The reproductive biology of Hydrocharis morsus-ranae. 11. Seed and seedling morphology

ROBIN W . SCRIBAILO~ AND USHER POSLUSZNY Department of Botczny, Urziversity of Guelph, Guelph, Orit., Canczdcz NlG 2WI

Received September 1 1 , 1983

SCRIBAILO, R. W., and U. POSLUSZN~. 1985. The reproductive biology of Hydrocharis morsus-rczncie. 11. Seed and seedling morphology. Can. J . Bot. 63: 492-496.

This study of seed and seedling morphology in the aquatic monocotyledon Hydrocharis morsus-rczncie L. (Hydrocharitaceae) represents part of a continuing study of sexual reproduction in this species. Scanning electron microscope studies of the seeds showed them to have testas covered with hollow spiralifom tubercles. Germination occurs when the radicle and cotyledon of the embryo from the exalbuminous seed elongate, splitting the tuberculate testa. The buoyant embryo then rises to the surface with the first foliage leaves emerging from a highly modified cotyledonary sheath. This makes early growth in the seedling look strictly hypocotyledonary. Both the radicle and cotyledon discontinue growth by the two-leaf stage in the seedlings. The young seedlings undergo several growth phases. At first they look Lernna-like in habit and then, subsequently, very similar to germinating turions of the same species. Several methods are discussed to help distinguish between germinating turions and seedlings. During a subsequent field study of a H. morsus-ranne population, only two germinating seedlings were discovered. Several reasons for the absence of seedlings in this population are discussed.

SCRIBAILO, R. W., et U. POSLUSZNY. 1985. The reproductive biology of Hydrochnris morsu.s-rnnne. 11. Seed and seedling morphology. Can. J . Bot. 63: 492-496.

Cette Ctude de la morphologie de la graine et de la plantule de I'Hydrochnris morsus-rnnne L. (Hydrocharitaceae) fait partie d'une recherche en cours sur la reproduction sexuCe chez cette espkce. Des I'Ctude des graines au microscope Clectronique 2 balayage montre que leur tCgument est couvert de tubercules creux de forme spiralCe. La germination se produit lorsque la radicule et le cotylCdon de I'embryon de la graine exalbuminke s'allongent, fendant le tCgument tuberculC. L'embryon flottable monte alors i la surface et les premikres feuilles Cmergent d'une gaine cotylCdonaire fortement modifiCe. Cette caractkristique donne I'impression que le debut de la croissance de la plantule est strictement hypocotylCdonaire. La radicule et le cotylCdon a d t e n t tous les deux de croitre lorsque la plantule a deux feuilles. Les jeunes plantules passent par plusieurs phases de croissance. Elles ont d'abord I'allure de Lemna et, plus tard, elles sont trks semblables aux turions en germination de leur espkce. Les auteurs discutent plusieurs mCthodes de distinguer les plantules et les turions en germination. Seulement deux plantules ont CtC dCcouvertes au cours d'une Ctude subsiquente d'une population d'H. morsus-rnnae. Plusieurs raisons pouvant expliquer I'absence de plantules dans cette population sont discutkes.

[Traduit par Ic journal]

Introduction 82" W. Secds wcrc obtained in Sc~tcmbcr 1983 from ~ollination

It is often assumed that vegetative propagation is so bagging experiments of fcnlalc flowers made (luring 21 study of the floral biology of this species (Scribailo and Posluszny 1984).

prevalent in many aquatic plant species, sexual reproduction is Obscrvations on morphological aspects of scccl germination wcrc of l imi ted ( S c u l t h o r ~ e 967). As a in many maclc throughout the study period using both a photo-dissccting !nitro- vegetatively propagating aquatics that also reproduce sexually, s,opc (zCiss DRC) a scanning clcctrOn nlicrOscopc (SEM), seeds little is known about sexual reproduction. Hyclrochnris tTlor.Yll.5- and seedlings prcparcd for the SEM wcrc fixed in f(,mlalill - acetic r-cltznc L. (Hydrocharitaceae) is a floating aquatic native to acid - alcohol (FAA), washed in 70%, cthyl alcohol, and transferrcd Europe which was introduced into Canada at Ottawa in 1932 to 100% ethyl alcohol. 'rhc material was then clitical-point clricd in an (Dore 1968). Its present range in North America includes much of Southern Ontario and parts of QuCbec and New York state bordering the St. Lawrence River (Catling and Dore 1982). The species spreads primarily by means of rapid clonal growth from turions during the spring months. Although flowering and seed set have often been noted in H . mor-sus-rntzc~e populations, both in E u r o ~ e and Canada. sexual re~roduct ion has been little studied, particularly in comparison to vegetative propagation (Cook and Luond 1982b). This paper on seed and seedling morphology in H . morsw-ratzne L. represents the second part of an investigation aimed at clarifying the nature of sexual reproduction in this species. 'The first part was a study of floral biology (Scribailo and Posluszny 1984). The objective of this paper is to describe in detail the morphology of seeds and seedlings, in part to aid in their identification in the field.

Materials and methods All seed collections of H. mors~1.s-rcirlcie were made at Rondeau

Park, Ont., Canada, locatccl on the shores of Lake Eric, 42"18' N ,

'Present address: Department of Botany, University of Toronto, Toronto, Ont., Canada M5S IAl.

Oniar 5PC-1500 critical-point dryer, mounted on rnctal stubs, and coated with gold-palladium {'or 5 rnin in aTcchron Hummcr I1 sputtcr coater. Obscrvations wcrc made using an E'rEC Autoscan SEM set at 10 kV.

Results and discussion Seed morphology

The seeds of H . tnorsus-rcltz~re are transversely elliptic in shape and are approximately 1 mm in length when fully mature (Fig. 1). The testas of the seeds are covered with knoblike tubercles of varying dimensions which give the seeds a spiny appearance (Fig. 2). Each tubercle is hollow and composcd of' a single tightly spiraIled thin band of wall material which is cemented between successive whorls with a waxlike substance. At their distal ends, the tubercles are enclosed by a niem- branous septum. When mature, the tubercles on the seeds are dark brown in colour. In immature seeds these structures have a geIatinous appearance and are practically transparent. Tuber- cles on the seeds were found to be quite springy and relatively unstable structurally, although they were more so on immature seeds. Consequently, any kind of manual manipulation or fric- tion on the seed coats caused the tubercles to uncoil (Fig. 3).

Can

. J. B

ot. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y U

nive

rsity

of

Que

ensl

and

on 1

1/24

/14

For

pers

onal

use

onl

y.

SCRIBAILO AND POSLUSZNY 493

Examination of a large number of sceds at various stages of maturity indicated that on most seeds, over 50% of the tuber- cles were in an uncoiled state. The tuberculate testas were easily removed surgically from the seeds and came off as a one piece flexiblc skin much likc the exocarp of a grapc. The inner surface of the testa was rnenibranous and covered with a cir- cular pattern with each ring corresponding to the basc of a tubercle.

Air drying of seeds caused the tubercles on the testas to collapse inwards, completely changing the extcrnal nior- phology of the seeds. After drying, the secd coats appeared reticulate rather than tuberculate (Fig. 4).

Several studies have examined the seed niorphology of H. rnorslrs-rcrntle in thc past (Richard 1812; Ascherson and Gurke 1889; Subramanyam 1962; Catling and Dore 1982; Cook and Luond 19820). Richard ( I8 12) considered the tubcr- cles (referred to as vesicles) as being linked by a hard but fleshy membrane and forming the outermost of thc integuments. He described the vesicles as spongy, oblong, cylindrical, rounded, and closed at the ends, each being very finely nieni- branous, transparent, and covered by transverse striations. This description does not differ from that given here except that we distinguish, as did Cook and Luond (19820), that each tubercle is coniposed of one tightly spiralled band of wall material. Richard ( 18 12) also recognized an inner integument composed of a very fine membrane which was attached to the outer integument but not the enibryo. Although we did not examine the integuments closely, two distinct layers were not immediately obvious. At the time of gerriiination the sced coat is a single fused structure, showing no evidence of the two integuments.

Catling and Dore (1982) described the seeds as sornctinics being muricate or merely with a rough, somewhat riclged or winged si~rfacc. We have never observed seeds of this type.

Within thc genus H~~d~.r)r.lzuri.s. H . rllrbirr (BI.) Backer also has a tuberculatc secd coat very similar to that of H. ~~rorslrs- rcirzcie. Seed structurc has never been described for H. ~ . I IPI>LI- lier-i (Dc Wildeman) Dandy (Cook and Luond 198211).

A brief literature survey indicated that many other genera within the Hydrocharitaceae have species with tuberculate seed coats. These species include Li~nrzohili~?i sporzgici (Bosc.) Steud. (as L. ho.scii Bosc.) (Montesantos 19 13), Elocleci grci~ra- te17si.s Hunib. and Bonpl. (as E. g~ryci~zrre~rsis Rich.), B/J.YLI alrbertii Rich. (Richard 18 12), BI~~vri he.rri~7rlrci Cook and Luond. Bly,vci oc'tri~zdrri (Roxb.) Planchon ex Thwaites (Cook and Luond 1983), Strcrtiotes trloirlcs L. (Cook and Urmi-Konig 1983), and Vallis~zerici rr~neric.cirrrr Michx. (Kaul 1976).

In addition. it is of interest that seeds of N~c.hci171ri1dra alter- 11lfo1icr (Roxb. ex. Wight) Thwaites (Cook and Luond 1982ci) and Appertiellri sp. (Cook and Triest 1982) look very similar to air-dried seeds of H. mor.sus-rri~za~ (Fig. 4). Since much taxonomic work is done froni herbarium sheets, it is possible that the testas of the above species may be ti~berculate in their natural state. A study of the Hydrocharitaceae using only fresh or liquicl-preserved seeds may reveal that tuberculate seed coats are more coninionplace in this family than presently thought.

Overall, it is hard to envisage that tubercuIate structures could have developed on the testas of Hydrocharitaceae with- out being of some adaptive value. Kaul(1976) has commented that the fenestrate tubercles of Vcillisrzerici ri~,zeric.arzci appear to function in anchoring the seeds to the bottom. Richard (1812) observed that the club-shaped tubercles on Elocleci gra~zcitetzsi.~ were often inflated at their exterior ends. It is possible that

these structures may function as flotation cleviccs for the seeds, although we observed no such function in H. r,lol:slt.s-rarzne. Van der Velde and van der Heijden (1981) recently proposed that similar structures on the sceds of N~r~zphoic1e.s peltriter (Gmel.) 0. Kuntzc may function in this way.

Seed clisp~r.scr1 Observations on the nature of fruit dehisccnce ancl seed dis-

persal were made from female flowers bagged during a floral biology study of H. rnnrs~r.s-,rr11ae (Scribailo and Posluszny 1984). These observations are fundamentally different froni those recorded by several other authors for this species.

Female flowers are drawn under water shortly after polli- nation by a rapid peduncle recurvation process (Scribailo and Posluszny 1984). In some marsh habitats where drawdown in the fall has greatly reduced water depth. the developing fruit may be pushed into the mud on the bottom, as suggested by Dore (1968). In most cases that we observed. however, plants were more than 30 cni from the bottoni. making this phenoni- enon quite unlikely.

Four to six weeks after anthesis, the fruits split open because of internal pressure of expansion from a mucilaginous sub- stance found in the fruit. This gelatinous mass holds the seeds together even when they sink shortly after liberation. In con- trast, Serbanescui-Jitariu (1972) reported that when riiature, fruits detach froni the mother plant, sink to the bottoni, and then split open to liberate the seeds. She also stated that the seeds do not stay on the bottoni long, but rise to and float on the surface, whereupon the testas of tlie seeds gelatinize, ini- . . tlatlng germination. We observed no evidence for the above sequence in any of the hundreds of seeds we examined. Our observations indicate that seeds remain on the bottom until germination commences. Germination occurs when growth of the eriibryo causes the testa to split and release the buoyant embryo which then floats to the surface. No secd with its tuberculate testa intact was ever found floating at the water's surface, although the separated split seed coats were occa- sionally found. sometimes months after germination had coni- menced. I t is interesting that Serbanescui-Jitariu (1972) never mentions the tuberculate sced coats of H . ~,ror.srr.s-rri~~ae.

Seecl gerr?zi~zritiorz rr11c1 seeclli~rg de~~elop~rzertt The first noticeable sign that gerniination is taking place

occurs when the small off-white enibryo honi the sced appears floating on the water's surface (Fig. 5). Our observations indi- cate that i t is the expansion of tlie embryo and the initial elongation of the radicle and cotyledon which cause the testa to split and release the buoyant embryo. The radicle of thc devel- oping embryo splits through the secd coat at the ~iiicropylar end. Embryos occasionaIly were seen to remain on the bottom until the added buoyancy of leaf growth enabled them to rise to the surface.

Young seedlings of H . rnonrus-rci~lrre have no endosperrn but have a massive cotyledon which functions as a storage organ (C, Figs. 6. 7, and 10). This cotyledon elongates slightly during the initial stages of seedling growth then seems to dis- continue growth entirely. The arrows in Figs. 5-7 indicate the direction of this cotyledonary elongation. The radicle of the developing embryo also elongates slowly but for a longer period than the cotyledon (R, Figs. 6 , 7, and 10). The root cap of the radicle becomes distinguishable very early in its growth (Fig. 6).

The first leaves emerge through a cotyledonary sheath (CS) in the embryo, with the first leaf (L , ) always emerging opposite

Can

. J. B

ot. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y U

nive

rsity

of

Que

ensl

and

on 1

1/24

/14

For

pers

onal

use

onl

y.

Can

. J. B

ot. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y U

nive

rsity

of

Que

ensl

and

on 1

1/24

/14

For

pers

onal

use

onl

y.

SCRlBAlLO AND POSLUSZNY

FIGS. 7- 12. Various stages in seedling and turion growth. Fig. 7. A scanning electron micrograph showing a later stage of seedling germination with both the first ( L , ) and the second leaf (L?) emerging and splitting the cotylcdon (C). Note the change in orientation of the seedling in this figure compared with Fig. 6. x26. Fig. 8. Surfacc view of seedlings at the one-leaf stage. X3. Fig. 9. Surface vicw of seedlings at the four- to five-leaf stage. X 1.7. Fig. 10. Sidc vicw of sccdlings shown in Fig. 9 with thc first truc root (TR) cmcrging characteristically from the medial rcgion of thc cotylcdon. Thc radicle (R) is still prcscnt and visiblc oppositc thc cotylcdon (C). x 1.7. Fig. I I. Early stagcs of turion germination, with scale lcaves (SL) still prcscnt. X 1.3. Fig. 12. Latcr stagcs of turion growth. X 1.2.

Serbanescui-Jitariu (1972) and ours is that she shows a ring of Pers., and Vnlisrzeria nrnericnna, which root on the bottom in anchoring hairs, which we never observed, as forming on the an epigeal fashion, anchoring hairs do form (Kaul 1976). radicle of H. morsw-ranne. Limrzobilrrn spongia and Strntiotes Within the Hydrocharitaceae, seed germination in H. nloides, which are both floating species like H. rnor-sus-rrlnrle, morsus-rnr~rle is most similar to that of Liirlrzobilrrn spongia. also produce floating seedlings without anchoring hairs The illustrations and descriptions provided by Montesantos (Montesantos 19 13; Cook and Urmi-Konig 1983). In other ( 19 13) for this latter species conform very closely to all aspects species of Hydrocharitaceae such as Ottelia alismoirles (L.) of seedling germination recorded here for H. morsus-rnnae.

Can

. J. B

ot. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y U

nive

rsity

of

Que

ensl

and

on 1

1/24

/14

For

pers

onal

use

onl

y.

406 CAN. J . BOT. VOL. 63, 1985

The seedlings of H. inor.r~rs-i.~iim~ can be said to be fairly typical of aquatic monocotyledons in that they arc exal- buminous, with food reserves stored in the large cotyledon (Tomlinson 1982). Also, there is characteristically an early onset of photosynthesis in the cotyledon and rapidly appearing first plumular leaves (Kaul 1968). The emergence of the first plumular leaves from a cotyledonary sheath, making early development essentially hypocotyledonary, is also quite com- mon in aquatic monocotyledons (Foster and Gifford 1975; Tomlinson 19821.

Seeclling ~.stcrI~li.shm~nt Throughout our attempts to locate seedlings at the Rondeau

study site in the spring of 1983, we found that prior knowledge of seedling morphology was often needed to distinguish between plants arising from turions versus those arising from seeds. Although there is little likelihood of confusing these two plant types at the one- to two-leaf stage, seedlings at this stage in their development can easily be confused with those of Letnntr ininor L. and Spi rod~kr polyrhiztr (L.) Schleiden because of their close similarities in leaf n~orphology. How- ever, an examination of the underside of the leaf of a plant will confirm its identity, since lelnnid species initiate roots directly from the leaf surface.

Once three or four leaves have tleveloped on seedlings, there is an increased likelihood of confusing them with plants arising from turions of H. inor.su.s-rtrr~re, primarily because the latter show such variability in their size and nlorphology. We fount1 several morphological characters useful in distinguishing between the two plant types. Seedlings tend to have cordiforn~ leaves (Fig. 9). while plants originating from tilrions tend to have renit'orm leaves (Fig. 12) which are more typical of those found on mature plants. Seedlings have a characteristically persistent cotyledonary sheath with a short radicle and single root always initiated medially at the base of the cotyledon (Fig. 10). In contrast, plants from turions have a knoblike pro- tuberance at their base (area of root development) which repre- sents the former point of attachment of the scale leaves from the turion. 'The scale leaves, or remnants of then], often remain attached at the bases of the turions for months after germination (Figs. 1 1 and 12). These plants usually also produce numerous roots from this region shortly after germination.

Only two seedlings of H. tilot-,s~~.v-iairue were found at Rondeau. Both of these were at an early stage of development and had two and three leaves, respectively.

This general scarcity of seed germination at Rondeau was unexpected for several reasons. Firstly, we hat1 estimated that a minimum of 250 seeds/m2 were produced in this area during the summer of 1982 (Scribailo and Posluszny 1984). Secondly, our preliminary observations have indicated that seeds strati- fied at 4°C for 4 months germinate readily. This temperature regime is typical of that that would be found at Rondeau over the winter months.

Although additional study is still needed, there are several plausible explanations that may account for the low numbers o f seedlings found. Many seedlings may have been overlooked since it was very difficult to identify them amongst the exten- sive and intertwined growth of germinating turions. Also, seed- lings may not be capable of competing with plants from turions which germinate and grow very rapidly and in profuse numbers early in the spring. Although we found only two seedlings germinating in the wild, the work of Serbanescui-Jitariu (1972) from Hungary has established that extensive seedling germi-

nation does occur in some populations. The descriptions of seed and seedling morphology provided in this paper will aid future researchers in their identification of seedlings of this species. More extensive ecological studies of H. r~~or-sus-rvirlcre would be of particular interest now to establish the fates of seeds in the wild.

Acknowledgements We would like to thank J . Scribailo for her helpful assistance

in the field. We would also like to express our gratitude to the Ministry of Natural Resources and in particular Al Woodliffe for making Rondeau Provincial Park accessible to 11s for this study. This work represents part of a Masters thesis submitted to the Department of Botany, University of Guelph, by R.W.S. and was supported by the Natural Sciences and Engineering Research Council of Canada (grant A 6260 to U.P.).

AsctlERsON. P., and M. GURKE. 1889. Hydrocharitaccac. Iir Dic Naturlichcn Pflanzcnfamilicn 11. Vol. I. Et1iretlI)y A. Englcrand K. Prantl. W. Engelmann, Lcipzig. pp. 238-258.

BENTHAM, G.. and J . D. HOOKER. 1883. Gcncra plantarum. Vol. 3. L. Rcevc and Co., London.

CATLING, P. M., and W. G. DORE. 1982. Status and identification of Hydrochtrris rr~ors~rs-r.trrrtre and Lir~~rrol~i~rrr~ sl)oirgicr (Hydrochari- taccae) in Northeastern North America. Rhodora, 84: 523-545.

COOK. C. D. K.. and R. LUOND. 1982~1. A rcvision of tlic gcnus Necl~c~rr~trr~dr~tr (Hydrocharitaccac). Aquat. Bot. 13: 505-513.

19821). A rcvision of the gcnus Hydrochtrris (Hydro- charitaceac). Aquat. Bot. 14: 177-204.

1983. A rcvision of the gcnus B/y.rcr (Hytlrocharitaccae). Aquat. Bot. 15: 1-52.

COOK, C. D. K.. and L. TRIEST. 1982. Apperriello a new gcnus in the Hydrocharitaccac. 111 Studies on aquatic vascular plants. Edited /IJ>

J. J . Syniocns, S. S. Hoopcr, and P. Compcrc. Royal Botanical Society of Belgium. Brussels. pp. 75-70.

COOK, C. D. K.. and K. URMI-KONIG. 1983. A rcvision 01' the gcnus Strcrtiorrs (Hydrocharitaccac). Aquat. Bot. 16: 213-249.

DORE. W. G. 1068. Progress of the European frog-bit in Canada. Can. Field-Nat. 82: 76-84.

FOSTER. A. S.. and E. M. G. GIFFORD. 1975. Comparative nior- phology of vascular plants. W. H. Frccman and Co.. San Francisco.

KAUL, R. B. 1968. Floral morphology and phylogeny irl the Hydro- charitaceac. Phytomorphology. 18: 13-35.

1976. Morphology of germination and cstablishmcnt of aquatic seedlings in Alisrnataccac and Hydrocharitaccac. Aquat. Bot. 5: 139- 147.

MONTESANT~S, N. 19 13. Morphologischc untl biologischc untcr- suchengcr iibcr cinigc Hydrocharidccn. Flora (Jcna, 18 18- 1965). 105: 1-32.

RICHARD, L. C. 18 12. Memoirc sur Ics Hytlrocharidccs. Mcm. CI. Sci. Math. Inst. Natl. Fr. 181 l(2): 1-88.

SCRIBAILO, R. W. . and U. POSL~SZNY. 1984. 'Thc rcproductivc biol- ogy of Hvtlroc/rtrris rr~orslls-rnrrtre: I . Floral biology. Can. J . Bot. 62: 2779-2787.

SCULTHORPE, C. D. 1967. Thc biology of aquatic vascular plants. Arnold Prcss, London.

SERBANESCUI-JITARIU, G. 1972. Contributions 2 la connaissancc du fruit ct dc la germination dc quclqucs rcprcscntants de la famillc dcs Hydrocharitaccac. Bull. Soc. Hist. Nat. Afr. Nord, 63: 19-28.

SUBRAMANYAM, K. 1962. Aquatic angiosperms: a systc~natic account of common Indian aquatic angiospcrms. Council of Scientific and Industrial Research, Ncw Delhi.

TOMLINSON, P. B. 1982. Anatomy of monocotyledons. Vol. VII. Helobiac (Alismatidac). Edited by C. R. Mctcalfc. Clarcndon Prcss, Oxford and Ncw York.

VAN DER VELDE, G. , and L. A. VAN DER HEIJDEN. 1981. ThC floral biology and sccd production of Nyrnphoides peltrctcr (Gmcl.) 0 . Kuntzc (Mcnyanthaceae). Aquat. Bot. 10: 26 1 -293.

Can

. J. B

ot. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y U

nive

rsity

of

Que

ensl

and

on 1

1/24

/14

For

pers

onal

use

onl

y.