The Structure of Anthoceros laevis in relation to its Water Supply

The Structure of Anthoceros laevis in relation to its WaterSupply

BY

IVOR ISAAC

(University College of Stcansea, and Botany School, Cambridge)

With seven Figures in the TextA NTHOCEROS LAEVIS is a very common member of the Anthocerotales,

* 1 growing in areas of abundant moisture, e.g. moist soil in fields, sides ofditches and streams, or even submerged. The plants are monoecious with thesex organs appearing on the dorsal surface. The prostrate thallus varies from5 to 15 mm. in width, the edges being somewhat lobed, and the plants branch^-ing dichotomously. The growth frequently becomes crowded, with the resultthat the edges overlap and the branches tend to become sub-erect. Thesporophyte is erect, attaining under favourable conditions a length of 15 to25 mm. The early stages of development of the sporophyte are accompaniedby some growth of the adjacent gametophytic tissue, resulting in the forma-tion of the involucre, which grows with the sporophyte for a time, reachinga maximum height of about 5 mm. With the progressive growth of the sporo-phyte this involucre remains as a collar around its base (Fig. i).

The mode of absorption and conduction of water by this rapidly growingsporophyte appeared to be a problem worthy of investigation, which involvedan examination of both the gametophytic and sporophytic anatomy.

Gametophyte.

The essential features of the anatomy of the gametophyte are well known.A somewhat regular layer of cells occur on the dorsal surface, followed by three tosix layers of large irregular parenchymatous cells with air spaces between them,and a ventral limiting layer of cells from which numerous smooth walledrhizoids develop. There are no scales on the ventral surface. Most of thecells of the thallus, especially those near the dorsal surface, contain a singlelarge chloroplast. As in the case of other species of Anthoceros, many inter-cellular cavities occur, filled with mucilage, opening to the ventral surface bynarrow slits, and occupied by the familiar Nostoc colonies. The presence of themucilage was proved by the application of 0-5 per cent, solutions of methyleneblue and toluidin blue respectively to sections of the thallus, resulting in apositive reaction by practically all the cells, including the rhizoids. The regionof the Nostoc colony showed the most abundant occurrence of mucilage.

[Annals of Botany, N.S. Vol. V, No. 18, April 1941.]

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SP.

th.

340 Isaac—The Structure of Anthoceros laevis in

A preliminary investigation of the path taken by liquid external to theplant was made by washing suitable specimens, freeing them from surpluswater by means of blotting-paper, and then placing them on prostrate wads of

filter-paper in shallow glass dishes containing 0-5per cent, solutions of 'vital' stains, such as vital red,methylene blue, neutral red, trypan red, pyrrol blue,and diamin black respectively, the last-named stainhaving been dissolved in a 1*0 per cent, solution ofsodium chloride. The plants were in contact on theirventral surface with the saturated filter-paper, butwere not immersed in the liquid, and each dish wascovered by an inverted glass pneumatic trough(Fig. 2) in order to maintain a moist atmosphere.After a few minutes, examination under the bin-ocular microscope showed that liquid had passedover the margins of the thallus on to the dorsalsurface, where it quickly spread. Later, sectionsshowed that the stain had entered the cells of thedorsal and ventral limiting layers and also therhizoids, while invariably the Nostoc colony andthe surrounding cells showed the presence of thestain before any coloration was apparent in theremainder of the internal tissue.

Further series of experiments were carried outin which the stains were replaced respectively by(a) o*i per cent, solution of potassium nitrate (sec-tions of the plant so treated being mounted in a dropof diphenylamine in concentrated sulphuric acidand examined at once for the blue colour charac-

teristic of the nitrate), and (4) o-i per cent, solution of ferric chloride (sectionsof these plants being mounted in a drop of ammonium sulphide solution, ablack deposit of ferric sulphide denoting the presence of iron). The results ofthese experiments again showed that the liquids had passed over the margins ofthe thalli on to the dorsal surface and penetrated into the cells of the dorsal andventral limiting layers, and into the rhizoids. It is, therefore, evident that inA. laevis absorption of water occurs over the whole surface and is not locatedto any particular region.

All of the previous experiments were then repeated with fertile thalli,and, in every case, examination within a few minutes of exposing theplant to the test liquid showed that the archegonia and antheridia weredeeply saturated, although within that brief period the internal tissueventral to the sex organs showed no trace of the presence of the stain, potas-sium nitrate, or ferric chloride respectively. Hence it appears probable thatin nature, as in the case of Pellia (Clee, 1939) the sexual organs receive ample

rh.

FIG. 1. Thallusof A.laevisbearing gporophyte. tp.,sporophyte; in., involucre;th., thallus; rh., rhizoida.


Relation to its Water Supply 341

water conducted in the form of capillary films over the external surface ofthe thallus. The spread of these external films of liquid probably facilitatesfertilization by carrying the swimming sperms along with them, and so aidingthem in reaching the oospheres, especially since the rapid entry of the testliquid into the neck canal of the archegonia suggests its rapid absorption bythe mucilage there.

The sporophyte.

The sporophyte of Anthoceros laevis is like an elongated spindle, with a3.0.

FIG. 2. Diagram of apparatus used, g.c, glass chamber; pt., plant; gd., glass dish;fp., filter-paper; /., liquid.

large bulbous foot embedded in the gametophyte, an intercalary meristematicregion, and a long green capsule. When young the sporophyte is green to thetip, but later it becomes brownish, and, as it ripens, it ruptures longitudinallyby slits which extend progressively downwards. The lower part of the sporo-phyte is surrounded by the aforementioned involucre of gametophytic tissuewhich, it is suggested, gives mechanical support to 'the weak intercalary zone'(Bower, 1935). This involucre is seen on close examination to be irregularlyridged, while its base is surrounded by a shallow moat-like depression in thethallus (Fig. 5). There is no cleft or mucilage layer comparable to that foundby Clee in Pellia at the base of the foot. The bulbous foot probably serves thedouble functions of anchorage and absorption, and the absorbing power ofthe basal region is increased by the papillate nature of the peripheral cells ofthe foot which impinge upon the gametophytic tissue (Fig. 3).

In the capsule the central columella consists of elongated thin-walled cells,which appear in transverse section as a square of 16 cells. A longitudinalsection shows that the end walls of these cells are very much oblique, the cellsdovetailing in a manner similar to that of fibres and tracheides (Fig. 4). Thearchesporium, as a single layer of cells, can be distinguished quite easily, evenin the meristematic region.

Outside the archesporial tissue occur about four to six layers of thin-walledcells. These cells on the outer region near the epidermis each contain twochloroplasts. Also, scattered throughout this parenchymatous tissue are seen



empty cells which are smaller than the normal ones in transverse section,but they are somewhat elongated longitudinally (Fig. 4). These cells formlongitudinal series, which appear to run independently, for no evidence oftheir direct contact with one another or with the epidermis was obtained.

pc.

FIG. 3. Longitudinal section through the sporophyte of A.laevis showingmeriitematic and foot regions, in., involucre; a.,archesporium; /., foot; pc., papillate cells; th., thaltus.

The epidermal cells are quite distinct from those underlying them. Theirouter walls are papillate, increasing the outer surface of the sporophyte.

In the region covered by the involucre, these epidermal cells are filled withdense protoplasmic contents, but higher up in the green capsule they areempty. It has been suggested by former workers (Campbell, 1924; Smith,1938) that the epidermal cells of A. laevis are strongly cutinized, but thewriter, testing with scharlach red, failed to find any trace of cutin. In surfaceview these epidermal cells are narrow and elongated with end walls which arefrequently oblique. Stomata, though present, are not numerous.

Longitudinal and transverse sections of the sporophyte were treated with


t Relation to its Water Supply 343

methylene blue and toluidin blue respectively, to test for the presence ofmucilage. The cells of the foot and columella, the scattered elongated cells ofthe parenchymatous region, and the papillate epidermal cells gave a positivereaction. The 'cutin', described by earlier workers, on the external wall of theepidermis appears to be really a mucilaginous layer.

FIG. 4. Longitudinal section through the mature sporophyte. ep.,epidermis; tp., spores in tetrads; c, columella; ef., elaters; nc., narrowelongated mucilaginous cell.

Experiments with vital stains, similar to those recorded above for the vegeta-tive and fertile gametophytes, were now carried out with thalli bearingdeveloping sporophytes, in order to trace the path of external liquid (Fig. 2).Examination every few minutes under the binocular microscope showed thatthe liquids passed quickly over the edges of the thallus on to the dorsal surfaceand soon fined the moat-like depression around the base of the involucre.From this they passed up externally over the involucre, the ridges of the latter



aiding the rise of the capillary films. Later, careful examination of sectionsshowed the stain passing down into the space between the involucre and thesporophyte. Some of the coloration was also seen in the cells of the involucre.Other sections showed the stain passing from the depression at the base of the

s.o.

T"-rh. TTT

FIG. 5. Diagram to show the path taken by water in reaching the sporophyte. Arrowsindicate the path taken, a., archesporium; c, columella; in., involucre; m.r., meristematicregion;/., foot; nc., Nostoc colony; t.o., sex organs; r., reservoir; rh., rhiroids.

involucre into the cells of the gametophyte towards the foot of the sporophyte.The moat-like depression seems therefore to act as a reservoir from whichwater can pass in two directions, viz. externally up over the involucre to theouter surface of the sporophyte, and internally through the cells of the gameto-phyte into the foot of the sporophyte. The internal supply to the foot of thesporophyte is also apparently augmented by liquid entering through theventral surface of the prostrate thallus and passing in towards the mucila-ginous cells of the foot, for, after a time, the stains could be plainly seen in thisregion (Fig. 5).

Although the vital stains could be clearly observed in the gametophytictissue surrounding the sporophyte, and could therefore be used as an indica-tion of the path of conduction to the sporophyte, they were never visible wit hinthe tissues of the latter, and so were useless for purposes of determination ofthe path of travel of water within the diploid generation. (This seems toindicate an interesting physiological difference between the two generations.)



For this purpose potassium nitrate and ferric chloride respectively weresuccessfully used. Sections of plants exposed to these liquids and treatedwith the appropriate reagents showed that the solutions penetrated into thethallus and passed very quickly through it to the dorsal surface and so reachedthe cells of the foot of the sporophyte before any other part of this generationshowed their presence. They also passed over the edges to the dorsal surfaceof the thallus, and could soon be detected passing up over the external surfaceof the involucre as well as penetrating into its cells. From the top edge of theinvolucre some of the solutions passed immediately into the empty mucilagi-nous epidermal cells of the sporophyte at that level, while tie remainderpassed down the cleft towards the foot. That which entered the sporophytepassed vertically upwards in the epidermal cells and obliquely upwards in theparenchymatous region, reaching the elongated mucilaginous cells of thisregion, and also the columella, and passing up in these. Although the solu-tions of salts were continuously passing in through the prostrate gametophyteto the foot of the sporophyte and from there to the columella, their level wasalways higher in the epidermal cells than in the cells of the columella. Insome transverse sections through the capsule it was seen that even the sporesand elaters had become saturated before the columella showed any signs of thepresence of the solutes. Also longitudinal sections showed that the reactionfor the presence of the solute was frequently given by isolated patches of thecolumella tissue, suggesting that its presence there was due largely to penetra-tion from the epidermal cells through the parenchymatous and spore-bearingregions, rather than to a direct rise in the columella after absorption by thefoot from the gametophyte. However, such a direct rise does take place,though comparatively slowly.

The times taken by the solution of potassium nitrate and ferric chloriderespectively to travel up the sporophyte were determined, and typical resultsare shown in Tables I and II.

It is clear from these figures that water can reach the tip of the sporophyteof A. laevis within reasonable time when ample supplies are available to thegametophyte.

An attempt was then made to determine the speed at which liquid rises to thetip of the sporophyte when the supply through the internal tissue of the gameto-

TABLE I

Length (cm.) ofPlant

Length (cm.) ofsporophyte.

i"5

0-7

1 6

1'42 - 0

Time(min.)

2 0

30

45

6080

Height (cm.) of nse ofpotassium nitrate.

o-2 (by internal conduc-tion)

°'S (by internal conduc-tion)

1-2 (by internal and exter-nal conduction)

1-4 (tip)2-0 (tip)



TABLE II

Plant.

2

345

Length (cm.) of8porophytc.

o-8

1-2I-21723

Time(min.)

15

3°406075

Height (cm.) of rise offerric chloride.

0-4 (by internal conduc-tion)

o-8 (tip)1-2 (tip)1-7 (tip)2-3 (tip)

phyte is eliminated. As much as possible of the prostrate thallus was cut away,leaving the involucres surrounding the lower region of the sporophytes and thetissue underlying the foot intact. Vaseline was then smeared over the bases ofthe plants and very nearly to the top of the involucres, after which the plantswere dipped into melted paraffin wax up to the level of the top of the vaselinedarea. The wax was then allowed to cool by immersing the whole in coldwater. The entry of the liquid through the underside of the gametophyte tothe base of the foot of the sporophyte was thus prevented. A number ofshort lengths of glass tubing were prepared, each of such an internal diameteras to accommodate these isolated sporophytes without damage. Each was fixedto a glass slide for support as shown in Fig. 6. The tubes were filled withpotassium nitrate solution and the plants, supported by glass wool, wereplaced in them, so that the level of the liquid was just up to the top of thewaxed area. The whole was then placed under a glass chamber to maintain amoist atmosphere. It is obvious that the only liquid available to the sporophytewas that which passed over the top of the involucre.

The times taken by the potassium nitrate to travel up the sporophyte weredetermined, and typical results are shown in Table III.

TABLE III

Length (cm.) of Time Height (cm.) of rise ofPlant. sporophyte. (min.) potassium nitrate.

1 0-7 15 o*2 up sporophyteo-2 down cleft

2 i-o 30 o-6 up sporophyte and tothe base

3 0-9 40 o-84 15 60 1-25 i-8 85 i-8 (tip)

A series of experiments was carried out with ferric chloride instead ofpotassium nitrate which gave similar results.

A comparison between Table III and Tables I and II shows that where thewater supply to the sporophyte can be regarded as entirely external—passingover the surface of the gametophyte—conduction to the tip was practically asrapid as in the cases where this external supply was augmented by absorptionby the gametophyte and passage through its tissues to the base of the foot.Where in these last experiments some liquid penetrated down the cleft



between the involucre and the sporophyte into the foot region, it could beseen to rise in the sporophyte in the cells of the columella.

In order to eliminate as far as possible this latter source of supply somesporophytes were dissected from the gametophytic thalli. Lengths of about

FIG. 6. Diagram to show apparatus used, g.c, glass chamber; sp., sporophyte;g.w., glass wool;ft., glass tubing; /., liquid.

3 mm. of the foot regions of these sporophytes were covered by coats of vase-line and paraffin wax. These were then placed as described above in tubesfilled with potassium nitrate and treated precisely as before. At intervalssections were cut and examined in order to ascertain the place of entry of thesolute and its rate of rise in the sporophytes. The solution clearly rose quiterapidly in the epidermal cells and in the series of mucilaginous cells in theparenchymatous region. It also passed down into the foot region, and tracesof it were also evident in isolated patches of the columella. Evidence wasobtained that the solute in the cells of the columella was due partly to aninternal ascent from the foot, and partly to transverse and oblique passagefrom the epidermal cells. However, the potassium nitrate had reached thetip of the capsule via the epidermal cells long before any trace of it wasapparent in the upper part of the columella, except in isolated patchesobviously resulting from diffusion in from the epidermis.

Typical times taken by the potassium nitrate to travel up the dissectedsporophytes were as indicated in Table IV.

Experiments using ferric chloride gave similar results.A comparison of Table IV with Table III shows that liquid reaches the

tip of the sporophyte quite as rapidly by the epidermis alone as by the epider-mis and columella together, so that, although the latter has some conductingpower, the epidermis and the mucilaginous cells of the parenchymatousregion are probably responsible for the upward conduction of the greater partof the necessary supply of water.



ant.i2

345

Length (cm.)sporophyte

0-7o oI "2

1*4i-7

TABLE IV

of Time(min.)

1535607080

Height (cm.) of rise ofpotassium nitrate in

epidermal cells.0-2 up and down to foot

1-2 (tip)1-4 (tip)1-7 (tip)

An attempt was then made to determine whether downward as well as up-ward passage of water is possible in the sporophyte. A number of capsuleswere cut away from the gametophytic thallus and their tips inserted throughholes of suitable dimensions in a thin slice of cork. The cork was then placedover a glass dish with the capsules normally oriented, their cut ends being afew centimetres above the water. The latter maintained a moist atmospherearound them. A small amount of cotton-wool, saturated with potassiumnitrate, was then placed on top of the cork, so that the tips of the capsules werejust in contact with it (Fig. 7). At intervals sections of them were examined,and it was found that the liquid had been conducted downwards in the epider-mal cells, passing from these into the internal tissue. Similar results wereobtained when cotton-wool saturated with ferric chloride was used.

The epidermal cells therefore appear to play an important part in the con-duction of ground water in the sporophyte of A. laevis. The mucilaginousnature of the walls of these cells together with their papillate form may alsocondition to some extent the absorption of atmospheric moisture. Water canthus reach the tip of the sporophyte of A. laevis without the aid of the game-tophyte, and, since the former also contains quite an appreciable amount ofchlorophyllaceous tissue, it appears probable that the main value of the game-tophyte may lie in its power of directing supplies of water and solutes to thebase of the sporophyte and in its support of the latter. If this be correct, andsupport and supply could be otherwise provided, the sporophyte might beexpected to live, grow, and develop sporogenous tissue, even if separated fromthe gametophyte at an early stage. This suggestion was strengthened by theobservation that when the gametophyte part of some plants kept in a coldframe died, turned black, and began to decay, the erect sporophytes continuedto live, grow, and produce mature spores.

This finding is in accordance with that of Campbell (1924) for Anthocerosfusiformis, the sporophytes of which he was able to keep alive for three monthswhen severed from the gametophytes, though they 'made very little growth,but ripened normal spores in a number of cases'. He suggested that the footin this case could absorb water independently of the gametophyte, 'and so thesporophyte has become practically independent of the gametophyte andreached a condition comparable to that of the Pteridophyte after it has estab-lished its first root'.



The writer dissected sporophytes of A. laevis of varying sizes until theywere free from the gametophytic tissue. Capillary glass tubing was cut intolengths of 2 cm. which were fastened into a bundle by means of an elasticband. The whole bundle was placed in a shallow glass dish into which Knop's

I.

FIG. 7. Diagram of apparatus used, gd., glass dish; cm., cotton-wool; tp., sporophyte;/., liquid.

culture solution was poured until the liquid had just risen to the tops of thetubes. The prepared sporophytes were then measured and one placed ineach tube supported by glass wool. The whole was then placed under a glasschamber in order to maintain a moist atmosphere. At the same time normalplants bearing sporophytes, which had also been measured, were placed inanother similar shallow dish containing Knop's culture solution, this dishbeing also enclosed in a glass chamber. The Knop's solution in each case waschanged three times a week. At intervals all the sporophytes, both attachedand unattached to gametophytes, were measured, and also examined to seewhen the characteristic splitting of the capsule occurred.

Though a number of the dissected sporophytes died after a few days,doubtless due to damage done to the foot region during the actual dissectionfrom the gametophyte, the majority survived for periods of weeks, duringwhich growth and development occurred.

The lengths of typical specimens of both these series are shown in Tables Vand VI.

It is evident from these figures that though the dissected sporophytesdid not grow as large and therefore did not produce as many spores as thenormal ones, yet they grew and gave rise to normal mature spores when com-pletely separated from the gametophyte at a very early stage.

This relative independence of the sporophyte of Anthoceros, together withits form, and the tendency to concentric zoning of its tissues suggest anapproach to the condition in the Psilophytales, especially since some of the


35° Isaac—The Structure of Anihoceros laevis in

Rhyniaceae were not dissimilar in size from some of the larger sporophytesof species of Anthoceros. The older conception of the parasitism of the

J

Time.

Beginning of

2 weeks3 »4 ,.

5 „

6 „

7 »

8 „

9 .,

io „

Time.

Beginning ofexpt.

2 weeks3 ..A*T »»

5 ,,6 „7 ,,

8 „

9 „

io „

No. i.o-8

I - I

i"31*4

(sporesformed)

i'4(sporesformed)Dead

—

—

—

—

No. i.0 5

o-81*2

i*7i*92-O

(sporesformed)

2-o(sporesformed)

2-o(sporesformed)

2-O

(sporesformed)

TABLE V.Dissected Sporophytes

Length (cm.) ofNo. 2.

o-5

o-6o-75o-8

o-9(spores

No. 3.o-4

o-60 9i - o

(sporesformed)

i -o

(sporesformed) formed)

0 9

(sporesformed)

0 9

(sporesformed)Dead

—

—

Dead

—

—

—

TABLE VI

sporophyte.No. 4.

o-7

i - oI - I

1 2 5

I - 4

I-45(sporesformed)

i-45(sporesformed)

i-45(sporesformed)

i "45(sporesformed)Dead

Normal SporophytesLength (cm.) of sporophyte.

No. 2.0-4

o-7I - I

i-7Si "9z-o

2 - 1

2 - 1

(sporesformed)

2"I

(sporesformed)

No. 3.O-2

o-6o-9

i-5Si-71-85

2 - O

2 - 1

(sporesformed)

2 - 1

(sporesformed)

No. 4.0-3

0-7i - o

i - 6

i-7Si "9

2 - 1

2 - 2

(sporesformed)

2 - 2

(sporesformed)

No. s.o-4

o-6o-8°'9S

I - I

I - I

(sporesformed)

I - I

(sporesformed)

I - I

(sporesformed)Dead

—

No. S.0-7

I - I

i -S

1*92 - 1

2 - 2

(sporesformed)

2 - 2

(sporesformed)

2 - 2

(sporesformed)

2 - 2

(sporesformed)

No. 6.o-8

I - I

I *2Si-35

i-4S

i-55

i - 6

(sporesformed)

i - 6

(sporesformed)

1-6(sporesformed)Dead

No. 6.o-6

i - o1-2

i - 7i "852-O

2 - 1

(sporesformed)

2 - 1

(sporesformed]

2 - 1

(sporesformed]

sporophyte upon the gametophyte in the Bryophyta, with the support whichit gives to the antithetic theory of alternation of generations, requires further



modification in the light of the present work, which, together with that ofClee (1939) and Bold (1938), tends to support the homologous view with itsconception of 'a fundamental similarity in capacity for self-nutrition of thetwo alternating generations' (Bold). Further work is in progress on the waterrelations of the sporophytes of other members of the Bryophyta.

SUMMARY

1. Anthoceros laevis is able to absorb water over the whole of the surface ofthe gametophyte and sporophyte.

2. The sex organs receive practically the whole of their water supply fromthese external sources.

3. The water passes up over the edges of the thallus on to the dorsal surfaceinto a shallow moat-like reservoir at the base of the involucre. Thence itpasses to the sporophytes, travelling up through the mucilaginous cells of theepidermis, and also down the cleft between the involucre and sporophyte,whence it reaches the foot. After absorption by the foot it slowly rises in thecolumella.

4. The external rise in the epidermis is sufficiently rapid to enable thetip of the sporophyte to be well provided with water even when disconnectedfrom the gametophyte, so long as supplies are available. Isolated sporophytesgrew and produced spores, living for nine weeks after separation from thegametophytes, when placed in culture solution.

5. The water absorbed by the epidermis gradually passes inwards viamucilaginous cells to sporogenous tissue and columella.

6. Although externally conducted water and solutes can provide all thenecessities for the development of the sporophyte of A. laevis, an appreciableamount does reach this structure internally as a result of rapid absorption bythe ventral surface of the gametophyte, and the passage of the absorbed solu-tions through the gametophytic tissue to the base of the foot. The rate of risein the sporophyte from this source of supply is, however, much slower thanthat due to the external liquid.

This work was carried out in the Department of Biology of the UniversityCollege of Swansea, and the writer wishes to extend his thanks to ProfessorF. A. Mockeridge for suggesting it, and for her very valuable help and criticismthroughout its progress.

LITERATURE CITED

BOLD, H. C, 1938: The Nutrition of the Sporophyte in the Hepaticae. Amer. Journ. Bot,551-7-

BOWER, F. O., 1935: Primitive Land Plants. London.CAMPBELL, D. H., 1924: A Remarkable Development of the Sporophyte in Anthoceros fuai-

formis. Aust. Ann. Bot., xxxviii. 473-83.CLEE, D. A., 1939: Morphology and Anatomy of Pellia ephiphylla in Relation to Absorption

and Conduction of Water. Ann. Bot., N.S., iii. 105-12.SMITH, G. M., 1938: Cryptogamic Botany, vol. ii.

966-18 A a


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The Structure of Anthoceros laevis in relation to its Water Supply