexual reproduction in a Pseudo-nitzschia species is
for P. subcurvata (Hasle) Hasle, which showed auxospore formation of a type consistent with other
pennate diatoms (Fryxell et al. 1991).
Until recently, it was believed that most diatom
species were monoecious, involving production of
Key index words: amnesic shellfish poisoning; auxospore;
diatom; Pseudo-nitzschia multiseries; P. pseudodelicatissima; sexual reproduction
During the past decade, diatom species of the genus Pseudo-nitzschia have been actively investigated,
partly because of their implication in amnesic shellfish poisoning (ASP) in humans, which is caused by
the neurotoxin domoic acid (Bates et al. 1989, Todd
1993). Investigations have touched on numerous aspects of the biology of this algal genus: nomenclature, taxonomy, morphology, distribution, bloom
dynamics (Bates et al. 1998), ecophysiology, toxin
production (Bates 1998), immunofluorescence features (Bates et al. 1993), and molecular biology
(Scholin 1998).
1
2
Received 26 June 1997. Accepted 8 November 1997.
Author for reprint requests; e-mail [email protected].
126
127
SEXUAL REPRODUCTION IN PSEUDO-NITZSCHIA
both male and female gametes by the same clone
(Wiese 1969, Drebes 1977). Therefore, investigators
did not seriously consider the possibility that clones
could be made to reproduce sexually simply by mixing them together. However, data obtained during
the last two decades indicate that some pennate diatoms are dioecious (production of either ‘‘male’’
or ‘‘female’’ gametes by two gametangia in different
clones), for example, Licmophora ehrenbergii, L. abbreviata, Striatella unipunctata, Haslea subagnita, and
Nitzschia longissima (Roshchin 1994, Roshchin and
Chepurnov 1996). Other pennate diatoms, for example, Synedra tabulata, Fragilaria delicatissima, Achnanthes brevipes var. intermedia, A. longipes, and
Nitzschia lanceolata, combine dioecious and monoecious types of mating (Roshchin 1994, Roshchin and
Chepurnov 1996, Chepurnov and Mann 1997). Although some of these species behave unisexually
during interclonal matings, particular clones may be
capable of limited intraclonal (monoecious) reproduction. Here we show that species of Pseudo-nitzschia have a dioecious type of mating and that allogamy
is their common mode of sexual reproduction.
MATERIALS AND METHODS
Clones of Pseudo-nitzschia pseudodelicatissima (Hasle) Hasle, designated as ND-1, ND-2, and ND-3, were isolated in May, April,
and February 1996, respectively, from plankton samples collected
in the Black Sea near Karadag on the southeast coast of the Crimean peninsula. Clones were maintained in exponential growth
by weekly reinoculation into fresh culture medium, prepared with
pasteurized (heated to 708–758 C) filtered seawater (about 18‰
salinity) enriched with nutrients (Chepurnov and Mann 1997).
Cells were grown in borosilicate petri dishes (9 cm diameter) containing 40 mL of medium and were exposed to natural light in a
temperature-controlled room at 19 6 18 C. In November 1996,
these clones were moved to the Gulf Fisheries Centre (Moncton,
Canada), where they were grown as described below. The culture
medium f/2 (Guillard 1975) was diluted with distilled water to a
salinity of 18‰ and estimated with a refractometer (model
A366ATC, Fisher Scientific).
Clones of P. pseudodelicatissima (CCMP-1562 and CCMP-1564)
were received from the Provasoli-Guillard National Centre for
Culture of Marine Phytoplankton (CCMP), West Boothbay Harbor, Maine; they were originally isolated in April 1993 by P. Hargraves from Bass River, Cape Cod, Massachusetts. The clones were
maintained in batch culture in Erlenmeyer flasks containing 75
mL of medium f/2 (29‰ salinity) at 208 C and a photon flux
density of 20–80 mmol·m22·s21 (12:12 h LD cycle) and were measured with a quantum/radiometer/photometer (model LI-189,
type ‘‘SA’’ quantum sensor, Li-COR, Lincoln, Nebraska).
Clones of P. multiseries (KP-103, KP-104, and KP-105) were isolated by K. Pauley from Cardigan Bay, Prince Edward Island, Canada, in December 1993 and were maintained in f/2 medium as
above. Scanning electron microscopy was used (Bates et al. 1989)
to verify the identity of each of the species studied.
Culture conditions suitable for sexual reproduction were essentially those used to maintain the stock cultures in a state of vegetative growth. The clones were kept in exponential growth at a
photon flux density of 20–120 mmol·m22·s21, either at 148 C and
12:12 h LD or at 208 C and 10:14 h LD.
Parent clones were mixed during the period of exponential
growth (days 3–5) in borosilicate petri dishes (5 cm diameter)
containing 15 mL of f/2 medium. Triplicate mixtures were made,
containing 0.15, 0.30, or 0.45 mL of each parental clone. The
mixed cultures were examined after 2–4 days for signs of sexual
reproduction. Cultures were observed in situ by light microscopy
(Dialux-20, Leitz) with a water-immersion objective (303) placed
FIG. 1. Schematic of a Pseudo-nitzschia auxospore, within which
an initial cell is formed. Measurements of the transapical auxospore diameter were made at ‘‘a’’; the position at which the auxospore diameter is equal to the zygote diameter is shown by ‘‘b’’.
directly in the petri dishes so as not to disturb the spatial relationship between the cells. Photomicrographs were taken using
XP2–400 (Ilford) film. Apical cell length was measured with an
ocular micrometer (precision was about one-quarter of an ocular
unit, or 0.8 mm).
The volume of the spherical gametes and zygotes was calculated
using measurements of their diameters. Auxospores were assumed to have a cylindrical shape, and their volume was calculated using the auxospore diameter and length of the initial cells
(Fig. 1).
RESULTS
Scheme of sexual reproduction. Gamete production
started 2–3 days after mixing either all clones of the
same species or certain pairs from different clonal
cultures. Mixture experiments therefore showed
that there were only two types (sexes) of clone, designated as ‘‘1’’ and ‘‘2’’. Each type was unable to
cross with clones belonging to the same type (intraclonal breeding not observed, except for one possible occurrence; see Discussion) but was able to
mate with clones belonging to the other type (interclonal breeding). Results of such crosses are
shown in Table 1. Therefore, P. multiseries clone KP105 was determined to be the opposite sex of clones
KP-103 and KP-104. For P. pseudodelicatissima, clone
ND-3 sexually reproduced with clones ND-1 and ND2. The two clones of P. pseudodelicatissima (CCMP1562 and CCMP-1564) could interbreed. All attempts to cross the ND- clones with the CCMPclones failed to result in auxosporulation in spite of
the fact that they are all P. pseudodelicatissima according to their morphology as seen by SEM (Fig.
2). The ND- clones were substantially wider (2.1–2.5
mm) than the CCMP- clones (1.4–1.6 mm), although
they were still within the acceptable range for P.
pseudodelicatissima (Hasle and Fryxell 1995). Some
gametangia in cultures of P. pseudodelicatissima (NDclones) and P. multiseries had deformities (cf. Subba
Rao and Wohlgeschaffen 1990), but this did not prevent them from entering into sexual reproduction;
however, most cells in the parental pairs were not
deformed.
128
NICKOLAI A. DAVIDOVICH AND STEPHEN S. BATES
TABLE 1. Results of crosses between all combinations of clones of Pseudo-nitzschia pseudodelicatissima and P. multiseries; 1 5 sexual reproduction
observed; 0 5 absence of sexual reproduction.
P. pseudodelicatissima clones
P. multiseries clones
Clone
ND- 1
ND-2
ND-3
CCMP-1562
CCMP-1564
KP-103
KP-104
KP- 105
ND-1
ND-2
ND-3
CCMP-1562
CCMP-1564
KP-103
KP-104
KP-105
0
0
1
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
1
0
1
0
The general pattern of sexual reproduction for P.
multiseries and P. pseudodelicatissima is shown in Figures 3–5, and the specific characteristics are summarized in Table 2. Pairing of parent cells (gametangiogamy), one from each of the two clones, was
the first stage (Figs. 3A, 4A, B). Gametangia contacted each other valve to valve, lying parallel. No
mucilage envelope or copulation tube was observed.
Pairing was possible both between two single cells
(Figs. 3A, 4A) and between one single cell and another still linked in a chain (Fig. 4B); the individual
cells and the chains are both motile (see MacPhee
et al. 1992). Pairs of single cells were most often
found in P. multiseries. Sometimes, more than one
pair could be formed within the chain. If cells forming a chain were small, they first had to become
separated from each other to pair with the larger
cells in the chain of the opposite sex because pairing requires close alignment of the cells.
The next stage was gametogenesis. Each cell in a
pair divided meiotically. The contents of the cells
then divided along the apical plane and after rearrangement (Fig. 3A) formed spherical gametes (Fig.
3B). Therefore, each gametangium yielded two morphologically isogamous nonflagellated gametes
(Figs. 3C, 4C), but their behavior was anisogamous
(physiological anisogamy). Although it was not obvious because of the close contact between gametangia, we could still conclude (on the basis of differences in cell size of each clone; see below) that in
each pair one gametangium produced two active gametes and the other two passive gametes (5 cis-anisogamy; Stickle 1986, Mann 1982a, 1993a). The
frustules opened completely, permitting both gametes in one cell to move by amoeboid action toward the passive gametes in the other gametangium
(Figs. 3C, 4C, D). This stage of sexual reproduction
was not documented in the CCMP- clones of P. pseudodelicatissima because it occurred less frequently.
Single gametangia (i.e. not in a pair) were occasionally found (Fig. 3E). Such cells may have been induced to form gametes as a result of their proximity
to another pair of cells undergoing gametogenesis.
Alternatively, the cells may have originally been
paired but subsequently became separated if, for example, the bonding between pairs was weak.
Only one pair of gametes fused at a time. Which
two gametes did fuse depended on the relative positions of the gametangia; each gamete fused with
the nearest one in the other gametangium. If the
two gametangia were well lined up with each other,
then both gametes in each gametangium could
FIG. 2. Scanning electron micrographs of (A) Pseudo-nitzschia multiseries (clone KP-103), (B) P. pseudodelicatissima (clone ND-1), and
(C) P. pseudodelicatissima (clone CCMP-1562). The central interspace, containing the central nodule, is indicated by an arrowhead in B
and C. Scale bar 5 1.0 mm.
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SEXUAL REPRODUCTION IN PSEUDO-NITZSCHIA
TABLE 2. Patterns of sexual reproduction in clones of the pennate diatoms Psudo-nitzschia pseudodelicatissima and P. multiseries, compared to
descriptions in the literature.
Number of gametangia
sufficient for auxosporulation
Relative position of gametangia
Existence of mucilage
envelope
Existence of copulation
tube
Number of gametes produced per gametangium
Rearrangement of gametes
Gamete morpohology
Gamete activity
Number of active gametes in gametangia
pair
Connection of auxospores with parent
frustule(s)
Disposition of auxospores relative to parent frustules
P. pseudodelicatissima
Possibilities
documenteda
Characteristic
b
(ND – clones)
P. multiseries
(CCMP – clones)
1, 2
2
2
2
Irregular, regular (contact girdle-girdle,
valve-valve, pole-pole)
Yes, no
Initially valve-valve, then
irregular
Regular, valve-valve
Initially valve-valve, then
irregular
No
No
No
Yes, no
No
No
No
0 , 1, 2
2
2
2
Yes, no
Yes
Yes
Yes
Isogamy, anisogamy
Isogamy, cis-anisogamy,
trans-anisogamy
1 1 0d, 1 1 1e,
2 1 0f, 2 1 2g
Isogamy
cis-anisogamy
Isogamy
cis-anisogamy
Isogamy
cis-anisogamy (?)
210
210
210
Yes, no
Yes, not strong
Yes
Yes, not strong
Irregular, regular (parallel, perpendicular)
Irregular
Regular: perpendicular
to parent frustule
Irregular
c
a
Geitler 1935, 1973, Wiese 1969, Drebes 1977, Round et al. 1990, Mann 1993a.
Sometimes, more than two gametangia take part in auxosporulation (see Geitler 1973, Roshchin 1994), but only one or two of them
is actually needed for the auxosporulation to be successful; one gametangium exists during automixis (pedogamy or autogamy) and
apomixis; two gametangia exist during modified oogamy and allogamy.
c No gametes produced during apomixis.
d One gamete produced per gametangium, where one is active and the other passive (anisogamy, type IIB; see Geitler 1973).
e If one gamete is produced per gametangium and both are active, then isogamy (type IIA); if two gametes are produced per gametangium and one is active in each gametangium, then trans-anisogamy (type IA1; see Mann 1982a).
f Two active gametes in one gametangium (cis-anisogamy, type IA2).
g Two active gametes in each gametangium (isogamy, type IB or IC).
b
eventually fuse successfully. However, if one gametangium was in a higher position relative to the other, then the widely separated end gametes were unable to fuse. Plasmogamy of two gametes to form a
spherical zygote (Figs. 3E, 5A) took only 1–2 min,
which is very short relative to the other stages of
reproduction. Sometimes, plasmogamy of the other
gamete pair was delayed for several minutes to
hours or did not occur at all, especially if the gametes were widely separated. Gametes were unable
to fuse if they had lost contact with their parent frustule; they apparently required a substrate for amoeboid movement. Many free-floating, single gametes
could therefore be observed in a culture (e.g. Fig.
3A–C).
The resulting zygotes then started to expand to
form auxospores (Figs. 3F, G, 4E, 5B), although not
necessarily synchronously. Sometimes, one auxospore enlarged relatively rapidly to its maximal size,
whereas the other was still only at the beginning of
expansion. As a rule, the two auxospores eventually
attained a similar length (Fig. 4E). The auxospores
remained attached to only one of the parent frus-
tules (Fig. 3F, H) (see below). Fully expanded auxospores contained two unfused nuclei and usually
four chloroplasts in a thin layer of peripheral cytoplasm (Figs. 3H, I, 4E). Most of the auxospore volume appeared to be occupied by a single vacuole.
Caps on the ends of the auxospore (Fig. 3H) represent the remnants of the wall secreted by the zygote (Mann 1993b) and could remain until formation of the initial cell. The restored, initial cell was
formed inside the auxospore (Figs. 3J, 5C) as a result of a two-step process of frustule construction.
Sometimes, the auxospores were slightly bent (Fig.
3I), giving rise to bent initial cells (Fig. 5C). The
entire process of sexual reproduction, from gamete
formation to the appearance of the initial cells, took
2–4 days.
Differences were observed among clones and between species in particular features of sexual reproduction (Table 2). The degree of attachment between a pair of parent cells or between a parent
frustule and the auxospores differed between isolates. Gametangia were most tightly attached in the
ND- clones of P. pseudodelicatissima, resulting in a
130
NICKOLAI A. DAVIDOVICH AND STEPHEN S. BATES
SEXUAL REPRODUCTION IN PSEUDO-NITZSCHIA
131
FIG. 4. Pseudo-nitzschia pseudodelicatissima (ND- clones). (A) separate pair of parent cells during rearrangement of gametes; (B) the
same stage as shown in A, but the pair forms part of a chain; note that rearrangement in the two gametangia is not synchronous and
may start first in either the larger or the smaller cell; (C) four gametes immediately before plasmogamy; (D) plasmogamy of two gametes
nearest to each other; (E) two large auxospores lying parallel to each other; they are closely connected with and perpendicular to the
mother frustule (arrowhead). Scale bar 5 20 mm.
high percentage of successful fusions of the two
pairs of gametes to yield eventually two auxospores
per gametangium (Fig. 4E). In contrast, for P. pseudodelicatissima clones CCMP-1562 and CCMP-1564
and P. multiseries, the parent frustules had only a
weak attachment. This resulted in gametogenesis,
but often without subsequent gamete fusion, or only
the first pair of gametes fused successfully, therefore
producing only one auxospore (Figs. 3H, 5C).
The weak attachment between the auxospores
and parent frustules sometimes led to an irregular
configuration between the two in P. multiseries (Fig.
3G) and CCMP- clones of P. pseudodelicatissima. In
the ND- clones of P. pseudodelicatissima, on the other
hand, the auxospores generally laid parallel with
one another and perpendicular to the parent frustule in the same plane (Fig. 4E).
In the case of P. multiseries and the CCMP- clones
of P. pseudodelicatissima, it was difficult to determine
what type of gamete activity (i.e. physiological isogamy or anisogamy) had taken place if gamete fusion
had not been observed prior to the auxospore stage.
However, when the attachment of the auxospores to
a parent frustule was strong, it was possible to ascertain the activity of the gametes. Because each clone
had a different cell size (see below), we could determine the clone of the parent cell to which the
auxospores remained attached. In such cases, the
auxospores were derived from gametes that had remained attached to the ‘‘mother’’ frustule, so those
gametes were passive. Therefore, even if the gametangia had lost contact with each other and if plasmogamy was not observed, the position of the auxospores relative to the frustule still supported the
conclusion that the gametes had displayed behavioral anisogamy. Furthermore, when the clones
studied had cells of a different size, it was possible
to determine which parent clone provided the active
←
FIG. 3. Pseudo-nitzschia multiseries. (A) paired gametangia with gametes just after rearrangement; the larger cell (clone KP-104) and the
smaller cell (clone KP-105) have contacted each other valve to valve; (B) one gamete of the pair shown in A has become rounded (arrowhead)
in the larger cell; (C) four rounded, morphologically isogamous gametes immediately before gamete fusion; (D) plasmogamy of the same
gamete pair as shown in C; (E) zygote and two gametes that have yet to fuse; a single cell with two gametes lies near the pair; these two
gametes, as well as those found separate from any other gametes in A and B, have no chance of taking part in reproduction; (F) pair of
growing auxospores, each of which has two unfused nuclei and four plastids located in a thin layer of cytoplasm near the cell wall. (G)
irregular position of auxospores relative to the gametangial thecae; (H) fully developed auxospore, showing caps at the distal ends (arrowhead); two other gametes have failed to fuse; (I) auxospore with a slightly bent shape; this expanded auxospore, like that shown in H, has
two unfused nuclei and four plastids; (J) initial cell (arrowhead) formed inside an auxospore envelope. Scale bars 5 20 mm.
132
NICKOLAI A. DAVIDOVICH AND STEPHEN S. BATES
FIG. 5. Pseudo-nitzschia pseudodelicatissima (CCMP- clones). (A)
two zygotes; (B) two expanding auxospores; (C) initial cell with
a slightly bent shape. Scale bar 5 20 mm.
(2) or the passive (1) gametes. Therefore, P. multiseries clone KP-105 could be shown to be of sex 1,
as could P. pseudodelicatissima clone ND-1 (Table 3).
No consistent morphological or behavioral differences could be seen between clones that produced
male and female gametes.
Change in cell size. The ND- clones of P. pseudodelicatissima were unable to reproduce sexually just after isolation in early 1996. About once a month
thereafter, the ND- clones were mixed to check their
ability to interbreed. Sexual reproduction was first
observed only when the apical cell length decreased
FIG. 6. Decrease in apical cell size of Pseudo-nitzschia multiseries
(clone KP-103) during growth in a culture under natural illumination and at a temperature of 188–228 C. Bars indicate 99% confidence intervals.
to approximately 80 mm. The cell length of all the
clones has since declined in culture. For example,
P. multiseries clone KP-103 decreased in length from
94.9 6 1.2 mm (6 SD, n 5 10) to 43.0 6 2.1 mm
(6 SD, n 5 10) during an 18-month period (Fig.
6), although the transapical width has remained
constant at about 5 mm. The cell length decreased
more rapidly during summer months than in the
winter, apparently as a consequence of the higher
growth rate under the prevailing natural irradiance.
By the time of our experiments in Canada, cells in
the various clones had decreased to the dimensions
given in Table 3, and sexual reproduction was possible. Parent cells of clones ND-2 and KP-103 (not
TABLE 3. Restoration of cell sizes (average 6 SD, n 5 30 unless otherwise indicated) after auxosporulation in Pseudo-nitzschia spp.
P. pseudodelicatissima
Characteristic
P. multiseries
(ND – clones)
(CCMP – clones)
Parent cell length in the
clones of opposite sex
(mm)
58.9 6 2.0
(KP-104, sex 2a)
38.7 6 2.3
(KP-105, sex 1a)
146 6 8.9
58.7 6 3.4
(ND-3, sex 2)
69.2 6 2.9
(ND-1, sex 1)
126.4 6 5.5
49.9 6 3.1
(CCMP-1562)
46.8 6 1.6
(CCMP-1564)
108.8 6 22.6
68–140
59–140
59–140
6.6 6 0.4
6.7 6 0.5
151 6 27
157 6 39
9.0 6 1.1
7.9 6 0.6
379 6 136
259 6 59
4796 6 1808
3656 6 1075
Initial-cell length
(mm)
Dimension range of
vegetative cells from
the literatureb (mm)
Gamete diameter
(mm)
Gamete volume
(mm3)
Zygote diameter
(mm)
Zygote volume
(mm3)
Maximal auxospore
volume (mm3)
a
b
Sex 2 5 active gametes (‘‘male’’); sex 1 5 passive gametes (‘‘female’’).
Hasle and Fryxell 1995, Hasle et al. 1996.
5.2 6 0.9
(n 5 10)
73 6 36
(n 5 10)
6.5 6 0.4
(n 5 10)
141 6 28
(n 5 11)
2804 6 909
(n 5 6)
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SEXUAL REPRODUCTION IN PSEUDO-NITZSCHIA
FIG. 7. Frequency distribution of apical length of initial cells
of Pseudo-nitzschia pseudodelicatissima (mixture of ND-1 3 ND-3)
(left; n 5 58) and of P. multiseries (mixture of KP-105 3 KP-104)
(right; n 5 75).
shown in Table 3) had apical lengths of 62.7 6 4.9
mm (6 SD, n 5 30) and 59.3 6 3.4 mm (6 SD, n
5 30), respectively.
Gametes of the opposite sex had a similar form
and dimensions (i.e. morphologically isogamous)
(Table 3). The volume of the zygotes was about
twice that of the gametes. Completely expanded
auxospores had a volume 13–20 times greater than
that of the zygotes. The length of the initial cells
formed within the auxospores was about twice that
of the gametangia in the ND- clones of P. pseudodelicatissima and even greater than this in the CCMPclones of P. pseudodelicatissima and in P. multiseries
(Table 3). Only the apical length was restored as a
result of auxosporulation; the transapical width in
parent and initial cells was similar. In the case of P.
multiseries, the cell length before and after auxosporulation was outside the range of dimensions reported in the literature for field-collected samples
(Table 3; Fig. 7). This suggests that a modification
of the species description is warranted. Except for
the small parent cell lengths in the CCMP- clones
of P. pseudodelicatissima, the remaining cell dimensions are within the range of published values (Table 3).
The frequency distribution of initial cell lengths
appears to be normally distributed (Fig. 7) and rather narrow (Table 3), giving, for example, a coefficient of variation of 4.4% and 6.1% in P. pseudodelicatissima (ND- clones) and P. multiseries, respectively. The major part (99%) of the normal distribution
is constricted in the range 6 3 s, and the large cells
generated lie within the rather broad range of 110–
143 mm for P. pseudodelicatissima (ND- clones) and
120–173 mm for P. multiseries.
Auxospores were characterized by a prominence
in their central region (Fig. 1); this was most pronounced in P. multiseries. The transapical width of
auxospores across the prominence was similar to the
diameter of zygotes, which testifies to the strictly bipolar growth of auxospores (Figs. 3H, I, 4E), as in
most other diatoms (Round et al. 1990). For example, in P. multiseries (KP-105 3 KP-104 mixture),
the dimensions were 9.2 6 1.0 mm (6 SD, n 5 30)
and 9.0 6 1.1 mm (6 SD, n 5 30) for the auxospores and the zygotes, respectively. We were unable
to further characterize the structure or composition
of the auxospore wall (cf. Mann 1993b).
Ecophysiological features of sexual reproduction. Sexual reproduction occurred under the same conditions that were normal for vegetative growth. Therefore, it was not necessary to manipulate the culture
conditions to induce sexuality, although the conditions may not have been optimized. Irradiance in
the range 10–120 mmol·m22·s21 was favorable for
sexual reproduction in clones of both species. However, it was virtually impossible to achieve auxosporulation by mixing parent clones that had already
reached stationary phase. The best auxosporulation
results were obtained by using exponentially growing (2–3 days after inoculation) parent cultures.
An abrupt change in salinity, that is, when parent
clones growing at 18‰ were mixed directly into a
medium of 29‰, did not prevent sexual reproduction in P. pseudodelicatissima (ND- clones). Normal
auxosporulation was observed 2–3 days after such
treatment, and the initial-cell size was essentially the
same as that in the 18‰ medium.
Sexual reproduction could proceed in the water
column as well as on the bottom of the petri dishes,
where many of the cells had settled. The frequency
of auxosporulation was different for each species.
Brief observations indicated that the most abundant
auxosporulation occurred in mixtures of ND- clones
of P. pseudodelicatissima, whereas it was sometimes
difficult to find auxospores in mixtures of CCMPclones of P. pseudodelicatissima, therefore explaining
the relative paucity of results reported for the latter
clones. In P. multiseries, the frequency of auxosporulation appeared to be greater for the mixture KP105 3 KP-104 than for KP-105 3 KP-103.
DISCUSSION
The number of diatom species in which sexual
reproduction has been observed is very small relative to the total number of species recognized
(Round et al. 1990) for reasons given by Mann
(1988). In the case of Pseudo-nitzschia spp., it would
be difficult to find sexually reproducing cells in the
field because 1) the entire process takes only 2–4
days (at least in petri dishes; it may take longer in
nature); 2) it may occur only once every 3 years (see
below); 3) the ratio of auxospores to vegetative cells
is low; and 4) the association between pairs of gametangia and between auxospores and gametangia is
likely so weak as to be disturbed during normal sampling. It is also not known how fragile the auxospores are or how well they preserve after collection.
Nevertheless, our documentation of the stages of
134
NICKOLAI A. DAVIDOVICH AND STEPHEN S. BATES
sexual reproduction in Pseudo-nitzschia spp. may
help others recognize such cells in field samples.
Making mixtures of different clones of Pseudonitzschia spp. permitted us to determine mating
types. Auxosporulation was observed only in
mixtures containing a pair of clones of the opposite
sex, not in separately growing clones, indicating that
intraclonal mating is rare or absent. It also shows
that the investigated Pseudo-nitzschia species exhibit
a dioecious type of mating. On one occasion, sexual
reproduction was observed in a mixture of two
clones (ND-1 and ND-2) of the same sex. Therefore,
Pseudo-nitzschia species may not be obligatorily dioecious under certain conditions, which remain to
be studied. Similarly, Chepurnov and Mann (1996)
observed that four clones of Achnanthes longipes behaved bisexually, but only at a low frequency. Dioecy, however, seems to be usual in pennate diatoms
(Roshchin 1994, Roshchin and Chepurnov 1996) in
spite of the recently held belief that diatoms were
mostly monoecious (Wiese 1969, Drebes 1977). Dioecious reproduction is not the exclusive type in all
pennate diatoms, and it can sometimes combine
with monoecious reproduction in the same species,
for example, Synedra tabulata (Roshchin 1987) or
Nitzschia lanceolata (Roshchin 1990, Roshchin and
Chepurnov 1996). There are also occurrences of
constitutional autogamy and pedogamy (Round et
al. 1990).
Our description of allogamous sexual reproduction in Pseudo-nitzschia spp. is contrary to the results
of Subba Rao et al. (1991) for the reasons given by
Rosowski et al. (1992) and Mann (1993a) but is consistent with a preliminary description given by Fryxell et al. (1991) for Pseudo-nitzschia subcurvata.
Among many other objections, Rosowski et al.
(1992) suggest that Subba Rao et al. (1991) did not
see sexual reproduction. Rather, they propose that
contaminating chytrids were mistaken for flagellated
gametes. The Pseudo-nitzschia spp. gametes we observed were morphologically indistinguishable from
each other and had no flagella, consistent with observations of other pennate diatoms (Mann 1982a,
Round et al. 1990, Chepurnov and Mann 1997).
The differences in gamete activity (each gametangium had either two passive or two active gametes;
Table 2) and gametangial dimensions (when a clone
had a different cell size, it was possible to determine
to which clone a cell in a pair belonged; Table 3)
in both species gave us the opportunity to verify hypotheses about diplogenotypic sex determination.
Gamete activity was not determined by gametangial
size. In P. pseudodelicatissima (ND- clones), the smaller gametangia produced active gametes, whereas in
P. multiseries the gametes of the larger gametangia
were active. Auxospores were connected solely to
the parent frustule that contained the passive gametes. Therefore, clones behaved unisexually, producing either ‘‘male’’ or ‘‘female’’ gametes only.
Because of the close contact between the game-
tangia, it was sometimes difficult to determine
whether gamete fusion was isogamous or anisogamous. The fact that the apical axes of gametangia
and auxospores bore a fixed relation at right angles
to each other in P. pseudodelicatissima (ND- clones)
and were often orientated irregularly in P. multiseries, suggests a IB1 and IC type of sexual reproduction, respectively, following Geitler’s classification
(Geitler 1973, Round et al. 1990, Mann 1993a).
However, the gamete behavior (two active or two
passive gametes produced by the same gametangium) and the connection of auxospores with the parent frustules of strictly ‘‘mother’’ gametangia (which
contain passive gametes) argue for the placement of
these Pseudo-nitzschia species in the type IA2 anisogamous sexual reproduction. Type IA2 is shown
mainly in araphid pennate diatoms (Stickle 1986,
Round et al. 1990, Mann 1993a). It is believed to be
primitive in pennate diatoms, suggesting a reduced
type of evolution of sexual reproduction patterns
and the origin of pennate diatoms from centric diatoms, in which gametangia are differentiated as
‘‘male’’ and ‘‘female’’ according to the activity of
the gametes produced (Drebes 1977, Round et al.
1990, Mann 1993a). Mann (1982a) suggested that
cis-anisogamous reproduction is the basic type for
araphid diatoms, although it has also been documented in evolutionarily advanced raphid pennate
diatoms, for example, in Mastogloia (Stickle 1986)
and Amphora (Mann 1993a). In Nitzschia longissima,
behavioral as well as morphological anisogamy takes
place (Roshchin 1994), and this occurrence could
be regarded as heterogamy, as in Grammatophora
(Roshchin 1994).
Pseudo-nitzschia is a geographically widespread genus restricted to marine environments (Hasle et al.
1996). Cells of this genus are capable of active motility, which is used to form stepped chains after vegetative division. This feature also appears to be adaptive for sexual reproduction, allowing the two gametangia to line up in close proximity with each other
to copulate. There may be a physiological requirement for the gametes to touch or to remain in close
proximity to a gametangium for gamete fusion to
be successful. Amoeboid movement of the gametes
requires such a substrate. In other pennate diatoms,
gametes are prevented from moving away from the
gametangia by a mucilage envelope (Mann 1982b)
or by the passage of gametes through a copulation
tube (Mann 1986). Such structures were absent in
P. multiseries and P. pseudodelicatissima.
During evolution, the copulative function in pennate diatoms is hypothesized to have been transferred from the gametes to the gametangia because
it was more economical to make fewer gametes that
no longer required flagella (Mann 1993a). However, because species of Pseudo-nitzschia inhabit the water column, gametangiogamy would be most effective only when cell abundance is high. This could
occur, for example, during a bloom when the rapid
SEXUAL REPRODUCTION IN PSEUDO-NITZSCHIA
increase in cell numbers favors their chance of cell
to cell encounters and also places them in a favorable physiological condition for auxosporulation.
This does not preclude the possibility that sexual
reproduction can also occur within cell clumps (Fryxell et al. 1991), on suspended particles (Buck and
Chavez 1994, Lee and Fryxell 1996), or at the sediment surface, although we have not had the opportunity to observe this.
The population dynamics of diatoms, normally
described by a change in total cell abundance, can
also be characterized by the appearance of new generations of cells having a large cell size arising from
auxosporulation (cf. Mann 1988, Jewson 1992a, b,
Crawford 1995). In P. multiseries, initial cells had an
apical length of 120–170 mm (Fig. 7). The timing of
the sexual phase in the life history of diatoms depends on the rate of decrease in cell size. This
mechanism has been described as an internal clock
for these algae, which have a cell life span in nature
shorter than yearly seasonal changes (Lewis 1984).
According to Figure 6, a reduction in size of about
2.5 mm per month, or about 30 mm per year, can
be estimated in culture. This compares with a decrease of 20–30 mm per year estimated for Nitzschia
sigmoidea (Mann 1988). Therefore, these initial cells
must grow in culture for about 3 years to reach the
sexually inducible phase in their life history, assuming that the suitable minimal length corresponds to
30%–40% of the maximal size (Geitler 1932, Drebes
1977). If the annual division rate of a diatom growing under optimal conditions in culture is assumed
to be faster than that in the sea, we estimate that P.
multiseries must grow vegetatively for at least 3 years
in the sea before entering the sexually inducible
phase. The time between periods of sexual reproduction for diatoms in nature ranges from 1 to 6
years (Mann 1988, Jewson 1992a, b) and possibly up
to 40 years (Mann 1988), although the latter is disputed (Jewson 1992b). Therefore, an estimated 3year cycle for P. multiseries is not out of the ordinary.
An understanding of the life history of P. multiseries may provide some insight into the reasons for
the interannual variability in bloom intensity and
toxicity of this species in eastern Prince Edward Island, Canada (Bates et al. 1998). For example,
knowledge of the cell size suitable for sexual reproduction and of the rate of its decrease would allow
one to predict when auxosporulation can take place.
This may be accomplished by measuring the size–
frequency distribution of Pseudo-nitzschia spp. cells in
field samples and by knowing the environmental
conditions conducive for sexual reproduction.
Changes in toxicity over the life history of the cells
could account for a year-to-year variation in bloom
toxicity. Preliminary experiments indicate that initial cells produce substantially more domoic acid
than the parent cultures, which have virtually lost
their toxicity over a period of several years. However, other questions remain. The vulnerability of the
135
various sexual stages to predation by animals and to
attack by viruses or fungi is unknown. It is also not
known whether a certain proportion of the population auxosporulates every year, which would give
a multimodal size–frequency distribution, as for
some other species (Mizuno and Okuda 1985, Mann
1988).
The genus Pseudo-nitzschia was recently revised
and separated from the genus Nitzschia because of
a set of morphological and molecular peculiarities
(Hasle 1994). However, it is virtually impossible to
distinguish between some morphologically similar
Pseudo-nitzschia species (e.g. P. delicatissima and P.
pseudodelicatissima or P. multiseries and P. pungens) using only light microscopy (Hasle et al. 1996). Therefore, electron microscopy is normally used to discriminate between these species. New methods of
discrimination have recently been developed, including a lectin-binding assay (Fritz 1992), an immunofluorescence assay (Bates et al. 1993), and molecular probes (Scholin 1998). It is now possible to
add to this list the ability for like diatoms to enter
into sexual reproduction, a major biological criterion of a species. This is made possible by a collection of clones now characterized as to their sexuality. However, it should be noted that a negative result does not necessarily mean that the species are
different; the cells must first be of a suitable minimal
size and the environmental conditions conducive
for sexual reproduction. Therefore, several successive trials must be made.
In the case of P. pseudodelicatissima, there was successful auxosporulation, separately, among the NDclones and among the CCMP- clones. This demonstrates that the cells were of a suitable size and that
the conditions were appropriate for sexual reproduction. Nevertheless, the ND- clones did not interbreed with the CCMP- clones in spite of the fact that
they are the same species according to their morphology as seen by scanning electron microscopy
(i.e. presence of a central interspace and one row
of square poroids between the interstriae; Fig. 2).
However, CCMP- clones differ from ND- clones by
1) deviations in the general pattern of sexual reproduction (Table 2); 2) the smaller transapical width
of the cells (Fig. 2); and, as a result, 3) a markedly
smaller volume for the gametes, zygotes, and auxospores (Table 3). These observations raise questions
concerning the taxonomic relationship between the
ND- and the CCMP- clones isolated from different
parts of world. They also suggest that more species
may exist in the genus Pseudo-nitzschia than have
been described according to the morphological systematics. Finally, this may explain some of the contradictory observations concerning the ability of P.
pseudodelicatissima to produce domoic acid in one
part of the world (Martin et al. 1990) but not in
another (Bates et al. 1998).
Our ability to induce auxosporulation in these
Pseudo-nitzschia species has important practical as
136
NICKOLAI A. DAVIDOVICH AND STEPHEN S. BATES
well as theoretical aspects. Up to now, clonal cultures of P. multiseries continued to decrease in cell
size, without sexually reproducing, until they eventually died. However, it is now possible to obtain new
clones of large initial cells of P. multiseries and P.
pseudodelicatissima by mating cells of opposite sex.
Such clones are important for studies on genetics
and toxicity and for rejuvenating cultures in cases
where it is not practical or possible to obtain new
clones directly from the field. Nevertheless, several
challenges remain to increase the percentage of
auxospores and initial cells formed and the survival
rate of newly isolated initial cells.
We thank C. Léger for expert technical assistance, D. O’Neil (Institute for Marine Biosciences, NRC, Halifax, Nova Scotia) for
providing the SEM photomicrographs, C. J. Bird (Institute for
Marine Biosciences, NRC, Halifax) for commenting on the identity of the ND- clones of P. pseudodelicatissima, and C. J. Bird, I.
Kaczmarska (Mount Allison University, Sackville, New Brunswick),
and two anonymous reviewers for improving the manuscript.
Funding by NATO Collaborative Research Grant OUTR.CRG
960380 is gratefully acknowledged.
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