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On the pollen morphology of picea and tsuga

species

Ronghui Ho & O. Sziklai

To cite this article: Ronghui Ho & O. Sziklai (1972) On the pollen morphology of picea and
tsuga species, Grana, 12:1, 31-40, DOI: 10.1080/00173137209427643

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 Grana 12:31-40, 1972
ON THE POLLEN MORPHOLOGY OF PICEA A N D TSUGA SPECIES
RongHui Ho and 0. Sziklai
(Received January 15, 1972)
ABSTRACT
Kong-IIui H o and 0. Sziklai (Faculty of Forcstry, Uni-
versity of British Columbia, B.C., Canada). Otr rhe pollen
rrtorphology of Picea and Tstrga species. Gram 12: 31-
grains of Picea oriertralis, P. sifcliettsis,
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40, 1972.--PoII:n
Tsirga Iieferophyllo, and T. ttierretisiatta were investigated
by means of light, scanning electron, and transmission
elcctron niicroscoov.
.< T h e fine structure of these four
spccies, the si.imesc pollen in P. si/chettsis, and the varia-
lions in wcci of T. nierfetisinria were demonstrated. Pol-
len morphdogy of T. trterretisiatta differed from that of
Picea, and even more from T. heferophylla. The diffcr-
ence in morphology of Tstrga spccies may have been du:
to varying rates of evolutionary change, to the modifica-
tion of reproductive structure for their continued existence,
or to introgrcssion. From the differences in pollen mor-
phology. thc authors consider that the two Tstrgu spccics
could be assigned to separate genera.
INTRODUCTION
Tsirga is a controversial genus, not only to paly-
nologists but also to biologists. The palynologists
arc struck by the complete difference in pollen
morphology between the mountain hemlock (T.
rnertcrlsiarici (Bong.) Sarg.) and the other hem-
locks, and biologists by the marked differenccs in
plant morphology.
Engelmann (1880) proposed dividing the genus
Tsiigo into two sections-Elitsirgo and Hespero-
pelice; a view which was also supported by Beiss-
ner (1909), and Dallimorc 6r Jackson (1923).
Lcmmon (1890, 1898) and Rydbcrg (1912)
separated them into independent genera. Ueno
(1957) suggested two subgenera rather than two
sections, o r genera.
The idea, that mountain hemlock is an in-
' This p a p r is I partof I thesis submitted by the senior
author to the University of British Columbia in partial
fulfilment of the requirements for the degree of Doctor of
Philosophy.
tcrgcncric hybrid betwccn western hemlock ( T .
Iiererophylla (Raf.) Sarg.) and Sitka spruce (Picea
SilChcrJsis (13ong.l Carr.1 Was presented by Campo-
Duplan 6: Gaussen (1948). Whether or not this
intergeneric hybrid is possibility is to qucs-
tion. The cxinc pattern of pollen is determined by
the sporophytc as as by the gamctophytc
(Iieslop-Harrison, 196s). Therefore a study of
thc topography of the pollen surface might pxmit
the scparation of various t m a and assist in the
interpretation of their rclationships.
MATERIAL AND METHODS
Pollen grains of two species each of the genera
Picen and Tsiiga were studied. The dates of pol-
Icn collection and sources are shown in Tablc I.
All the pollen grains werc stored in closed vials
and placed in a desiccator with calcium chloride
at 6 2 ° C in the cold storage.
Two size mcasuremcnts were made on the
grains which were biconvex in shape (Fig. 1 B).
In the case of saccate pollen, the corpus and the
cntirc grain were measured for height, length,
width and total length (Fig. 1 A). Fresh pollen
grains were mountcd in distilled water. A hun-
drcd, randomly selected, turgid grains from
each spccies wzre measured. Pictures were also
taken to measure the angle of thc saccate at-
tachment. This angle was designated u (Fig. 1 A).
Specimens werc prepared for the scanning clcc-
tron microscopc (SEM) by cvaporating carbon
onto the fresh pollen grains followed by pal-
ladium-gold alloy (Pd : Au - 40 :60) (wire diam-
eter 0.2 mm). The thickness of each film was
about 100 A. Thc specimens were scanned at an
angle of 45' at 10 KV in the SEM (Cambridge,
Type 961 13, Mark 2 A, England).
G r m a IZ
 32 RorigfIiti Ro arid 0. Sziklai

Table I. Dotes orid soilrces of polleri collections of Picca old T ~ u g aspecies

Date of
Species Common name collection Source

P. orientalis Oricntal spruce Apr. 17, 1970 Univ. of B.C.

,P. sifcliensis Sitka spruce hlay 5, 1970 Univ. of B.C.
T. lief eropliyh Western hemlock hlay 25, 1968 Univ. of B.C.
T. nrerfensimm Mountain hemlock June 2, 1970 Seymour hlt.. Vancouver

The carbon replica technique for the transmis- cm in a vacuum evaporator. The replica was ex-
sion electron microscope (?‘EM) was more clabor- amined at 50 KV in a Hitachi HS-7S electron
ate. Fresh pollen grains were sprinkled on the grid microscope.
with Fornivar film which was wetted with acetone.
The film was then softened with acetone and al-
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lowed to dry. Grains were removed from it, leav-
ing impressions in the film. The impressions were
coated with a carbon film 300 A thick. Thc
Formvar film was dissolved away leaving the
carbon replica. No more than 3 cm of Pd-Au al-
loy wire was used to shadow the replica. The
metal was deposited through grid bars at a dis-
tance bctween the source and target of 14-15
RESULTS AND DISCUSSION
I . Polleri Size
Pollen grains of oriental spruce (Figs. 2, 6, 7),
Sitka spruce (Figs. 5, 8, 9) and mountain hemlock
(Figs. 10, 11, 33) are saccatc, whilc those of west-
ern hemlock arc biconvcx when wet (Figs. 19,
20) and saucer-shaped when dry (Fig. 24).
Pollen sizes of the four species examined are
shown in Table 11. Of the saccatc type, the meas-
urements of corpus and total length are, in
decreasing order, Sitka spruce, oriental spruce
and mountain hemlock. The corpus length of
mountain hemlock pollen showed thc least varia-
tion.
Sitka spruce pollen was found to be 70 x 78 t t
(IVodchouse, 1959); mountain hemlock meas-
ured 4 5 x 7 8 ~ 7 0p (Erdtman, 1965), 64 [ i
(Campo-Duplan, 1950), and 70 x 55 x 68 i t (Ucno,
1958); wcstern hemlock was 60 p (Campo-
Iluplan, 1950), 60.5 x 75.5 p (Ho 6; Sziklai,
1971), and 65 p (Ueno, 1957). Thc corpus of thc
saccate-type pollen is elliptical (the two short axes
are unequal in length). Three measurements there-
fore should bc considered necessary. Biconvex
pollen types on the other hand require only two
measurements. From all previous reports and thc
present data, thc difference in measurements may
be attributable to thc effects of different treat-
ments, mounting procedures, mounting media,
environmcntal conditions and ccotypic variations.

Fig. I . (A) Pollen of saccate type. (D) Pollcn of bicon-
vex type. 11, Height; I , length; 11, total length; w, width;
a, angle of saccate attachment.
Grarra 12
2. Polleri Strirctirre
( A ) Light riiicroscupe strrdicx
Pollen grains of the two spruce species were
characterized by their ovoid-conical form, with
 Polleri rriorphology of Picen cirrtl lsrrgo 33

Table 11. Argle of snccrrs attaclirrrerit (irr degrees), orerage pollerr size (x’) (irt rrricroris), stnrrrlnrtl tlerintiuri
(+S.D.), nrirl cuLjficierrt of rarintiori ( & C . V . ) of P i c a arid Tsuga species

Picea orieiifolis Piccn sircl1ensis Tsiigo iiierrriisinim Tsiign-hefcroph)llnlln

31 31 49

Anglc ... .f &S.D. &C.V. k.5.D. &C.V. s +S.D. + c . v . .f &S.D. +C.V.

~~~

Corpus height 59.4 4.7 7.9 71.7 4.2 5.9 60.2 3.9 6.5 - - -
Corpus length 74.0 3.4 4.6 84.7 3.4 4.0 65.1 0.7 1.1 73.1 3.3 4.5
Corpus jvidth 56.4 4.1 7.3 72.0 3.1 4.3 61.9 3.2 5.2 59.0 2.4 4.1
Total length 85.3 4.2 4.9 105.2 6.0 5.5 70.0 2.9 4.1 - - -

thc sacci broadest at the base. T h e curves of the pollen was also indicated by H o S: Sziklai (1971).
cap and the outer wall (sexine) of the sacci However, Ueno (1957) reported that it was a
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formed a continuous, essentially unbroken, line
(Figs. 2, 3, 5).
The sizc of the saccae was found to be more
variable in oriental spruce pollen, (Figs. 2 and 3)
than in Sitka spruce, and in addition Sitka spruce
containcd siamese pollen (Fig. 4) at a frequency of
about one in a thousand.
T h e size of thc pollcn sacci varied substantially
in mountain hemlock. Most of the grains had two
~ v e l l developed sacci (Fig. lo), but in some of
them sacci werc either reduced (Fig. 1 l), vestigial
(Fig. 12), or absent (Fig. 13). Abnormal grains
were found with thrce sacci (Fig. 14) or with a
single saccus, either surrounding (Fig. 15), un-
frilled (Fig. 16), o r frilled in form (Fig. 17). The
pollen grains of this species were divided into four
classes o n the basis of saccus variation. T h e fre-
quency of each class is shown in Table 111. Some
variation in saccus size in henilocks with saccate-
type pollen have also been noticed by Campo-
Duplan (1955) and Ucno (1957).
The saccate attachment angle is shown in Ta-
ble 11. T h e two spruce species examined have the
same degree of angle. T h e s a c 5 of mountain
hemlock, however, were displaced further to-
wards the distal face than those of spruces (Fig.
lo), hence the angle was larger. Ucno (1960)
reported an angle for Picen spccies ranging from
25 to 43O, and for Tsrrgn (Ilesperopcirce) spccics
of 49O.
T h c western hemlock has a unique pollen
morphology. T h e puffy frills, with sonic varia-
tions, encircle the equatorial arca (Figs. 18, 19,
20, 21). Pollen with o r without a narbe was found
cqually common (Figs. 19, 20). T h e charac-
teristic presence of a narbc in western hemlock
3 - i25sns
Characteristic of Eiitsirgn pollen. This does not
find support in the present study, because all the
pollen grains are not ornaniented with a narbc.
( B ) Electron riiicroscopic stirdies
T h e depth of focus of the SEhl has enabled us to
study the shape of the dry pollen grains and to in-
vestigate the fine structure of the surface topo-
graphy. However, it is likely that surface replicas
for T E h l will be necessary for a study at high
resolution.
In spruce the arched corpus of the dry pollen
grain encloses the germinal furrow, whereas the
sacci are not contiguous (Figs. 6, 8). This was
found to b: characteristic for dry grains of both
spruces and was evident in the photomicrographs
of Engclmann spruce pollen presented by Martin
S: Drcw (1969). T h e shape of dry grains of nioun-
tain hemlock is diffcrcnt from that of the spruces
since the sacci in this case bend inwards and en-
close the germinal furrow (Fig. 22). On the other
hand, dry pollen grains of western hemlock hnvc
a c o n c w e distal face and are saucer-shapcd (Fig.
24).
Table I l l . Freqrrericy of polleri grnirrs witli iiorrr1n1,
restigiol or nbrrorrrrnl strcci, arid without a sncc~is,iri
Tsuga mertensiana
Pollen with sacci Pollen
Ivithout
Normal Vestigial Abnormal a saccus
Total no. of
pollen counted I484 30 12 6
I’ercen t 96.6 1.9 0.8 0.4
Grotin 12
 34 RorigHiri H o and 0 . Sziklai
The sculpturc typc on the corpus and sacci of
pollen of both spccics of sprucc cxamincd was
found to be microverrucate (Figs. 6, 7, S, 9), as
described for Engelmann spruce pollcn by hlartin
& Drew (1969). Oriental sprucc pollen had a
.smooth marginal area (Figs. 6, 7), a fcaturc not
found in Sitka spruce (Fig. 8). On the cap of Sitka
spriicc pollcn, a faintly markcd triradiatc strcak
was occasionally observed (Fig. 9).
From the TEM photomicrographs, the granular
surface of the spruce pollen was found to be in-
tersected by ribs (Figs. 37, 28). hlinutc protru-
sions arosc, either from the ribs or from thc
granular surface. Thc areola formed by the intcr-
cccting ribs was shown only in Sitka spruce (Fig.
Downloaded by [109.163.234.2] at 12:18 31 May 2016
28). Yamazaki S: Takeoka (1959) desxibxl the
topography of Japanese spruce pollen as a “c3uli-
flower-type” structurc which was ornamented with
spinulcs.
Hcmlock pollcn sculptiirc was also microvcr-
rucatc in typc, but with morc densely packed
granules (Figs. 22, 23, 24, 26). A singlc spinulc
(rarely two) was found on the crest of granules of
western hemlock pollen (Figs. 24, 26), but not on
mountain hemlock pollen. Erdtman (1957) reached
the same conclusion. A groove in the equatorial
area (Fig. 25) and a narbe, which was a cluster
of outstanding granules with spinulcs (Fig. 26).
wcrc observed in the pollen of wcstcrn hemlock.
The wavy surface of the granulcs on mountain
hemlock pollen, as revealed by TEM photomicro-
graphs, was delimited by the ribs, and minute
protrusions arosc either from the ribs or from
Figs. 2, 3. Pollcn of oriental spruce ( x 300). (2) Turgid
grain. (3) Pol!en with rcduced s x c i .
Figs. 4, 5 . Pollen of Sitka spruce ( x 300). (4) Siamcsz
pollen. ( 5 ) Turgid grain.
Figs. 6, 7 . SEhI photomicrographs of oriental sprucz pol-
len ( x 650). (6) Pollen with the smooth marginal area.
(7) Showing the cap of thc grain.
Figs. 8, 9. SEhl ph2tomicrographj of Sitka spruce pJllcn
( x 650). (8) Showing the arched corpus and th2 rough
marginal surface. (9) Pollen with faintly marked triradiatc
strcak.
Figs. 10-17. Pollen of mountain hemlock ( x 450). (10)
Turgid grain. (11) Pollen with reduced sacci. (12) Pollen
with vestigial sacci (arrow). (13) Pollen without saccus.
(14) Pollen uith three s x c i . (15) S h m i n g the distal face
of pdlen \\ith a saccus which encircles the equatorial
area. (16) Pollen with an unfrilled form of thz saccus.
(17) Pollen with saccus in frilled form.
Gratm 12
the granular surface (Fig. 29). However, the sur-
face of both granulcs and spinules on wcstcrn
hemlock pollcn was smooth and intcrscctcd by the
ribs (Fig. 30).
3. The ReIationslrips of Picca and Tsuga
The pollen morphology of the mountain hcmlotk
differed from that of the spruce spccies, and cvcn
more from that of the westcrn hemlo-k. It is
possible that both thc saccate and the biconvex
typcs of pollen in thc hcmlo-ks might havc evolved
from a common ancestor which possessed a single
girdling saccus resembling that of Cordnires. The
following factors niay havc contributed to thc
cvolution, cventually, of sntcate pollen by the
reduction of frills and of biconvcx pollen by the
reduction of sacci.
(1) Morphological changes of differcnt parts
of plants do not prorced at the same ratc during
thc ,course of evolution. Speciation arises through
the accumulation of many different gcnctic
changes, adaptation to diffcrcnt habitats, and sub-
sequent genetic isolation. Since mountain and
wvestcrn hemlock occupy different habitats, the
difference in their pollen morphology may be the
result of long cvolutionary dcvclopment.
(2) Pollen morphology may havc bccn modified
as an adaptation to a ncw cnvironnient as a prc-
rcquisitc for the continued existence of the species.
The pollination me-hanisms of mountain and
wcstcrn hemlocks arc different (Doylc, 1943,
and certain changcs in pollcn morphology may
Figs. I S - 2 1 . Pollen of ibcstern h m l o c k ( x 450). (18)
Shoaing the distal face of the pollcn. (19) Pollcn with
narbe. (20) Pollen withmt narbe. (21) Pollcn with a thick
puffy frill.
Fig. 22. SEhl photomicrograp:i. Pollen of mmntain h:m-
Icck. shapc of dry grain ( x 1000).
Fig. 23. Pollen of mountain hemlcck showing the dcnsely
packed granule; ( x 2 000).
Fig. 24, 25. Pollen of western hemlock ( x 1 000). (24) Dry
grain of sauccr shape. (25) A groove in the equatorial
area.
Fig. 26. Pollen of western hcmlock showing dctails of the
narbe and frill ( x 2 000).
Figs. 27-30. 1EhI photomicrographs of the fine surfac-
structure (all x.8 OOO). (27) Oriental spruce. (28) Sitka
spruce. (29) hlountain hemlock. (30) Western hemlock.
Abbreviaricns: A, areola; j , frill; G, groove; N, narbc;
P, protrusion; r, rib;
S, spinule; TS,triradiate streak.
 Pollen rworpliology of Piccn nrrd l’sirga 35
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Gram 12
Downloaded by [109.163.234.2] at 12:18 31 May 2016

36 RorigHiri Ho and 0.Stiklai

 Polleri rtiorphology of Picea miti Tsirga 37
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Grnrin I t
 Downloaded by [109.163.234.2] at 12:18 31 May 2016

38

Grrttm
I.?
RorigHlri H o and 0..Sziklai

have been neccssary for pollination, pcrmination
and the orientation of the pollen on thc nuccllus.
This changc, in turn, niay have facilitated pollcn
iub:: travcrse of the nuccllus and incrcascd the
chances of fertilization, and may thus be essential
for the succcssful reprodiction and survival of
the spccios.
(3) This modification may have bccn due to
introgrcssion, which might havc rcsultcd in cer-
tain changes of the genetic makc-up and in modifi-
cation of the pollen morphology.
Carnpo-Duplan EC Gausscn (1948) proposd
that mountain hcnilock was an intcrgencric hybrid
bctwccn western hcnilock and Sitka spruc2. Duf-
lield (I 950) qucstioned this pmsibility, sincc su:h
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hybrid swarms have not bccn found within ihe
arca of their natural distribution. Chemical sinii-
larities b:twecn these two genera havc b x n meas-
ured and were not found to be indicativc of
hybridization (Tayler Sr Wood, 1971).
Tsirgn jcjjrcyi has long b x n acccptcd as a
hybrid between mountain and western hemlo:k
(Dufficld, 1950). Howcvcr, Taylor (1971) studied
the phytochcniical relationships in the genus
Tsirga and concluded that “chemical hybrids” of
these two spccics, although rare, did occur.
Further studies, such as DNA-hybridization,
cheniotaxonomical invcstigation and artificial hy-
bridization, are nccdcd to shcd morc light on the
rclationships bctwcen Tsirgn and Piccn sp:ci:s.
Based on anatomical and morphological stud-
ies, Campo-Duplnn & Gausscn (1948) found that
mountain hemlock rcscmblcd wcstcrn hrmlock
in nine out of forty characteristics invcstigated.
Thcsc two species differ not only in pollen nior-
phology but also in habitat, plant morphology,
pollination mc:hanism and polyphtnolic com-
pounds. The authors recommend the classification
of mountain and western hemlo-k as two scparatc
genera, as was formcrly proposed by Lcmmon
(1890, 1898) and Rydbzrg (1912), rather than
simple separation into two sections or subgcxra.
ACKNO\VLEDGEMENTS
The financial suppsrl of British Columbia Forest Producls
Ltd., in thc form of a Forest Gmetics Fellowship, is
gratefully acknowledged. Grateful thanks are cxtcnded to
hir hl. D. hleagher for providing the hemlock p A k n
samples, and to hlr L. Veto for technical advicc on clec-
tron microscopy.
Pollen rtiorpliology of Picen nnd Tsirgn 39
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Grmm 12
 40 RoiigHiti 110 arid 0.Sziklai

Yamazaki, T. & Takeoka, hl. 1959. Electron microscopc Roiiglliri Ilo

investigations on the surface of the pollen membrane, Oscnr SziLlai
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Cnlrodn
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Grriirn 12