Bangor University DOCTOR of PHILOSOPHY Biosystematic and Cytological Studies of Mosses and Liverworts. Abderrahman, Salim Moh'd

Bangor University

DOCTOR OF PHILOSOPHY

Biosystematic and cytological studies of mosses and liverworts.

Abderrahman, Salim Moh'd Mansur

Award date: 1981

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TEXT IN ORIGINAL IS CLOSETO THE EDGE OF THE PAGE BIOSYSTE14ATIC'AND'CYTOLOGICAL', STUDIES'OF

'MOSSES'AND 'LIVERWORTS

A thesis

Presented for the &gree pf-

PhiloSophiae Doctor

in the University of Wales

Salim Moh'd Mansur Abderrahmano B. Sc. Biology & Ed. Cý (Kuwait University)

School of Plant Biology,

University College of North Wales,

Bangor M. K. ), I hereby declare that the work herein, now submitted as a thesis for the degree of Philosophiae Doctor of the University of

Wales, is the result of my own investigations and that all references to ideas and work of other resefrchers has been specifically acknoFledged.

Candidate

Supervisor

I hereby certify that the work embodied in this thesis has not already been accepted in substance for any degree, and is not being concurrently submitted in candidature for any other degree. Ht, Candidate 7wý Date t ..... ý Sunmary

Biosystematic investigations have been carried out on members of the Hypnum cupressiforMe, aggregate which are variable morphologically and on Atrichum undulatum which is variable cytologically. Cultivation experiments have shown that variation in gametophyte plants of the

Hypnum cupressiforme, aggregate is partly genotypic and-partly environmental and that there are four distinct species within the aggregate.

H. cupressiforme sensu stricto, H. jutlandicum, 1j. mammillatum and

H. vaucheri. Within H. cupressiforme s. s. there are three varieties, var. cupressiforMe, var. lacunosum and var. resupinatum and within

H. mammillatum are two varieties, var. mammillatum and var. filiforme.

These conclusions are supported to some extent by physiological and electrophoretic studies but-the aggregate is cytologically uniform.

In Atrichum undulatum there, are haploid, diploid and triploid cytotypes which are indistinguishable both in field material and after cultivation, morphological variation being both genetical and environmental. Only a single taxon can be recognized although there are measurable differences in DNA content between the three cytotypes,

There are significant differences in the relation frequencies of the three cytotypes in different parts of Britain.

a Acknowledgments

I am greatly indebted to Dr. A. J. E. Smith for valuable guidance# supervision and help throughout the study; to Professor J. L. Harper for the facilities provided in the School of Plant Biology, to

Dr. ý X. Newton for her help and advice.

I would like to thank Dr. C. Gliddon, Dr. J. Wilson and Dr. D. Shaw for their help in the experimental design and interpretation of the results included in 3.1,3.1,4.1 and 8.2.

My thanks are also due to:

The numerous field collectors named in Appendix. 4.

R. Oxley and C. Whittaker for their statistical advice.

Dr. J. Prijddle for his linguistic help.

Mr. W. Niville who developed the photographic films

Mr. D. A. Davies for his help in using the SEM.

The technical staff in the School of Plant Biology

I also wish to record my thanks to my parents for their encouragement and support and to my wife for her encouragement and patience.

I am very grateful to Xr. Dawood Mussad Al Saleh for his generous I financial aid throughout the course of the work and to Mr. Xoh'd Amin

Hindi for his co-operation. CONTENTS Pacre

Chapter' l Hypnum: cupresaiforme aggregate Introduction

Chapter 2 Hypnum. cupressiforme. aggregate Variation in morphology 2.1 Materials and Methods

2.2 Study of living and herbarium specimens.

2.3 Characters selected for the study of 9

cuRressiforme s. l.

2.4 Spore Wall Ornamentation 17'

2.5 Cytological investigation 19

2.6 Variability within taxa of H. 'eupresdifdrme S-1. 26

2.7 Numerical approach 30

Chapter 3 Variation in non-morphological characters

3.1 Introduction 35

3.2 Analyses of Carotenoid Pigments 36

3.3 Isozymes 37

3.4 Analyses of Amino Acids 41

Chapter 4 Physiology and Ecology of H. cupressiforme aggregate

4.1 Drought tolerance 43

4.2 Ecology and distribution of H. cuRressiforme s. l. SO

Chapter 5 Conclusion and Description of taxa

5.1 Conclusion 53

5.2 Description of taxa 55

5.3 Key 59

Chapter 6 Variation in Atrichum, undulat=

6.1 Introduction 61

6.2 Cytological investigation 00 CONTENTS(cont'd) Patre

Chapter 7 Morphological variation of cytotypes'

7.1 Materials and Methods 73

7.2 Characters selected for the study of 74

A., undulatum cytotypes.

7.3 Variability within cytotypes of 79

A. undulatum

7.4 Numerical approach 81

Chapter 8 Variation in non-morphological characters

8.1 Isozymes in three cytotypes of A., undulatum 85 85 8. Z DNA content of nuclei 90 8.3 Heterochromatin bodies in A. undulatum

Chapter 9 Distribution and Conclusion 95 . 9.1 Distribution of A. undulatum cytotypes

9.2ý Conclusion 103

References 105 119 Appendix 1 Analysis of carotenoid pigments 122 Appendix 2 Comparison of chromosome length of haploids

diploid and triploid Atrichum undulatum using

Tukey's interval estimate 125 Appendix 3 Comparison of DNA content of three cytotypes

of Atrichum undulatum using Tukey's interval

estimate 127 Appendix 4 Localities and chromosome numbers of specimens

of Atrichum, undulatum, used in this study. -I-

I 'CHAPTER 1

apn='d, aptda9ifdMe agSregaýe

Introduction (Smith, There are eleven species of Hypnum recognized in Britain

1978a; Ando and Townsend, 1980),, * Hypnum bambergeriý H. 'callichroum,

hamulosum H. imponens H. jutlandidum, H. lindbergii, H. cupressiforme, 11. _ There H. mammillatum, H. revolutum, Hý'uncinulatum and H. vaucheri. are in a further five European species that have not been, found Britain

(Corley et al., 1981). Eight of the British species show little

morphological or ecological variation and have posed no taxonomic

problems, but the other three$ 11. cupressiforme. H. jutlandicum, and has been H. mammillatum are a very-different matter and: their treatment

a matter of controversy. Thus Wilson (1855) says about H. cupressiforme solo "Extremely "extremely variable Dixon and Jameson (1924) say; (1954) variable", Rilstone (1948) described it a's very variableg Nyholm

"a Watson (1968) says: strongly variable species ... says: "often most variable British moss" and in (1971) he says: regarded

as the most variable British moss Smith (1978a) says: is "a very polymorphic species the nature of variation within which

Ando (1979) "this is the most unknown ...... says species one of Crum Anderson (1981) variable mosses ...... and and state:

"Exceedingly common and notoriously variable in Europe ......

1j. cupressiforme 9.1. (in the broad sense and which includes

H. cupressiforme HO jutlandicum and H. mammillatum) has been variously

divided into varieties and sub-species and in some instances species

have been segregated from it. Some of the taxa have been treated

uniformly by most authorities whilst othershave ranged in taxonomic status -2-

from worthless form to species depending upon the author concerned.

Treatment in Britain by Wilson (1855), Braithwaite (1895-1905)t

Dixon and Jameson (1924), Richards and Wallace (1950), Warburg (1963) and Smith (1978a) is shown in Table 1. The opinion of sixteen British bryologists gathered during preparation of The British and Ilish laoss

Flora (Smith, 1978a) is shown in Table 2. From Table 2 it is evident that if views of the majority of the British bryologists asked are accepted, then var. ericetorum should be treated as a species, var. mammillatum as a sub-species and var. filiforme, lacunosum and resupinatum as varieties with var. lacunosum and var. tectorum being regarded as synonymous.

Treatment by other European bryologists has also varied.

The opinions of bryologists in Finland (Koponen et al. 1977),

Fennoscandia, (Nyholm, 1954), Poland (Ochyra and Szmajadav 1978)t

Scandinavia (jenseng 1939; Damsholt et al. 1969)q Germany (Du'll, 1977) and France (Guillamount, 1949; Doignon 1950,1963) are shown in Table 3.

Some authorities treat H., vaucheri as a variety of H. cupressiforme

(e. g. Wijk et al., 1963; Ando, 1972a; Crum and Anderson, 1981)9 others treat it as a distinct species (e. g. Ando, 1976; Smith, 1978a).

There is a record of H. vaucheri Lesq. from Yarmouth in Wilson (1855) but there has clearly been some confusion as he says it is intermediate between H.. crassinervium (Cirri2hyllum crassinerium) and H. piliferum-

(C. piliferum). He is probably referring to H. vaucheri Rabuh

C. tenuinerve (Lindb. ) Wijk & Marg. ) a plant not recorded from

Britain. -3.-

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Table 2. Showing the opinion of sixteen British bryologists about

the treatment of Hypnum cupressiforme s. l.

Taxon Species Sub- Variety Forma species resupinatum 3 2 10 1 filiforme 2 - 13 1 mammillatum 5 6 5- ericetorum 8 3 5 tectorum 1 12 2 lacunosum 3 10 2

Ten out of the sixteen British bryologists suggest that lacuposum and tectorum belong to a single taxon. -6-

Table 3. The treatment of European bryologists to Hypnum'cupressiforme s. l.

FINLAND (1) mammillatum, resupinatum, ericetorum, filiforme,

lacunosum, vaucheri

FENNOSLANDIA (2) filiforme, lacunosum, resupinatum, vaucheri,

mammillatum, ericetorum

POLAND (3) cupressiforme, ericetorum, lacunosum, ma=illatumt

resupinatum, subjulaceum ( H. cupressiforme forma)

SWEDEN (4) resupinatum, mammillatum, ericetorump vaucheri,

DENMARK (5) mammillatum, jutlandicum

DDR (6) filiforme, mammillatum, lacunosum, resupinatump

jutlandicum, vaucheri

FRANCE (7) tectorum, filiforme, mammillatum

FRANCE (8) tector=, uncinatum ( H. cupressiforme forma)v

filiforme, reSuPinatum, mammillatum, subjulaceum,

ericetorum

FRANCE (9) imbricatum, elat=, tectorum, longirostrum,

resupinatum, filiforme, cuspidatum ( H. cupressiforme

forna), imponens, mexicanum (. H. cupressiforme forma),

ericetorum.

1. Koponen, Isoviita & Lamares (1977); 2. Nyholm (1954);

3. Ochyra & Szmajda (1978); 4. Jensen (1939); S. Damsholt et al. (1969);

6. Dull (1977); 7* Guillamont (1949); 8. Doignon (1950);

9. Doignon (1963) -7-

The taxa that have. been recognized in Britain since Wilson (1855) are:

var. cupressiforme

var. elatum

var. ericetorum

var. filiforme

var. lacunosum

var. ma=illatum

var. resupinatum

varo tectdr=

H. , cupressiforme var. elatum was reduced to synonomy with

R. -.cupressiforme var. lacunosum, by Limpricht (1904), var. tectorum reduced to a forma of var. lacunodum by Podpera (1954), var. ericetorum and var. mammillatum are treated as species by Nyholm (1954) and

Smith (1978a), var. filiforme is, reduced to synonomy with H. mammillatum by Smith (1978a).

Clearly . the status, of the taxa of H. cupressif6rme s. l. is controversial and it has been the aim of this study to investigate the nature and range of variation, between and within taxa as. well as assessing the status of the various taxa. -8-

CHAPTER 2

"'VAriation'in morphology

2.1 Materials and Methods

For convenience the name H. cuptesaiforme s. l. (sensu lato) is used to cover all taxa of the aggregate in, general descriptions etc.

Several approaches were used inthe study of H. cupressiforme Sol*

These were:

1. Studies of field and herbarium specimens

2. Cultivation experiments

3. Phytochemistry

4. Spore wall ornamentation

5. Cytological investigation

6. Drought tolerance

7. Ecology and distribution

2.2 Study of living and herbarium s2ecimens

Material of H. cupressiforme s. l. was collected in the field.

Each gathering was divided into two parts, one of which was cultured. the other preserved asan herbarium specimen* Herbarium material from

Bangor (U. C. N. W. )$ Glasgow (G. L. ) and Edinburgh (E) was studied.

Living material was cultivated at the School of Plant Biology,

Pen-y-Ffridd Field Station, Bangor. The specimens were grown in an unheated glasshouse in a mist unit on peat-based potting compost in

10 inch (25 cm) flower pots. The pots were covered with transparent polythene hoods to prevent cross-contamination. The plants were unshaded and watered with tap water (pH 7.5). As all the cultures were grown in a limited area (250cm x 120cm) at the same time, it could be assumed that growth was under uniform conditions. It was found that three months growth was adequate for the purposearequired.

r Samples were taken from living material before and after culture under uniform conditions were dried for later examination in the laboratory. Twenty f ive samples with f ive replicates f rom each CEthe gatherings was found to give satisfactory results.

The samples were dissected and the plants mounted ia. gum, chloral and examined using a Leitz "laborlux" microscope. Measurements were made with an eyepiece micrometer .

2.3 Characters selected for the study of He' tjTrts8ifdrme*s. l.

In the past relatively few morphological characters have. been used for the discrimination of, the taxa within H. cupressifdrme s. l.

(see for example, Dixon, 1924;, Nyholm, 1954; Smith, 1978a). These include gross morphology, branching pattern, shape of pseudoparaphylia.? leaf shape, leaf apex shape, nature of the leaf margin,, nerve strength, basal-cell shape, angular cell morphology, leaf cell size, capsule shape, posture of capsule, operculum shape and peristome structure. The following characters were studied and found to-be of use.

1. Gross morphology

(a) Plant size

(b) Branching pattern

2. Leaf morphology and anatoMZ

(a) Leaf length

(b) Leaf curvature

(d) Nerve strength

(e) Nerve length .

(f) Cell size

(g) Angular cell shape -16-

(h) Angular cell wall thickness

(i) Angular cell size.

3. Sporophyte

(a) Seta length

(b) Capsule posture

(d) Lid shape

(e) Lid length

(f) Spore size

Other characters sometimes thought to have some taxonomic value include plant colour and nerve width and which show a great degree of variability within H. cupressiforme s. l. were discarded after a preliminary study.

Measurements of gametophyte characters were made on field material before and after culture. Measurements of sporophyte characters were made on herbarium and field gatherings only as culture was not long enough for the growth of sporophyte. Plants were identified using the nomenclature of Smith (1978a) and additionally var. filiforme, which Smith (1978a) treats H. as a synonym of mammillatum , was recognized. 1. Gross morphology

(a) Plant size: plants were scored for three categories of sizeý large,. medium and small. All plants except var. filiforme came within the first two categories and intergraded. 70% of var. filiforme was small but after culture only 34% of plants remained small. It would appear that plant size, which is of some importance in discriminating var. filiforne, is partly an environmental effect and is of questionable taxonomic value. -11-

(b) Branching pattern: plants were scored for three categories of

branching pattern : irregular, loosely pinnate and densely pinnate.

All plants except H. Jutlandidum, fell within the first two categories

and intergrade. 88% of H.' Jutlandicum, gatheri. ngs were densely pinnate and

almost the same proportion (84%) remained so after culture. This

character appears to be. genetically determined and of good taxonomic value. I (c) Pseudoparaphyllia small, leaf-like structures surrounding

branch primordia and found at the base of young branches. Three categories

were scored: narrow, medium and wide. He vaucheri has much wider

pseudoparaphiglia than He cupressiforme s. l. (see Fig. 7d). Other plants

came within the first two categories with 63% of them with medium width

pseudoparaphylia. This character is valuable for discriminating H. vaucheri.

2. Leaf morphology and anatomy

(a) Leaf length,: reference to Figs-la-6a and Table 4 shows that

there is a considerable degree of overlapping. The amount of variation

in leaf length among all plants of H. cupressiforme, under uniform

conditions is markedly lower than that in the wildsuggesting high

environmental effects. In gatherings named var.. filiforme the amount

of variation is almost the same in plants under both conditions whilst

a slight increase in leaf, length is shown (see Table 4), indicating

very low environmental effects.

(b) Leaf curvature: Three categories were scored: slightly curved,

curved and strongly curved. H., mammillatum tends to have strongly curved

leaves and almost all plants retained the character after culture it is suggesting genetically determined. This character, moreovero has in discriminating. a good value between HO'mammillatum and var. -cupressiforme. All plants of H. cupressiforme came within the first two categories, did this not change after culture, taxa intergraded with one anothir and It with var. cupressiforme. would appear that leaf curvature

rk I I iN Fig. 1. Morphology of leaves and capsule of var. cupressiforme:

leaf ; angular cells;

cells ; (f) capsule lid;

(g) spores. TE I, - Fig. 2. Morphology of leaves and capsule of var. resu2inatum-o

leaf ; (b) angular cells;

(e) cells; (f) capsule lid;

(g) spores. CD

C)c

I Fig. 3. Morphology of leaves and capsule of var. lacunosum:

(a) leaf ; angular cells; (c) pseudoparaphylium (d) capsule ;

(e) cells-, (f) capsule lid

(g) spares. a0 Fig. 4. Morphology of leaves and capsule of H. mammillatum:

leaf ; (b) - angular cells ;

(e) cells ; (f) capsule lid ;

spores. a

d ('S

Ii U- '5 Fig. 5. Morphology of leaves and capsule of var. filiforme:

(a) leaf; (b) angular cells ;

(e) cells; (f) capsule lid ;

spores. (1S

0(: D Fig. 6. Morphology of leaves and capsule of Ho jutlandicuTn. *

(a) leaf; angular cells;

(e) cells; (f) capsule lid;

(g) spores. (2ý\,i D

(.1

(2) C b

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EI In'EV c) 0

Fig. 7. Morphology of leaves of H. vaucheri:

(a) leaf ; angular cells ;

which is of importance in discriminating 1j. mammillatum from- other plants

is not environmentally influenced and of good taxonomic value for

manmillatum.

(c) Leaf apex shape: two categories were scored: curved downwards 1 and straight. All plants except var. resupinatum have leaves curved

downwards at the stem apices, and were distinguishable. 96% of the 1 gatherings named var. resu2inatum had straight leaves at the stem apices,

but only 50% of the gatherings. remained thus which suggests that leaf

apex shape is not a reliable character in taxon discrimination as it is

phenotypically variable. This character is of little taxonomic value.

(d) Nerve strength: three categories. were scored:. not-detectable,

distinct and very distinct. All plants except H. vaucheri and some

gatherings of H. mamillatum had distinct. nerves. H. vaucheri-tends to

have a much more distinct nerve. than other plants. 10% of. the gatherings of H. mammillatum had no. nerves. This character is of good taxonomic value for discriminating H. vaucheri but is of no use within

H. cupressiforme sol.

(e) Nerve Length: H* ma=illatum, which possesses very short nerves

in comparison with other plants, shows a sharp decrease in the amount of variation under uniform conditions (see Table 4), Other plants show a considerable degree of overlapping. In plants referable to H. mammillatum nerve length is evidently affected by environmental factors and, as with other taxa, is of little taxonomic value.

Cell Size: This is a variable character, Plants. corresponding to H. mammillatum and var. filiforme have, shorter, cells (30-75 jim and

28-44 pm) compared with other plants (28-90 pm), As can-be seen from

Fig. (4e) and Fig. (5e) these two taxa are more or less distinct from the others of Ho cuRressiforme s. l., bothýbefore and-after culture (see Table 4). -13-

It is evident thatcell size-is markably affected by environmental conditions (Table 4), an interesting point as cell size is often regarded as a stable character.

(g) Angular cell shape: this character was scored for three categories: irregular, irregularly quadrate and irregularly rounded.

All plants except var. resupinatum had irregular andjor irregularly rounded angular cells. Almost all plants named var. resupinatum had regularly quadrate angular cells (see Fig. 2b), but only 55% of them retained this after culture which suggests that-this character is genotypically variable and of little taxonomic value.

(h) Angular Cell wall thickness: three categories were scored: thin, medium and thick. Three groups were formed based on these categories, the first groups with thin walls consisted of var. filiforme

(see Fig. 5b). Var. resupinatum and H. jutlandidum formed a second group with medium cell walls (see Fig. 2b; 6b). He mammillatum and var. cupressiforme formed a third group with thick walls (see Fig. 4b; lb). Var. lacunosum had a range of cell wall thickness. Observations after culture show that 52%, 65%, 80% and 72% of the gatherings of var. filiforme, var. resupinatum, H. jutlandicum and Ho''mamillatum respectively retained their original wall type. Clearly this character is genetically variable.

(i) Angular c2ll size: this is a variable character. All plants except Ho vaucheri overlapped and-were indistinguishable. H. vaucheri has very short angular cellsin comparison with other plants (see Fig. 7b).

The amount of variation among plants corresponding to*H. mammillatum, var. cupressiforme and vari r"u inatum, produced under uniform condition is almost the same of that in wild plants (see Table 4) suggesting genetic uniformity and little environmental effects. On the other hand the amount -14-i

of variation in the other three'taxi'showed a sharp decrease after culture suggesting marked environmental effects. Size of angular cell is a useful character for rec. ognizing H. vaucheri.

3. Sporophyte

Seta Length: This is is tendency a) a variable character, -There a for var. lacunosum to have a relatively short seta and for H. jutlandicum to have a relatively longer one. Other plant show a complete overlap and discrimination between them using this character is not possible.

b) Capsule posture: three categories were, scored-. inclined, intermediate and erect. Three groups were formed, the first group with inclined capsules was made up of var. 'cupressiforme, var. filiforme and

H. jutlandicum (see Fig4. ld, 5d, 6d). It was found that 82%, 82% and 89% of these plants respectively had inclined capsiýles. A second group was. made up of var. lacunosum in which 84% of the gatherings fill within the second category (see Fig. 3d). The third group with erect capsules contained H. mammillatum and var., resupinatum in which 86% and 84% of their gatherings respectively had erect capsules (see Fig., 4do 2d).

c) Capsule__Length: there is a, tendency for He jutlandicum to have the largest capsules (Fig. 6d) ard for Var. f ilif orme and var. ' resupinAtum to have the smallest ones (see Pigs 2d, 5d). Other plants overlapped and were indistinguishable. This character may be ofsome taxonomic value.

d) Lid shaRe: three categories were scored: lid mammillate, rostrate and subulate. Lid shape is a. good character for. separating %'mammillattim and var.. resupinatum from other taxa of H. cupressiforme s. l.

H*'mammillatum has a mamillate lid and var. 'resupinatum a subulate one

(see Figs. 4f, 2f). Other plants, have a rostrate lid and. were indistinguishable from one another. -15-

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e) Lid length: this is a variable character. All taxa overlapped

and were indistinguishable from one another. It is clearly of no taxonomic

value (see Figs. lf-6f).

f) Spore Size: spore size., of H. dupressiforme s. l. ranges from

11 to 24 M. The spores of var. ''filiforme tend to be smaller (11-16r=)

(see Fig. ýg) than those of other plants, but otherwise the character has

Jittle systematic value.

As no one character by itself, except for lid shape in the case

of H. mammillatum is discriminatory, it was clearly necessary to see if

all the characters taken in combination would separate the taxa.

2.4"'Spord Wall Ornamentation

Introduction

The spores of H. *cupressiforme range from 11 to 24 um. Surface

structures are not easily visible with the light microscope. The surface

of moss spores is often described simply as either smooth or papillose (Koponen, 1977). Although Erdtman (1965) noted the presence of large and

small spores in some Macromitrium species (e. g. M. crenulatum and

M. criotitrium) he made no mention of possible difference in surface structure.

The limits of light microscopy, in the study of moss spores and the

possibilities of using the Scanning Electron Microscope (SEM) have been

shown by Boroi & Jarai-komlodi (1975). McClymont (1955) showed the importance of observing-ornamentation in fully mature spores. He studied

spores from a range of Bruchia, species and could recognize two distinct

types of ornamentation. A similar observation with Orthottichum species

was made by Hirohama (197-7)v The'spores of British species of Polytrichum.

whi6h. all look similar under'the light'microscope, can be. distinguished

under'. the SEHby ornamentation (Dickson, 1969,1973). Denizot (1971)

showed in a similar way that Lunularia sPeciis are clearly ornamented I -

at the higher magnification of SEM. The study of the ornamentation of spore walls of the Bryum capillare group under SEX shown. Considerable differences (Syed, between one species and another which are of taxonomic significance

1973)'. Sorsa & Koponen (1973) studied the spores of fourty two species of the family Mniaceae by both light and SEM. Smith 1974 described three major differences groups of spores in the family Polytrichaceae and suggests that the between Polytrichum and Polytrichastrum are fundamental. Eight North American spocies of Eucalypts. can be distinguished on the bas-is of the spore morphology (1979) SEM (Vitt and Hamiltong 1974; Horton & Murray, 1976). Ramsay gathered data on the spores of eighteen Macromitrium species in Australia and examined daemelli several populations of some species (e. g. six of M.watsii, five of M. and seven of Macroccma tenue) as well as several capsules from each population to determine the reliability of surface ornamentation as a taxonomic character, she found that the general pattern that emerged was that although the papillae on the perine areas are densely distributed on the small -spores and more widely spaced on the large, there appears to be no absolute distinction between the in two types. From the investigation mentioned above, spore morphology mosses in may be useful in their classification and in some cases in identification in spite of the note of Koponen (1977) that ultrastucture is useful mainly the classification of taxa above specific level.

Materials and Methods

For SEM work, untreated spores are often taken directly from herbarium specimens, (Taylor et al. 1974, Saito and Hirohama, 1974), and this can give satisfactory results.

Results and Discussion

Although there are differences in size between the spores'. of some taxa of, H. cupressiforme s. l., spore wall ornamentation of H. 'rhamillatum,

jutlandicum lacunos 9avar. 'eupressiforme, var' um , var, resupinatum and var. filiforme shows no clear differences between them (see Fig. 8) (Va.

.' iL

I- i', t. 4 a b

ý Wl

p. -

C d

'; c4,

Fig. 8. Spore wall ornamentation of H. cupressiforme s. l..

(a) var. cu2ressiforme, (b) var. lacunosum

(e) H. jutlandicum; M var. filiforme

All x 15000

L. ". jjj . .C "T' -- -ý19-

Spore size has been discussed before (p., 17) and used in the numerical analysis. Ph-otomicr. ographs in plate 8 show that the spore walls are papillose and of the simple type in all the taxa examined. It would appear that the papillae are of almost equal size, with irregular rounded tips and more or less evenly distributed on the walls.

Spore wall ornamentation appear to be of no taxonomic value in the

11. cupressiforme complex. More transmission electron microscopic studies are necessary before any significant conclusions can be drawn.

2.5 Cytological investigation

Cytological observations on H. cupressiforme s. l. were carried out on six taxa: H. mammillatumg H. jutlandicum, var. cupressiformes varo resupinatum,, var. filiforme,. and var. lacunosum.

Observations on somatic chromosomes were made on meristematic (1: 1: 1) tissue of gametophytes. About 0.5cm of shoot apices were fixed in acetic acid : absolute alcohol : cbloroform for 2 hours or more

(Ramsay, 1969) . Fixed ma:terial was stained in a saturated solution of aceto-orcein in 45% acetic acid, for 12 hours or more at room temperature,

The apex was transferred to a drop of aceto-orcein on a slide. The shoot was macerated with a brass rod and a cover-slip added. The cover- slip was tapped a few times with a needle handle. Pressure applied to cover-slip to spread the chromosomes. After removing surplus stain by filter paper, the cover-slip was sealed with rubber solution.

Chromosome numbers and source of the specimens from which they were obtained are listed in Table 5. Table 6 gives chromosome counts from H. cupressiforme

The in s. l. by other authors. results Fig. 11 , are similar to those obtained'by Ramsay (1969), from British material. Garnetophytic chromosomes from the six examined taxa-are similar in number, morpholgy and position of the centromere. The longest chromosome in 'Ramptophypt mitosis is distinct. It is half quite almost one and a times length of the second -20-

Table 5. Showing chromosome numbers and source of Hypnum cupressiforme. s. l.

used in the present study.

Taxon Locality (n -

10 H. mammillatum Cwm-y-Glo, Caernarfonshire Nant Gwynant, Caernarfonshire 10 Aber Valley, Aber, Caernarfonshire 10 Treborth Botannical Gardeng Bangor. 10 Betws-y-Coed 10 H cupres sl fo rme 10 Var. cuýressiforme Treborth Botannical Gardent Bangor . Aber Valley, Aber. 10 Tyn-y-Mynydd, Anglesey 10 Nant Gwynant 10 H Cupmssiforme Var. lacunosum Aberffraw, Anglesey 10 Bwrdd Arthur, Anglesey 10 cu pres si forme Var. resimpinatum Maes-y-Geirchen, Bangor 10 Glanrafon Hill, Bangor 10 Penmynydd, Menai Bridge, Anglesey 10 (Smith)*

H. jutlandicum Old Quarries, near Llanberis 10 Quarry near Llanberis 10 Pengilfachs near Llanberis 10

Var. filiforme Aber valley, Aber 10 , Treborth Botannical Gardens 10

*All gatherings collected by the author unless otherwise stated

p -21-

for Table 6. Showing the chromosome counts reported in the literature

ii. cuRressiforme 6.1s

Author Taxon Chromosome Source Number

Vaarama, 1950 Hypnum 10 Finland 16 Rocky Mountains, Anderson & cupressif6rme N. America Crum, 1958 10 India Chopra & Kumar, 1967 11 Ukraine Visotskya, 1967 10 Great Britain Ramsay, 1969 10 Caucasus Visotskya, 1969 10 U. S. S. R. Visotskya, 1970 10 U. S. S. R. Petisova and Visotskya, 1970 10 U. S. S. R. Lazarenko et al. 1970 11 U. S. S. R. Lazarenko et al. 1971 10 Japan Inoue, 1971 10 France Wighs 1972 10 Sweden Tsutsumi et al., 1973 10,10+1 Austria Bryan, 1973 H CuPrýsslforme Var. cupressiforme 10 Great Britain Smith and Newton, 1966 10 Tasmania Ramsay, 1974 Smith H. mammillatum 10 Great Britain and Newton, 1966 10 Turkey Nyholm and Wigh, 1973

H cup reb sifo rme Var. 10 Great Britain Smith and resupinatum Newton, 1966 -22-

longest with sub-terminal centromere. Four metacentric and three

sub-terminal chromosomes' followed'. while the two smallestp of almost

equal size, are metacentric.

British material of H. 'dtipressiforrae s. l. has been studied by

Smith and Newton (1966), Newton (1968) and Ramsay (1969). Meitotic

studies of H. mammillatumvar. cupressiforme and var. resupinatum by

Smith and Newton (1966) showed that there are ten bivalents and that

variation in size of the chromosome complement is not very marked.

Meiosis in H. mammillatum, var. cupressiforme and varo r6dupinatum

was studied by Newton (1968) who found ten bivalents, generally large

showing a wide range of size. Ramsay (1969) showed that there

are ten bivalents in the msiotic complement (Fig. 9g). Of mitotic chromosome

from gametophyte cells the longest is quite distinct.

The haploid number, n- 10 has been reported by several authorities

(Vaarama, 1950; Chopra and Kumar, 1967; Ramsay, 1967; Vysotska, 1970;

Inoue, 1971; Bryan, 1973; Tsutsumiet al. 1973); . 11 was reported by

Lazarenko et al (1971) from the U. S. S. R. and n 10 +lm. by Bryan (1973)

from a single gathering from Austria. The count n* - 16 was reported by

Anderson and Crum (1958) from a single population from the Canadian Rocky

Mountains, but Smith and Newton (1966) say: "Professor P. W. Richards

(personal communication) suggests from field observations that the north American plant called He cupressiforme may not be conspecific with the European plant and the difference in chromosome number supports this".

Vaarama (1950) examined'meiosis in Finnish material and found ten bivalents, three large, in addition to threa medium size and four smaller ones. This differs from British material (see 'Fig. 9h).

Indian H. cupressiforme has n' - 10 (Chopra and Kumars 1967). The large, complement contains three three medium-sized and four small %W4.10 50 IV? % I 0404060 46 a dpp

) (4ý 44 // ellý01o ý f ?10 10 46 oo

o1 wli

g n

Fig. 9. Illustrations of meiotic chromosomes of H., cgressiforme s. l. I by:

(a) Anderson & Crum (1958) (b) Chopra & Kumar (1967);

(e) Vysotska (1970) M Lazarenko, Visotskaya, Lesnyak &

Mamatkulov (1970)

Ramsay (1969) ; (h) vaarama. (1950) - -23-

is bivalents. This agrees with Finnish material (Vaarama, 1950) but different from British material ýsee Fig. 9b).

Bryan (1973)'reported n- 10 from all her gatherings, from Austrian first material except one which had n- Ia. + lm- She found the chromosome to be a large conspicuous bivalent and in this it resembles British material (see Fig. 90 -

Ramsay (1974) examined material from Tasmania and found, ten bivalents in in at metaphase-1. The smallest bivalent disjoined early some and bivalent others was late separating. Such behaviour of the smallest was not reported from the British material but otherwise Tasmanian and

British plants are very similar (Fig. 9d).

Russian material was studied'by Lazarenko at al. (1971) who reported n- 11. It would appear from t1mir illustration (Fig. 9f) that there are last eleven bivalents, three large, seven of similar medium size and the (1970). one small. N- 10 was reported by Vysotska It would appear from her illustration (Fig. 9e) that there are three large and seven smaller of almost equal size. The first report disagrees with that of the British material in both number and morphology of chromosomes

(see Fig. 9f), but as in both report the presence of three large bivalents (1967) they agree. with those of Vaarama ' (1950) and Chopra and Kumar from Finnish and Indian material respectively.

Mitotic karotypes illustrated by various authors are as follows:

Ramsay (1969) found ten chromosomes in the gamatophyte calls with the

longest quite distinctl being one ahd. a half times the length of the second longest. It had a subterminal centromere. The next seven consist of four metacentric and three with subtarminal centromares, while the two smallest (of almost equal size)-are metacentric (Fig. l0a). ýý0. % lopoll. «. I 9jftb::)

Fig. 10. Illustrations of mitotic chromosou*s of H. cuRressifozme s. l. by:

(a) Ramsay (1969) (b) Wigh (1972);

1 diamm I)Ijr)(111

Fig. 11. Ill, ustration of gametophytic mitoses of H. 'cuBressiforme. -24-

Gametophyte mitosis of Swedish material was examined by

Tsutsumi et al. (1973) who repbrted'n - 10 according to the formula from formula n- 10 V+ 3V-+ 2J + 2v +i+ vo It would appear this

that the first six chromosomes are large. The first four with median

or submedian centromes and the other two'are with sub-terminal ones.

The rest are small consisting of two median or submedian, one terminal British and the last iaedian or submedian. Comparative formula at

and Swedish material would be:

British material n- lo: J+ 4V + 3J + 2v

Swedish material n' 10: V+ 3V + 2J + 2v +j+v

Hungarian material studied by Wigh (1972) had n- 10 with three

long chromosomes of similar size and seven others difficult to classify

(see Fig. 10b). The three large chromosomes are similar to those

found by Vaaroma (1950) and Chopra and Kumar (1967).

Wigh (1972) found the chromosomes of var. lacurlosum from

Czechosolovakia. to be similar to those of his Hungarian populations

of H. cupressiforme. (1973) Turkish material reported on by Nyholm and Wight had n with three long chromosomes and seven small (Fig. 10c).

In Japan* Inoue (1971) studied gametophyte mitosis of H. cupressiforme

and reported n- 10 with four median or submedian (Fig. 10d). The first

is the longest. The next two, chromosomes had subterminal centromeres, while the remaining for small chromosomes were two. median or submedian

one subterminal and the last one is an m-chromosome according to the. formula:

10: V+ 3V + 2J +, 2v ++

Comparative fomulae of British and Japanese material ýare: - --

-25-

British'matarial nw 10 :J+ 4V + 3J + 2v

Japanease. n- 10 V+ 3V + 2J + 2v, + j+m

(1973) except that The latter report agrees with that of Tsutsumi et-Als

the last chromosome which was reported from the Swedish material as (1971) be median or submedian is considered by Inoue to an ur-chromosome in longest This agrees with the British material the presence of the

chromosome. in It would seem that there is considerable variation chromosome there morphology within H. cupressiforme and that cytologically are

two groups, plants with one large bivalent or chromosome:

Ramsay (1969) reported from British material

Bryan (L973) reported from Austrian material

Ramsay (1974) reported from Tasmanian material

Tsutsumi et al - (1973) reported f rom Swedish material

Inoue (1971) reported from Japanease material

and plants with three large bivalents or chromosomes:

Vaarama (1950) reported from Finnish material

Chopra, and Kumar (1967) reported from Indian material

Lazarenk et al. (1971) reported from Russian material

Vysostska (1970) reported from Russian material

Wigh (1972) reported from Czechoslovakian material

Wigh (1972) reported from Hungarian material

Nyholm and Figh (1973)ýreported from Turkish material

Whilst the Japanese system of chromosome formulae is stylized and

their labelling chromosomes H or h (heterochromatic) is open to criticism

(Ramsay, 1969; Newton, 1977; Smith, 1978b), it is nevertheless a useful

way of rapidly compari. ng Karyotypes. Karyotype formulae of mitotic -26-

chromosomes illustrated by various authors are as follows:

Inoue (1971) ,a- 10 :V+ 3V + 2J + 2v +j+m

Tsutaumi (1973) n- 10 V+ 3V + 2J + 2v +i+v

Ramsay (1969) 10 : J+ 4V + 3J + 2v

Wigh (1972) 10 : 3(1 or V) +7Q or v)

Wigh (1973) n- 10 :3 (J or V) +7Q or v)

2.6 Variability within taxa, of H. cupressiforme s. l.

1. Var. cuRressiforme

Leaf length increased in culture (see Table 4, Fig. 12a), but the variation in length decreased. The increase in length is similar to that in H. mammillatum. All the other taxa show decrease in length variation in culture. This character is highly influenced by enviro=ental factors.

Nerve length varies in a similar way (see Table-40 Fig. 12b).

The increase in its length is also similar to that in HO mammillatum

All the other taxa also show decrease in nerve length variation in culture, suggesting that nerve length is highly influenced by environmental conditions.

Cell size is almost the same under both conditions as it is in

H. mammilatum (sea Table 4, Fig. 12d). The variation in size decreased in culture similar to all the other taxa except var. filiforme where it increased, suggesting that cell size is slightly affected by environmental factors and the variation is partly genetic in nature.

A slight increase and a slight decrease in angular call size (see Table 4, Fig. 12c)$ and variation in culture similar to that in var., resupinatum suggesting that angular call size is slightly affected by environmental conditions and the'variation is partly genetic in nature. Variation in othei'charactars, 'did rjot'jjdjcEtd -thatýmTdifferenceý -27-

between wild population are genetic.

2. Var. 'resupinatum

There is a slight increase in leaf length and a decrease in length

variation (Table 4, Fig. 13a). All the other plants show decrease in

length variation in culture, suggesting that leaf length is highly

influenced by e'nvironmental factors.

Nerve length increased slightly in culture and the variation in

length decreased slightly (Table 4. Fig. l3b), suggesting that nerve

length is slightly influenced by environmental conditions and the

variation is partly genetic in nature.

A slight increase in call size and a slight decrease in size variation

in culture (Table 4, Fig. 13d), suggesting that cell size is slightly

influenced by environmental factors and the variation is partly

genetic in nature.

Angular call size slightly increased and the variation in size

slightly decreased'in culture (Table 4, F,ig. 13c). This is similar

to that in var. cupresgiforme, suggesting that angular cell size is

slightly influenced by environmental factors and the variation is

partly genetic in nature* t About half the plants remained after culture with straight

leaves at the stem apices suggests that the apex shape is not a reliable

character in taxon discrimination and it is environmentally variable. Other did-not characters, show remarkable differences before and after culture.

3. Var. lacunosum

Leaf length is almost the same in field and cultured material (see Table 4

Fig. 144. The in le. variation ngth, de.creased, in culture. Leaf length H. jutlandicum filiforme -in and var. is almost the same in both cultures -28-

in (Table 4). All the other taxa show decrease in length variation

length influenced by culture, suggesting that leaf is slightly

variation is in nature. environmental factors and the partly genetic in A slight increase in nerve length is similar to that 14b)- H. jutlandicum. and var. filiforme (Table 4t Fig- ,var. resupinatumg The decrease in length'variation'is*similar to all'the'othet taxal

length is influenced by suggesting that nerve slightly environmental in factors and the variation is partly genetic nature.

is (Table 4, Fig. 14d). Cell size decreased slightly and this unique

The variation in size decreased similar to all the other taxa. except is var. filiforme where it increased, suggesting that cell size slightly is influenced by environmental conditions and the variation partly

genetic in nature. in An increase in angular cell size and a decrease in size variation culture is, similar to that in var. cupressiforme and var. resupinatum

(Table 4, Fig. 14c) suggesting that angular cell size is highly influenced

by environmental factors.

Other characters did not show a remarkable difference between wild

population and probably are genetically determined.

4. Var. filiforme

Leaf length is almost the same in both cultures (see Table 4, Fig. 15a).

This is similar to that in var. lacunosum and H. jutlandicum. The

variation in length is slightly decreased'in culture. All the other taxa

diow adecrease in length after culture, suggesting that this character is

slightly affected by environmental conditions and the variation is partly

genetic in nature. '

A slight increase in nerve length is similar to. that in var. resupinatum in var. lacunosum and H. 'Jutlandicum (Table 4, Fig. '15b)'. The variation -29-

length decreased in culture as in all the other taxa,, s uggesting that . nerve length is slightly influenced by environmental factors and the variation is partly genetic in nature.

A sharp increase in cell size is unique (Table 4, Fig. 15d).

The variation in size increased sharply and this is also unique, suggesting

that this character is highly influenced by environmental factors.

The size of angular calls is almost the same in both culture similar

to that in H. mammillatum and H. jutlandicum (Table 4, Fig. 15c).

The variation in size decreased in culture similar to all the other taxa except H. mammillatum where it is almost the same, suggesting that this character is slightly influenced by environmental conditions and the variation is partly genetic in nature.

5. H. mammillatum

Leaf length increased in culture and the variation in length decreased

(Table 4, Fig. 16a). This is similar to that in var. cupressiforme.

Var. resu2inatum showed a slight increased in length, suggesting that leaf length is highly influenced by environmental conditions. Nerve length varies in a similar way (Table 4, Fig. l6b). Call size before and after culture is almost the sams (see Table 4, Fig. 16d). The variation in size decreased in culture similar to all the other taxa except var. filiforme where it increased, suggesting that cell size is slightly affected by environmental factors and the variation is partly genetic innature

Angular callsize was found to be almost the same size before and after culture with the same amount of variation (Table 4, Fig. 16c), suggesting that the variation is genetic in nature. 0

Fig. 12. Histograms illustrating variation of var. cupressiforme

before culture (a-d) and after culture (e-h):

(a) leaf length; (b) nerve length;

Note that the Y axis is not identical in all cases.

Character (a) in m; for (b), 1 unit = 0.1 mm;for (c) & (d)s 1 unit = 26 ym. BEFORE CUL11TRE AF Tt R CU LTURE

6.6

3.0 a 2.0 )

1.1

I 1. a

I 6.0

2.4

4.8 a

14 16 It, 13

U I

I Fig. 13. Histograms illustrating variation of var. resupinatum

before culture (a-d) and after culture (e-h):

(a) leaf length; (b) nerve length

Note that the Y axis is not identical in all cases.

Character (a) in mm;.for (b), 1 unit = 0.1 mm; for (c) & (d), 1 unit = 26 BEFORE CULTURE AFTER CULTURE

418 LI

2.8

1.8 1.0

7 I

4 I

I, 13

4 a

d h

I i

a 30 aW fA Fig. 14, Histograms illustrating variation of var. lacunosum

before culture (a-d) and after culture (e-h):

(a) leaf length; (b) nerve length;

Note that the Y axis is not identical in all cases.

Character (a) in mm;for (b), 1 unit = 0.1 mm;for (c) & (d), 1 unit =. ?6, ym. BE FORE CULTURE AFTER cTiLTTTPE

4.8 4.4

Ll La

3.8

1.0

b f a I p

I a

1.6 Fig. 15. Histograms illustrating variation of var. filiforme

before culture (a-d) and after culture (e-h):

(a) leaf length; (b) nerve length;

(d) (c) angular cell size cell size .

Note that the Y axis is not identical in all cases.

Character & (d), 1 unit = 26 (a) in mm; for (b), 1 unit = 0.1 mm; for (c) BEFORE CULTUTRE AFTER CULTURE a

4.0 4.4

3.4 3.4

1.0 3.0

1.9 I's

f b

g C to 6.4

3.0

2.0

1.0

d h

13 S

a Fig. 16. Histograms illustrating variation of H. mammillatum

before culture (a-d) and after culture (e-h): (a) leaf length ; (b) nerve length ;

for (c) & (d), I 26 ym. Character (a) in mm; for (b), I unit = 0.1 mm; unit = 13EFORECULTURE AFTER CULTURE

S a

3 S

I. f

C a la 10

I 1

$ 4 Fig. 17. Histograms illustrating variation of He jutlandicum

before culture (a-d) and after culture (e-h):

(a) leaf length ; (b) nerve length

Note that the Y axis is not identical in all cases.

(d), 1 26 F. Character (a) in mm; for (b), 1 unit = '0.1 mm; for (c) & unit = BEFORECULTURE AFTER CULTURE

12 f

4 4

%.I

4.6 S S.6

4.6

1.0 I -30-

IL. illtlandicum Leaf length before and after culture is almost the same, as in var. lacunogum and var. 'filifdrme (Table 41 rig. 17a). The variation in length decreased in culture as in all the other taxa, suggesting that leaf length is slightly affected by environmental conditions partly genetic in nature.

A slight increase in nerve length is similar to that in var. 'resupinatum, var. lacunosum and var. filiforme (see Table 4, Fig. l7b). The variation. in length decreased in culture as it does in all the other taxag suggesting that this character is highly influenced by environmental factors.

An increase in cell size and a decrease in size variation in culture is similar to that in var. resupinatum (Table 40 Fig. 17d). The decrease in size variation is siuilar in all the other taxa except var. filiforme where it increased.

Angular cell size is almost the same before and after cultures as it is in H. mammillatum and var. filiforme (table 4, Fig. 17c). The variation in size decreased as ij all the other taxs, except H.' mammillatum where it is the same, suggesting that this character is partly genetic in nature.

2.7 Numerical approach

As quantitative characters overlap between taxa of H., E22ressiforme, it might be that the qualitative characters are the important ones and to investigate this a numarical technique incorporating both types of characters used. Ordination methods have been found useful in a variety of taxonomic contexts (Sneath and Sokal, 1973). -31-

A commonly used ordination technique$ principal component, analysis (PCA), is s=etimes, used'. in a-taxonomy (e.. g. Smith, 1970;

Rahman, 1972; Lewls and Smith, '1971). The choice of a numerical method was limited by the type, bf variation found in HO''cuoressif2rme, continuous and multistate discreteý PC& can be applied to continuous data but not to multistate discrete characters, Discrete characters with only two states can be. acc odated to give satisfactory results by coding 0 for the first state and 1 for the second as if, the characters are continuous. Sneath'and Sokal, (1973)'have shown, multistate discrete characters can be accommodated'as dichotomous variables. However, they advise caution when applyi. ng this. method; in small problems it may be possible to calculate the best dichotomy directly by-simple enumeration, but in large problems it, is, computationally impractical,. for example, if the colours green and blue, haVeýbeen, divided into, numerbus different shades'-using a colour, chatt. PCA: cannot be. used satisfactorily where there are multistate discrete characters (Hill and Smith, 1976). On the other hand correspondence analysis. (CA) can be, applied satisfactorily to data with multistate'discretia-.. ýhiracters. Smith, and Hill (1975) used

CA in an investigation of luropearkIllota soecies. But CA cannot be applied'toýa continuous data. Therefbre a, technique which, combines

PCA and CA is needed'. An exteniion, of PCA combining these two. requirements is described by Hill and Smith-. '(1976) and programmed by Hill 1976, in

ALGOL for use on the'U. C. N. W. -.Computer', the program bei. ug, called, MIXORD.

MTXORDwas applied'as followat

to herbariumi, material including II. 'VAudheri z

to-hetbaiium,. and-field material vithout. Hývaqcheri I (iii) to wild materialýbefbre 6ulture

to wild material after culture. -32-

As indicated below H.' vaucheri is very distinct and the second

analysis was run after the'Aremoval of H.''vaticheri data which may. have

affected the discrimination of the H. 'cupresgifdrme taxa.

In Figs. 18-26 it can be seen'that there are four distinct groups.

These. correspond to H. vaucheri (Figs. 18920), 11. mar=illatum, (Figs. 18*

20,21,22,24,25),, H. jutlandicum (Fig. 24) and var.. filiforme

(Figs. 22,23,26). Two groups remain distinct after culture and these

correspond to H. mammillatum and H. jutlandicum (see Figs. 27,28), but var. filiforme overlaps after culture with other plants (see Figs. 270

28,29).

Reference to Figs. (18920) show H. vauchdri to be a homogenous

and distinct group. Three gametophytic characters, pseudoparaphylia, nerve ýtrength and angular cells are discriminatory. Ando (1976) has

listed twelve gametophytic and sporophytic characters distinguishing

H. vaucheri from H. cupres6iforms. As I was unable to examine the

sporophyte of H. vaucheri I found the other gametophytic characters, epidermal cells of stem, leaf margin, laminal cells, lower laminal calls and perichaitial leaves showed a considerable degree of overlapping.

On the above evidence H. vaucheri should be treated as a good species

and not a variety or subspecies of H. cupressiforme.

H. ma=illatum shows some overlap with the other taxa examined in quantitative characters. Two quantitative and two qualitative characters

are to some extent discriminatory. These are nerve length and cell size.

H. mammillatum has much shorter nerve whilst cell size. tends to be short but shows, some overlap with the other taxa (see Table 4). The mamillate lid and strongly curved leaves"are discriminatory. H,, mammillatum has been treated by several authors as a variety of H, 'cu2ressiforme (Wilson, 1855.

Braithwaits, '1895-1905; Dixon and Jameson, 1924; Jensen,, 1939; -33-

Richards & Wallace, 1950; Warburg, 1963; Koponen, 1977). others have 1969; treated' it as a distinct species' (Nyholms, 1954; Damsholt, et. al .

Smith, 1978a). Figs. 18,20,21,22,24,25. and Figs. 27,28 after culture

dhow H. mammillatum as a homogeneous and distinct group and therefore

it it would be more appropriate to treat as a species than a variety.

Overlapping between H. mammillatum- and var. filiforme after culture is occured. The reason for this intergrating because of the presence of intermediate plants and the intergradation of various characters* in the Usually ! j. mammillatum and var. filiforme are distinct the shape of intermediate capsule lid, but the presence of plants with lids of an nature, make this character of little value.

In Britain var. filiforme was treated as a variety of H. cupressiforme by Wilson (1855)9 Braithwaite (1895-1905), Dixon & Jameson (1924),

Richards and Wallace (1950). Smith (1978a) treats this taxon as synonym of H. mammillatum. In Europe it has been variously treated as a variety 1977; of 11. cupressiforme, (Nyholm, 1954, Doignon, 1963; Dull Koponen et al;

1977), and as a distinct species by Guilmont (1949). Because of the occurrence of intermediate plants between it and H. mammillatum it would seem that var. filiforme is closely related to H. mammillatum rather than to H. cupressiforme. It should be treated as a variety of H. mammillatum.

It is evident that H. jutlandicum is distinct both before and after culture (Figs. 24,27,28), supporting the opinion of the sixteen

British bryologists as given in Table 2. The distinguishing features are densely pinnate branches and a very, long sata. Holmen and Warneke (in

Damsholt et al., 1969) introduced the name H; JutlandicumIto replace

H. ericetorum. (B. S. G) Loesq. which is a later homonymof H. ericatorum, Brid. Fig. 18. Ordination of morphological characters for H. cupressiforme s. l. herbarium and field material. Key to symbols:

(v) Specimens referred to H. vaucheri

(A) Specimens referred to var. cupressiform

(D) Specimens referred to var, resupinatum

(M) Specimens referred to var. lacunosum

(9) Specimens referred to var. filiforme

(0) Specimens referred to H. mammillatum

(A) Specimens referred to H. jutlandicum

Note that axis I plotted against II.

F-I

0%Cb DIP C4300 D. A D. I V, So: 1> MD. . ýo

40 0 00 o. A 000 044 00404 oc%-0

-60 60

Fig. 19. Ordination of morphological characters for H. cuRressiforme

s. l. herbarium and field material. Key to symbols:

(T) Specimens referred to H. vaucheri

(A) Specimens referred to var. cupressiforme

(Cj) Specimens referred to var. resupinatum

(a) Specimens referred to var. lacunosum

(0) Specimens referred to var. filiforme

(0) Specimens referred to H. mammillatum

(A) Specimens referred to Ho Jutlandicum

Note that axis I plotted against III. Fig. 20. Ordination of morphological characters for H. cupressiforme s. l. herbarium and field material, Key to symbols:

Specimens referred to H. vaucheri

Specimens referred to var. cupressiforme

Specimens referred to var. resupinatum

(a) Specimens referred to var. lacunosum

(0) Specimens referred to var. filiforme

(0) Specimens referred to H. ma=illatum

(A) Specimens referred to H. jutlandicum

Note that axis II plotted against III-ý

I 12ý Fig. 21. Ordination of morphological characters for H. cupressiforme

s. l. herbarium and field material. Key to symbols:

(A) Specimens referred to var. cupressiforme

Specimens referred to var. resupinatum

Specimens referred to var. lacunosum

Specimens referred to var. filiforme

(0) -Specimens referred to H. mammillatum

(A) Specimens referred to H. jutlandicum

Note that axis I plotted against II. Gatherings of'H. ''v6ucheri have been omitted. 8('4 8I I-I 2 8 0 co

e 1 Fig. 22. Ordination of morphological characters of H. cupressiforme

s. l. herbarium and field material. Key to symbols:

(A) Specimens referred to var. cupressiforme

([I) Specimens referred to var. resupinatum

(M) Specimens referred to var. lacunosum

(0) Specimens referred to var. filiforme

Specimens referred to H. mammillatum

(A) Specimens referred to H. jut andicum

Note that axis I plotted against III. Gatherings of H. 'vaucheri have been omitted. Fig. 23. Ordination of morphological characters for H. cupressiforme

s. l. herbarium and field material. Key to symbols:

(A) Specimens referred to var., cupressiforme

(a) Specimens referred to var. resupinatum

(M) Specimens referred to var. lacunosum

(0) Specimens referred to var. filiforme

(0) Specimens referred to H. mammillatum

(A) specimens referred to H. jutlandicum

11 against III. Gatherings of H. vaucheri have Note that axis plotted rý been omitted. B

6 Fig. 24. Ordination of morphological characters of H. cupressiforme s. l.

field material. Key to symbols:

(A) Specimens referred to var. cuDressiforme

(0) Specimens referred to var. resupinatum

(M) Specimens referred to var. lacunosum

(0) Specimens referred to var. filiforme

(0) Specimens referred to H, mammillatum

(A) Specimens referred to H. jutlandicum

have Note that axis I plotted against II. Gatherings of H. vaucheri been omitted.

Fig. 25. Ordination of morphological characters of H. cu2ressiforme s. l.

field material. Key to symbols:

(A) Specimens referred to var. cuRressiforme

(a) Specimens referred to var. resupinatum

(0) Specimens referred to var. lacunosum

(0) Specimens referred to var. 'filiforme

(0) Specimens referred to H. mammillatum

(A) Specimens referred to H. jutiandicum

Note that axis I plotted against III. Gatherings of H. vaucheri have been omitted.

Fig. 26. Ordination'of morphological characters of He cupressiforme s*le

field material. Key to symbols:

(A) Specimens referred to var., ey2ressiforme

(13) Specimens referred to var. resupinatum

(M) Specimens referred to var. lacunosum

(0) Specimens referred to var. filiforme

Specimens referred to H. mammillatum

(A) Specimens referred to H. jutlandicum

have Note that axis II plotted against III. Gatherings of'H. 'VaUcheri been omitted. at 8

l. Fig. 27. Ordination of morphological characters of H. cupressiforme s,

after culture. Key to symbols;

(A) Specimens referred to var. cupressiforme

(a) Specimens referred to var. resupinatum

(0) Specimens referred to var. lacunosum,

(0) Specimens referred to var. filiforme

(0) Specimens referred to H. mammillattim

(A) Specimens referred to H. jutlandicum

Note that axis I plotted against II. Gatherings of'H. ''vaucheri have been Witted. . Fig. 28. Ordination of morphological characters of He cupressiforme s. l.

after culture. Key to symbols:

(A) Specimens referred to var. cupressiforme

(13) Specimens referred to var. resupinatum

(a) Specimens referred to var. lacunosum

(0) Specimens referred to var. filiforme

(0) Specimens referred to H. mammillatum

(A) Specimens referred to H. jutlandic=

Note that axis I plotted against III. Gatherings of H. 'vaucheri have i- been omitted. s. I. 8

80 ...... 20 40 60 Fig. 29. Ordination of morphological characters of H. cupressiforme s. l.

after culture. Key to symbols:

(A) Specimens referred to var. ey2ressiforme

(M) Specimens referred to var. resainatum

(M) Specimens referred to var, lacunosum

(*) Specimens referred to var. filiforme

(0) Specimens referred to H., mammillatum

(A) Specimens referred to H. jutlandicum

Note that axis II plotted against III. Gatherings of'H. 'VAutheri have been omitted. 0

6 -34-

The situation concerning the remaining three taxa of H. cupressiforme s. l.

is much less clear and it is clear from Figs. 18-29 that there is considerable

overlap. The erect capsule with-subulate beak of var. ' resupinatum is distinctive although the upward pointing leaves, usually regarded as

a character, grade into the type found in var. CUpressiforme in culture.

Var. resupinatum is probably best treated as a variety of H. cupressiforme.

Var. lacunosum is also distinctive in its sporophytet with a short + seta and erect capsule although overlapping in gametophyte characters with var. cupressiforme. Again it is probably best treated as a variety of H. cupressiforme. -35-

CHAPTER 3

Variation in Non-Morphological Characters

3.1 Introduction

Mosses have a comparatively simple organization, and therefore the number of morphological diagnostic characters available for taxonomic work is rather low andmoreover, these characters show a relatively large degree of variability between and within taxa (Krzakawa, 1977), Thus recent modern taxonomic methods such as phytochemistry and isozyme, studies may provide additional means to investigate variability.

Several phytochemical methods of analysing plants have proved of value in both orthodox taxonomy and biosystematics. These methods seem to be the best alternative where genetical experiments are not possible

(Smith, 1978b). Bryophyte phytochemistry is reviewed by Huneck (1974) and Suire (1975). Use was made by Koponen (1968) of the so called

"Colour Substances" in cell walls of members of Mniaceae to group species.

A comparison was made of a cyclic sugar alcohol, in two Plagiochia and two Jamesoniella species (Lewis, 1970). The study of the distribution of unsaturated fatty acids in mosses wig applied by Anderson et al. (1974).

Lewis and Smith (1977) found that seven British species of Pohlia formed three distinct groups on the basis of their carotenoid pigments.

Isozymes were used in studies on interspecific variability of four

Pellia species (Krzakowaq 1977). Investigation of peroxidase isozymes in natural populations of Plagiochil. 1 asplenioides revealed very distinct isozyme polymorphism and geographical races in this species. Investigation of isozymes of twenty-one populations of Conocephalum conicum showed differentiation, of this species into three groups (Krzakowa, 1977). In an attempt to find more charactersinthe H. Q=elsiforme s. l. carotenoid -36-

'pigments and amino acids were analysed and isozymic, studies carried out.

3,2 Analyses of Carotenoid Pigments,

Differences in colour occur between some populations of H. cupressiforme

S. 1. and these were investigated by analysis of carotenoid pigment content.

Materials and Methods

The pigment content of six taxa of H. cupressiform s. l. was investigated using thin layer chromatography (Davies, 1965). The procedure followed in this analysis is given in Appendix 1. Two developing solvents were used, 20% ethyl acetate in methylene chloride and 15% methanol in benzene.

Results and Discussion

The chromatograms are shown in Figs. 30 and 31. With ethyl acetate as the solvent, the taxa examined fall into three groups on the basis of the number and position of pigments:

1. H. mammillatum, var. filiforme and H. jutlandicum, 7 spots

2. var. cupressiforme and var. resupinatum, 5 spots

3. var. lacunosum 9 spots

Using benzene as the solvent:

1. H. mammillatum and var. filiforme 7 spots

2. var. cupressiforme and var. resupinatum. 5 spots

3. var. lacunosum 9 spots jutlandicum, 4. H. 8 spots

There is a close similarity in the number and position of spots in

H. mammillatum and var. filiforme and in var. cupressiforme and var. resupinatum. This supports the suggestion that var. filiforme is close rbCD

PPC) CD

rp pa C"-=ý P

abCd0f

Fig. 30. Chromatographic patterns and spot colours of He cupressiforme

s. l. after spraying with antimony trichloride. Developing solvent: 15% methanol in benzene, Key to symbols: p purple pp pale purple

rp reddish

g grey

rb reddish brown pg pale grey

Where,

(a) var. lacunosum; (b) He jutlandic=;

(e) vare cupressiforme (f) var. resupinatum. P 01

P db C: D rb e c--> pp c::: > C)

P CD 0 bo cz:>

a b Cd f

Fig. 31. Chromatograph ic patterns and spot colours of

H. cupressiforme s. l. after spraying with antimony trichloride.

Dev eloping solvent 20% ethyl acetate in methylene chloride.

Key to symbols: p - purple pp - pale purple rb - reddish brown b - blue g - grey bg - bluish grey db - dirty brown

Wherep

(a) var. lacunosuml; (ýb) H, mammillatum;

(e) var. cupressiforme; M var. resupinatum,. -37-

to H. mammillatum and these two are quite distinct from the other jutlandicum., 11. cy2ressi-forme s. l. taxa, as is also H. supporting the is argument that they should be given specific status. Var. resupinatum

obviously close to var. Earessiforme but the affinity of var. lacunosum

is less clear with the e. a. solute. There are four spots additionalto those of

11. cupressiforme but the other spots are similar indicating similarity.

lacunosum is distinctive it is With the benzene solute var. , suggesting

less clearly related to var. cupressiforme and var. resupinatum.

3.3 Isozymes

Isozymes are considered to be the product of genes which segregate

in a Mendelian manner and they are therefore very useful as genetic

markers at the molecular level (Conklin and Smith, 1971). Systematists

exploiting electrophoretic methods of isozyme separation have discovered

particularly important characters in distinguishing species and clustering

them into species groups (Krzakowa, 1981). In recent years, studies on

genetic variation in natural hepatic populations based on electrophoretic

allozymic variations have shown that their patterns of variability are

of the same characters as those of higher plants (Krzakowa, 1977;

Krzakowa, and Szweykowski, 1979; Krzakowa and Szweykowski, 1977a). Taylor

and Elliott (1972) found two months of mild conditions were required for

Eurhynchium to re-establish normal activities of dehydrogenases after a

period of dryness or intense cold, Krzakowa (1977) used peroxidase

isozyme patterns as evidence for geographical races in Pellia endiviifnlia.

Isozyme electrophoresis has been used in investigation of interspecific

differences (Krzakowa and Szweykowskil, 1977a, b; Krzakowa and Szweykowski,

1980). Krzakowa (1981) used isozyme differences as charactersseparating

Pellia megaspora from the other Pellia taxa. She concluded that there are -38-

two groups of Pellia. peroxidase isozyme phenotypes, one composed of

Depiphylla, P. Wesiana and P. borealis. The other of P. endiviifolia. from

Europe, L. endiviifolia from Japan and P., megaspora. The phenotypes - within the second group differed supporting the suggestion that there

are three taxa presents

Materials and Methods

The method used is a modification of that of Gliddon (1976).

Electrophoresis of the Xauthein Dehyarogenase (XDH) system was carried

out on horizontal-slab, polyacrylamide-gels. The gel boxes used (LKB)

allow separation over a distance of up to 10 cm., with a choice of

sixteen or twenty-four samples being run concurrently. The gels were

polymerized in forms and transferred to the boxes. The gel surface was

cooled by a plate in which water at 30C was circulated. Temperature

control was achieved by means of an accurate electronic thermostat$

controlling heater immersed in the bath. The cooling facilities, with

the accurate thermostat combined with the large reservoir of cooling

water and high circulation rates, gave excellent temperature control

and therefore allowed good reproducibility of results.

Electrical connection of the gel was via wicks of absorbent

material. Both electrode compartments had platinum wire electrodes

extending the full width of the gel to minimise differences in the

electrical field across the gel. Pockets for the application of samples

were made by inserting a toothed plastic formen, into the unpolyrerized

gel mixture and removing it after the gel has polymerized.

Preparation of Gels

The gels were polymerized, at A maximum one day prior to uses

The gel strength was 7-.5% acrylamide and the components of the mixture were: -39-

Acrylamide monomer (BDH) 4.6 gm,

N-methylene bis-acrylamide (Eastman) 0,2 gm.

N, N, N1, Nl tetramethyl-ethylene diamine (Sigma) 0,14 ml

10% Ammonium persulphate solution 0.6 ml.

All ingredients were dissolved in 65 ml of the appropriate buffers

Tris/citric acid pH 7.0. The solution was made up, as above, adding the ammnium persulphate last and poured into the gel formen. The gel was allowed to polymerize at room temperature for 45 minutes. The appropriate buffer was then added, the gel placed on the cooling-plate and cooled for at least one hour after which it was ready for use.

Buffer System

A buffer system was chosen for the gel and electrode buffer that wculd give satisfactory results for different taxa, This is to minimise differences in treatment between plants. The buffer system used was as follows:

Tris/citric acid, pH 7.0

16.35 gm Sigma 7-9 buffer

9.04 gm Citric acid'(anhydrous) made up to 10 litres. The buffers are used at the same strength for both gel and electrode buffer.

Preparation of Plant Material

About 10 mg of fresh shootwere taken, washed in distilled water and placed in a grinding tube with 25 ul of grinding solution. The grinding solution was freshly made up of at least every two weeks and 0 kept in the dark at 2-4 C. The grinding solution was made up as follows:

0.1% Triton X-100 (W/V)

0.1% mercaptoethanol (V/V)

10% sucrose (W/V) -40-

in the appropriate buffer, Tris/citric acid, pH 7. The purpose of

the detergent was to improve cell distruption during grinding and the

mercaptoethanol was used to protect the cell soluble enzymes from the

effects of oxidizing agents (e. g. phenols) released in grinding. The

sucrose made the extract more dense and this caused it to layer when

applied to the gel. The plant material was ground up in the grinding

solution by hand over ice. The ground samples were then centifuged

at 10,000 xg for 3 minutes at OOC. 15 Ul of-the supern atant was then

removed with a Finpipette and applied to the sample pockets on the

gel.

Enzyme Stain

Details of the stain applied and which gave satisfactory results

for the different taxa examined areas follows:

Xanthine Dehydrogenase (XDH)

Stain buffer: 0.1 H pyrophosphate/glycine, pH 9.0.

Stain : 100 mg Hypoxanthine (Sigma) dissolved in 2 ml

1M NaOH. #

1g Glycine (to neutralize NaOH)

40 mg NAD

30 mg NBT

5 mg P14Safter 30 minutes incubation at 370C in

100 ml incubated at 370C until a satisfactory bands appeared.

Results and Discussion

Four different (XDH) isozyme phenotypes were distinguished (see

Fig, 32), These are: -

var. cupressiforme,, var. lacmosum and var. resupinatum with a

single large band. abcdf

Fig. 32. Diagram showing the observed xanthein dehydrogenase isozyme band patterns in H.. cupressiforme.

(a) H. mammillatum; (b) var. cupressiforme,;

(e) var, lacunosum; (f) var. resupinatum. z -41-

2. `H, Jutlandicum with a single small band.

3. H. mammillatum with two adjacent small bands.

4. var. filiforme with two bands, one large and one small.

Three varieties, var. cupressiforme, var. lacunosum and var. resupinatum are apparently closely related genetically. 11. mammillatum and He jutlandicum are clearly distinct. Whilst there is some similarity between He mammillatum and var, filiforme in that one band of each

coincide, there is another in each taxon which do not, supporting the hypothesis that there are two genetically distinct taxa involved.

3.4 AnalXses of-Amino Acids

According to Davies and Heywood (1963) the techniques of paper chromatography have been applied extensively to amino acid analysis, but the number of acids which have been characterized is quite small, around fifty, and the occurrence of perhaps half of these may be so general that they are of no particular systematic value. Refined techniques have made the identification of individual components feasible and these may have potential taxonomic significance, but so far the results have been disappointing although the restricted distributions shown by some of these acidsmay prove valuable. Huneck's (1974) and

Suire's (1975) reviews contain many tables which show the presence and absence of different chemicals in mosses. The aim of this studywas to investigate the presence of different amino acids in the six taxa of

H. cupressiforme, and the value of amino acid for the status of these taxa, -42-

Materials and Methods, in The six taxa. of H. cy2ressiforme s. l. were grown under glass a - in moist unheated unit Pen-Y-Fridd Stationt Bangor, Tap water of pH

7.5 was added daily in order to maintain a moist atmosphere under the

glasshouse. Materi. al was removed from this unit after at least

two weeks. Leaves were dissected from the stem and cut into fine

sectionsg which were homogenized and extracted with 2 ml 50% aqueous

ethyl alcohol at room temperature. After centrifugation the supernatant

was evaporated under reduced pressure. The residue was dissolved in

water (10 ml) and the resulting solution was added to the column of the

analyser in I ml quantities (Ilider and John, 1973). A Technicon Auto 0 Analyser utilizing a standard Dowex 50 column at 60 was eluted with

sodium, citrate buffer. The limit of sensitivity for amino acids was

10 n mol.

Results and Discussion

The following thirteen amino acids were found in every taxon:

Aspartic acid, Tyreonine, Serinel Glutamic acid, Glycine, Alanine,

Cystine, Valine, Tyrosineq Phenyl alanine, Lysine, Histidine and

Arginine.

Analysis of amino acids of six taxa of H. cuRressiforme s. l.

suggests that there are no differences between them in the type of amino

acids. This indicates that this character has no systematic value in

distinguishing between the taxa examined. More biochemical studies are needed before any conclusions can be reached. -43-

CHAPTER4

Physiology and_EcoloBZ of H. cupressiforme aggregate

4.1 Drought Tolerance

Introduction

is- One of the most important factors capable of causing stress

drought. Bryophytes differ from higher plants in that individual

shoots exert little control over water loss to dry air, while the (Hinshiri cytoplasm itself shows a high degree of desiccation tolerance

and Proctor, 1971). It is well established that many bryophytes can withstand dehydration to approximately 10% of their fresh weight and

subsequently return to a near normal rate of respiration and photosynthesis or dehydration'(Tucker et al., 1975). Recovery is not instantaneous, taking a period of hours or days and it may never be complete (Dilks and Proctor, 1974). Drought tolerance can be assessed from the effects of desiccation on rates of oxygen evolution. The highest degree of drought tolerance is generally found in bryophytes

from dry habitats(Clausen, 1952,1964; Dilks and Proctor, 1974)o

Intraspecific, adaptation to desiccation has been investigated by Lee and Stewart (1971) who reported that resistance was less in plants from wet than from dry habitats in Calliergonlcuspidatum, Climacium dendroides and Hypnum cupressiforme. Comparative experiments on plants cultivated under similar conditions are particularly important in this type of investigationt as desiccation resistance varies seasonally within the population (Hosokawa and Kubota, 1957; Dilks and Proctor, 1976). There have been relatively few detailed studies of seasonal differences but some were found by Ocfii (1952) in Dicranium japonicum and Polytrichum formosum material collected in Septemberg January and April. Hosokawa and Kubota (1957) found that all of their species from Japanese beech -44-

forests survived desiccation longer when collected in winter (November)

than in summer (August). In addition, Hinshiri and Proctor (1971)

reported that Anomodon viticulosus was more tolerant to desiccation in

April than in January. Lee and Stewart (1971) also found that

Callierszon cuspidatum, from a wet habitat was more tolerant to desiccation

in winter than in summer, while for material from a dry habitat the

reverse was true. Six bryophyte species showed clear evidence of

seasonal variation in desiccation toleranceg as measured by net assimilation

rate following 24 hours re-moistening. These speciess Plagiochila spinulosa, EXIocomium splendens, Scorplurium. circinatum. Tortula ruraliformis,. Racomitrium aquaticum and Andreaea rothiis showed low desiccation tolerance in autumn (October) and winter (January) and increased tolerance in spring and summer (Dilks and Proctor, 1976)..

The aims of this study were to investigate whether there is any correlation between net assimilation rate and resistance to desiccation in four taxa of H. cupressiforme s. l., to study the seasonal variation in each taxa and to determine whether it is influenced by genetic or environmental factors.

Materials and Methods

The material used in the experiments was collected on 4th October,

1979. and 10,12th January and 5th July, 1980, each from two separate areas, the material was kept after collection for at least one week in in polythene bags the laboratory. Details of the collection sites are as follows: -

Hypnum mammillatum. On fallen trees, in shadiBd humid habitato Trebortho

near Bangor.

On treL trunks, in shaded humid habitato Aber

Valley. -45-

U4tS"n ftj4fnM; -H , Treborth vdr. E113-forme. On vertical tree trunks, in shaded habitats,

and Aber Valley, H cupressif orme Arthur, var. Iacunosum. Limestone, in exposed habitat, Bwdyrr Anglesey.

Sandy in exposed habitat$ Aberffraw, Anglesey. H cup res- if o rm var. cupressiforme. On trees, in shaded habitat, Aber Valley and

Traborth. H cupressiformo var. M-upinatum. On rocks, in shaded habitat, Glanrafon Hill, Bangor

and Maesgeirchen.

The method used to desiccate the mosses was similar to that

described by Proctor (1972). Cut shoots were placed in desiccatm

hydrated CaCl 6H20, humidities over calcium chloride 2 giving relative

of approximately 32% at laboratory temperatureo

The ability of the plants to recover from desiccation was assessed

by dehydrating the plants for up to 150 days and then returning them

to moist conditions for 24 hours before measuring 02 evolution with

the Gilson respirometer. The dark respiration was estimated at 200C

by placing the material in the bottom of a Warburg flask with 2 cm3 0f

water and 0.5 cm3 of 10% potassium hydroxide in the central-well. The

flasks were covered with alluminium foil to exclude light. A second

set of Warburg flasks were prepared with no KOH in the central-well and

were illuminated from below with a bank of xenon lights which gave a

light intensity 260 2 of watts/m .A measure of photosynthesis was then

obtained. All results are based on oven-dry weight. The results shown

are the mean of five replicates. To examine the recovery of the plants

after dehydration they were transferredtomunheated mist unit aý

Pen-Y-Fridd Field Station, Bangor and re-examined after at leasttwo

months recovery. -40;-

Results

Drought resistance in all the taxa, examined declined with increasing desiccation time as shown by the decrease in oxygen evolution with increased desiccation (Figs. 33a - 37a). Drought resistance by appeared to be lowest in material collected in October as shown the in low photosynthetic oxygen evolution which was 50% lower than material collected in January. Oxygen evolution in October material was between 2.2 - 4.3 (average 3.9) pl. 02 /gm. dry weight/hour after ten days of continuous desiccation. A higher resistance to desiccation was found in material collected in January where the photosynthetic oxygen evolution was between 3,9 - 8.6 (average 6.5 ul. ) after ten days of continuous desiccation. The highest resistance was found in material collected in July where the oxygen evolution was 20% higher than that in January. In July the 02 evolution varied between 5.2 -

10.8 (average 8.8 jil. ) (Figs. 33a - 37a). After ninety days of continuous desiccation the 02 evolution from material collected in October was also 50% lower than material collected in January and was between

0.7 - 1.8 Ul. with an average 1.35 Ul. A higher resistance was found in material collected in January where the 02 evolution was between

I-4.1 Ul. with an average 3.2 pl. of oxygen (Figs. 33a - 37a).

The highest resistance was found in material collected in July where the 02 evolution was 20% higher than that in January and varied between 1.9 - 4.5 Ul. (Figs. 33a - 37a). In plants collected in January there was a rapid decrease in oxygen evolution after ninety days of continuous desiccation H. mammillatum and var. filiforme (Figs. 36a, 37a) and after sixty days in var. CUpressif-o, rre (Figs. 33a). This rapid in found decrease 02 evolution was in plants collected in October after Fig. 33. Changes in oxygen evolution of var. Mressiforme

dehydrated for 150 days hydrous CaCl 24 up to over a 2 and rehydrated hours before measurements comenced. Material was collected from

Aber Valley (-) and Treborth (--) in July (4), January (1:1) and

October (0) and the hydration treatment started immediately (Fig. a) or material was cultivated in glasshouse for two months before the start of dehydration (Fig. b). CD

LU C=f'

20 40 60 80 1WO 170 1F. Q 'tu

OAYS

=9 ID

-T-

C14 4

-- -a- 2

14G 160

0A "-"'3' . Fig. 34. Changes in oxygen evolution of var. resupinatum dehydrated for up to 150-days over a hydrous CaCl2 and rehydrated 24 hours before measurements commenced. Material was collected from

Glanrafon Hill (-) and Maesgeirchen in July (A), January (c) and

October (0) and the hydration treatment started immediately (Fig. a) or. material was cultivated in glasshouse for two months before the start of dehydration (Fig. b). a

U.j ar C4

loo 120 140 160 20 40 60 80

OAYS

LU cr 4

140 160 20 1.0 ao 80 100 120

DAYS Fig. 35. Changes in oxygen evolution of var. lacunosum

for 150 days hydrous CaCl 24 dehydrated up to over a 2 and rehydrated hours before measurements commenced. Material was collected from

Bwrdd Arthur (-) and Aberfraw (--) in July (,&), January (0) and

October (0) and the hydration treatment started immediately (Fig. a)

or material was cultivated in glasshouse for two months before

the start of dehydration (Fig. b). 12 N%

=9 10

6 C

LU CP 4

20 40 60 80 100 120 140 160

DAYS

M la

cI LLJ C:f4

.i ti ea 80 100 170 eto 160

DAYS Fig. 36. Changes in oxygen evolution of H. mmnmillatum

for 150 days hydrous CaCl 24 dehydrated up to over a 2 and rehydrated hours before measurements commenced. Material was collected from

(-) Aber (---) in (4) January Q3) Treborth and Valley July , and

October (0) and the hydration treatment started immediately (Fig. a) or material was cultivated in glasshouse for two months before the start of dehydration (Fig. b). 12

U.1 Cýo 4

20 40 60 so 100 12U 14U 1 Ou

DAYS

0 12 4

-A-

=m 10 zý

6 zz > LLJ c: r 4

N 43 6a 30 100 120 140 160

Ld-nAYS , 10 Fig. 37. Changes in oxygen evolution of var. filiforme

150 days hydrous CaCl 24 dehydrated for up to over a 2 and rehydrated hours before measurements commenced. Material was collected from

Treborth (-) and Aber Valley (--) in July (A), January (C3) and

October (0) and the hydration treatment started immediately (Fig. a) or material was cultivated in glasshouse for two months before the start of dehydration (Fig. b), 12 a

1: 9 10

LU d" 4

ILO 160 N 40 60 80 100 120

DAYS

\ C) "no uj 0

-KO 160 -47-

twenty days desiccation in var. cy2ressiforme while in material of var. cupressiforme collected July showed a similar (Fig. is pattern after sixty days desiccation 33a). This pattern

not as readily apparent in the other taxa (Figs. 34a - 37a).

After culture under similar conditions all plants were most

sensitive to desiccation in October, became less sensitive in

January and they reached their maximum drought tolerance in July

(Figs. 33b - 37b). The lowest value for 02 evolution from material

collected in October after ten days desiccation was 3.9 -5 Ul.

(average 4.5 111.) which was 25% lower than that of material collected

in Januaryq where oxygen evolution between 4.3 - 11.7 jil with an

average 7.6 Ul. The highest resistance was found in material

collected in July when the 02 evolutiop was 15% higher than that in

January and varied between 5.9 - 12.3 Ul. (average 10 ILI. ). After

120 days desiccation the 02 evolution was lowest in material collected

in October which was 15% lower than that in Januaryq and varied between 0.8 - 1.9 Ul. with an average L4 Ul. The resistance increased

during January where 02 evolution was between 1.7 - 2.1 Ul. with an

average 1.9 Ul. The highest resistance was found in material collected in July where 02 evolution was 20% higher than that in January (Figs. 33b

37b).

All taxa showed in each season a different pattern of change in photosynthetic oxygen evolution both before and after culture under glasshouse conditions, except var. filiforme where an almost similar pattern was found (Fig. 37a, b). The same pattern of change in oxygen evolution was found for material of each taxcncollected from separate localities except var. lacunosum where material collected from a sandy -48-

from limestone habitat showed a higher drought tolerance than that a

habitat (Fig. 35a)., This difference was not detectable in plants

that had been cultivated in the glasshouse (Fig. 35b).

Discussion

Tn all experiments dark respiration was much slower than

photosynthesis even though there is often an initial stimulation of ýomose (11440); respiration on remoistening. This agrees with observations of

Hinshiri and Proctor (1971); Dilks and Proctor (1974). Thereforep this

had little effect on the pattern of photosynthetic oxygen evolution

under our conditions.

The seasonal differences observed in response to desiccation

(Figs. 33a -'37a), agrees with the findings of Dilks and Proctor (1976)

on six bryophytes. The occurrence Of seasonal variation in resistance

'to desiccation was already noticed ýy Ochi (1952) in Dieranum, japonicum

and Polytrichum attenuatum. In October (autumn), resistance to drought

its lowest level is reaches where'O 2 evolution 50% and 70% lower thV

that in material collected in January and October after 10 and 90 days

desiccation, increasing during January (winter) and reaches its

highest resistance in July (summer)l where 02 evolution is 70% and 20%

higher than that in material collected in October and January after 10

and 90 days desiccation. This may relate to the physiological state of

the plants at a specific time of year and as all taxa, might have a

similar growth cycle, little difference can be detected between plants*

These results are similar to those obtained by Dilks and Proctor (1976) on

six bryophytes. In contrast these results disagree with those obtained by Hosokawa and Kubota (1957) who reported that the resistance of

epiphytic mosses to desiccation was hýgher in winter and lower in summer. -49-

The causes of the seasonal changes in tolerance to desiccation are not known. However, Schwabe and Nachmony-Bascomb (1963) found that the high level response in Lunularia was unequivocally photoperiodic. The high of photosynthetic oxygen evolution during the summer suggests resistance to drought during this season. The absence of the rapid decrease pattern in oxygen evolution during January in var. lacunosum during and var. XesuRinatum indicates high resistance to drought winter, lacunosum, similarly the absence of this pattern in H. mammillatum " var. high var. resLiRinatum and var. filiforme during October and July suggests resistance during autumn and simmer. The presence of the rapid decrease pattern in oxygen evolution after 90 days of continuous desiccation in material collected in January in H. maMj1-1a= and var. filiforme- suggests similar physiologies in these two taxa and low resistance during winter. The fact that all plants when grown under similar conditions showed their lowest resistance to drought in October (autumn), and highest resistance in the summer, suggests a degree of genetic uniformity between the plants. The production of a, different pattern of oxygen evolution in each season after culture by all plants except var, filiforre

(Fig. 37a, b), suggests that there is-a physiological adaptation in var. filiforme. This agrees with Longton (1979) who reported that the morphological variation of H. cupressiforme_is in part genetically determined.

A similar pattern of oxygen evolution was produced from material collected from two separate localities of each taxonin each season except for var. lacunosum collected during, July from sand dunes which showed a higher drought resistance-,. than that from a limestone habitat#

This agrees with the results of Lee and, Stewart (1971) who demonstrated in

Callierg2r cuspidatumo Climacium dendroides and Ho cupressiforme,, thus the -50-

is pattern of oxygen evolution in var. lacunosum influenced by the

type of habitat (at least in summer). The absence of this situation

after culture suggests that it is not genetically determined.

4.2 'The'Ecolog'and'distribution of H. cupressiforme s. l.

The order Hyprobryales is among several orders of mosses described

by Miller(1979) which have the potential to adapt to changing climate.

Longton (1979) says "Richards also referred to unpublishedwork by

Chamberlain which indicated that many characters distinguishing the

varieties of H. cupressiforme are genetically fixed so that these taxa

may also be regarded as ecotypes".

The six taxa studied are morphologically and ecologically variable.

1j. cu2ressiforme s. l. is widely distributed in different parts of the

northern and southern hemisphere except the tropics. The ecology and

distribution of H. cupressiforme s. l. has been discussed by Ando (1972b,

1979) and Doignon (1950,1951). The wide distribution and range of

habitat suggest a high degree of ecological tolerance. There is,

however, some correlation between habitat and morphological form.

All forms of H. Suj2ressiforme are dioecious and there is some

evidence (Gemmello 1950) that dioecious species are more frequent and

genetically variable than monoecious ones. This would appear to be

particularly true of H. cupressifome, s. l. with its wide ecological

tolerance and range of morphological forms. H. cuEressiforme s. l. is

the cormonest and most variable species in the British moss flora

(Smith, personal communication), Habitat relationships of the taxa of

cupressiforme s. l. in Britain are as follows: -

H. cu2ressiforme var. cupressiforme

This variety occurs on rocks, walls, bark, logs and sormtimes on hand-packed soil; never on peat or leaf litter, It is found both in Ireland. Fig. 38. Distribution of var. cupressiforme in Britain and in Britain and Treland. resupinatum I Fig. 39. Distribution Of var. pig. 40. Distribution of var. lacunosum in Britain and Ireland. in Britain and Ireland. Of 11 m-ammilatum Fig. 41. Distribution . in Britain and Ireland, Fig. 42. Distribution of var, filiforma

i 0

43. Fig. Distribution of H. jutlandicum in Britain and Ireland. -91-

exposed and sheltered habitats, especially where acidic, and the' altitudinal range is from sea level to 1230 m.

var. cupressiforme is widely distributed in the British Isles.

It is less frequent in the Midland. It would appear from Fig. (38) that in Ireland it is distributed near the coasts.

H. cupressiforme var. resy2inatum

This plant is found, usually in shaded situations, on tree branches and on rocks and in rock-revices. It is the variety most commonly encountered in maritime habitats (e. g. Cornwall, S. W. Ireland)#

Apparently neutral to calcifuge, a lowland plant.

Fig. (39) shows the distribution of var. resupinatum. It would appear it is less widespread than var. cupressiforme and especially in the north-east and some parts of the Midlands. It is also less abundant in Scotland than var. cupressifoLme. It is scattered in many places in Ireland.

3. H. cupressiforme var. lacunosum

A calcico le from chalk and limestone grassland, dune slacks, calcareous rocks and walls, very rarely trees. Plants from acid habitats are smaller and intergrade with var. cupressiforme. Ranging from sea level to 1000 m.

Fig. (40) shows that var. lacunosum is less common than both var. cupressiforme and var. resuRinatum. It is less frequent in the east and the north-east of England. It is scattered in Scotland and Ireland.

4. H. mammillatum

In similar habitats to var. cy2ressiforme. probably as common as$ if in not more so western parts of Britain and Ireland, but certainly overlooked, apparently rare in drier and exposed habitats and in lowland -. 12-

Britain. Ranging from sea level to ca. 1000 m.

Fig. (41) shows the distribution of H. mammillatum. It would

appear it is widely distributed in the south-west of Englandq north-

west of Wales and scattered in Scotland and Ireland.

5. Var. filiforme

Forms thin and sometimes extensive mats, usually on vertical tree

trunks, more rarely on vertical rock faces, in shaded humid habitats,

common in woodland habitats in western and northern Britain and in

Ireland, rare elsewhere. Neutral or calcifuge. A mainly lowland plant.

Fig. (42) shows the distribution of var. filiforme. It would

appear that it is frequent in the south and north of Britain, less widely

distributed in the Midlands and the east coast of England. It is

scattered in Ireland.

6. H. jutlandicum

On peaty soil and leaf litter on heaths, moorland and coniferous woodland, occasionally on tree boles and porous rocks. Strongly calcifuge.

Fig. (43) shows the distribution of H. jutlandicum. It would appear it is widely distributed except in south-east and central of

Ireland. It is abundant along the coasts and in the north of Ireland. -53-

CHAPTER 5

Conclusion and DescriEtion of Taxa

5.1 Conclusion

Cultivation experiments with the gametophyte plants of the

Hypnum, cu2ressiforme aggregate have shown that variation is partly J69- genotypic and partly environmental. There are four distinct species within the, aggregate, 11. cupressiforme sensu stricto, H. jutlatdicum,

H. mammillatum and H. vauche'ri. H. vaucheri is clearly distinct from

other taxa in three main characters, wider pseudoparaphyllia, shorter

angular cells and very distinct nerves These results are in line with

the conclusions of Ando (1976) who listed twelve gametophytic and

sporophytic characters distinguishing vaucheri from HO cupressiforme, 'R. I was unable to examine the sporophyte of H. va.ucheri and other

characters showed a considerable degree of overlapping with

H. cupressiforme aggregate. H. jutlandicum is also a discrete taxon both before and after culture. The branching system and seta. length are discriminatory characters, it being densely pinnate branched and having very long setae (up to 4.3 cm nerve of H. 'mammillatum is much shorter than in other taxa and although cell size shows some overlap with the other taxa it can be used to distinguish it from H. cupressiforme which possessesionger cells. The mamillate lid and a strongly curved leaf are also discriminatory characters. H. mammillat= is a discrete taxon and best treated as a distinct species. Plants of var. filiforme, may intergrade with H. manmillatum . Usually the lid of capsule and plant shape are distinctive, but intermediate plants occur. It seems that var., filiforme is closely related to H. maromillatum and best treated as a variety of it rather than Of cupressiforre. _H, The remaining three taxa appeared to overlap with each other especially in culture. The subulate beak and erect capsule of var, resupinatum are distinctive, although the upward pointing leaves, usually regarded as a -54-

discriminatory characterare not maintained in culture, It is best

treated as a variety of H. 'cuprdasiforme. Var. 'lactnoaum with a short

seta and erect capsule appeared. to be distinct. On the other hand it

shows a considerable degree of. overlapping in the gametophyte characters

with the other taxa, aad3*sbest treated as a variety of H. cupressiforme.

This taxonomic treatment does not receive any support from

cytological data. The aggregate is cytologically uniform. The only

general information on moss variability in relation to breeding system

is given by Gemmell (1950) who mentioned that dioecious species are the more widespread and that there are more varieties-described from dioecious

species than monoecious species. He suggests that this is related to

greater variability from. outbreeding.. H. cupressiforMe is dioecious

and. hence outbreeding. - It would seem from the degree of variability and the taxonomic complexity within the H. cupressiforme

complex that evolution is actively occuring. -55-

5.2 DescriRtion of Taxa

Hypnum cupressiforme Hedw.

Dioecious. Plants slender to robust,, stems prostrate, irregularly

pinnately to pinnately branched, branches prostrate to ascending;

pseudoparaphyllia. Lanceolate-subulate. Leaves appressed and

overlapping and straight to strongly curved and pointing in one direction,

concave, narrowly lanceolate, in mid-stem 0.9 - 2.8 mm long, apex

acuminate to filiform, margin plane or recurved below, sub-entire or

denticulate towards apex; nerve very short and double or wanting;

basal cells narrowly rhomboidal with rounded ends, incrassatet often

thick-walled incrassates irregular- I porose, angular cells to strongly quadrate in shape, hyaline to yellowish, middle angular cells mostly

12 - 23 U wide, cells in mid-leaf 30 - 102 Um. Capsule errect or

inclined, obloid to sub-cylindrical, straight or curved, 1.1 - 3.4 mm

long; seta 1.2 - 4.5 cm; lid with rostellate to subulate beako 'lid

1.2 - 2.2 am, beak 0.2 - 0.5 mm; penistome teeth longitudinally striate

below,. papillose or smooth above, bordered or not.

Var* cupressiforme

Plants slender to medi= sized. Leaves weakly falcato-secund to

falcato-secund, 1.2 - 2.5 = long, ovate to lanceolate, gradually

narrowed to long slender acumen, margin usually plans; angular calls

strongly incrassate, irregularly quadrate, middle angular cells mostly

17 - 23 um, cells in mid-leaf 54 - 102 Um long. Capsule inclined,

obloid to sub-cylindrical, curved or straight, 1,5 - 2.8 = long, lid

rostellate to rostrate, 0.5 - 0.9 mm long; spores 13 - 20 um. Fruit

occasional to frequent, autumn n- 10. ý Green or pals green patches on

rocks, walls, bark, logs, soil, etc., in sheltered or exposed habitats -96-

especially where acidic, very common.

Var. resy2inatum (Tayl. ) Schimp.

Plants slender. Leaves ± straight, usually directed upwards

especially at stem and branch tips, 0.6 - 1.4 mm long, margin entire;

angular cells i quadrate, thick-walled, hyaline, mid-leaf cells 50 - 85 Jim

long. Capsule erect, sub-cylindrical, straight or slightly curved

1.5 2,3 mm long; lid 0.8 - 1.3 mm long with subulate beak; spores

15 23 um. Fruit frequent, autumn. Pale green patches on barks logs,

rocks and walls in slightly shaded habitat, frequent.

Var. lacunosum Brid.

Plants robust. Leaves ± straight and imbricate to falcato-secunds

1.5 - 3.0 mm long,, concavep ovate to ovate-oblong to oblong-lanceolate

and tapering to channelled apex, margin recurved below, plane or inflexed

above, sub-entire to denticulate towards apex; angular cells quadrate to rounded-quadratesincrassate, cells in mid-leaf 30 - 100 Um long.

Capsule ± erect, sub-cylindrical, straight or slightly curved; lid rostrate, seta very short. Fruit rare, autt=, winter, Dull green to glossy, yellowish-green to golden brown patches, on usually basic soil, rocks, wall tops, etc., common,

H. mammillatum. (irid. ) Loeske

Dioecious. Plants very slender to medium-sized or filiform, stems procunbent or creeping, irregularly pinnately branchedl branches procumbent to ascending, spreading to parallel with stem. Leaves falcato-secund, ovate to lanceolate, gradually tapering to channelled acumen, margin plane or slightly recurved below, sub-entire to denticulate; nerve very short and double or wanting; basal calls narrowly rhomboidal with rounded ends, incrassate, porose or notp angular cells i irregularly quadrate to quadrates strongly incrassateg -57-

inclined, cells in mid-leaf 30 - 65 um. Capsule erect or oblong- 1.4 2.3 ellipsoid to sub-cylindricall straight or slightly curved, - mm long; lid mamillate 0.3 - 0.5 = long; outer peristome teeth longitudinally striate or finely papillose above; spores are variable in size 12 - 23 Um.

Fruit apparently common in larger forms, autumn n- 10. Yellowish- green to green patches on bark, fallen logs, rocks and wall topsp shaded conditions,, rarely on soil. Common.

Var. Tnammillatum

Plants slender to medium sized, stems procumbent or creeping, irregularly pinnately branched, branches procumbent to ascending, I spreading to parallel with stem. teaves falcato-secund, ovate to lanceolates gradually tapering to channelled acumen, margin plane or slightly recurved below, sub-entire to denticulate, nerve very short and double or wanting; basal cells with rounded ends, incrassate, porose or not, angular cells i irregularly quadrate to quadrate, strongly incrassate, cells in mid-leaf 3.0 - 6.5 um. Capsule erect or inclined, straight or slightly curved, 1.4 - 2.2 mm long; lid mamillate 0.3 - 0.5 mm long; spores variable in size, 12 - 23 M. Fruit co=on, autumn, n- 10. Green patches on bark, rocks and wall rops, shaded conditions.

Common in western and northern Britain but rare elsewhere except in humid habitats.

Var. filiforme

Plants extremely slender, procumbent, stems longj distantly pinnate, with long ± straight i parallel, almost filiform branches, forming very low patches. Leaves small, regular, falcato-secund usually denticulate; nerve short;, angular cells ± irregular, incrassate, cells in mid-leaf 28 - 44 um. Capsules small, inclinedisometimes i erect, -58-

spores small, 11 - 16 um. Fruit rare-jj - 10. Yellowish-green patches on

trunks of trees, rarely on vertical rock. Co=on in woodland in western

and northern Britain, occasional elsewhere.

ji. jutlandicum Holmen & Warncke

Dioecious. Plants madiunr-sized, stem greenish, procumbent to

ascending, densely and regularly pinnately; pseudoparaphyllia

lanceolate-subulate to triangular with subulate apex. Leaves not

crowded, slgihtly overlapping, falcato-secund to ovate, tapering to I acuminate to filiform apex* margin plane, sub-entire to denticulate

basal - towards apex; nerve very short; cells narrowly rhomboidal,

incrassate, angular cells incrassate, rounded-quadrate, cells in mid-

leaf 40 - 70 um. Capsule inclined, obloid to shortly cylindrical,

curved, 1.2 - 1.5 mm long, outer peristome teeth vertically striate or

smooth above; lid rostrate; spares 12 - 15 pm, Fruit rare* winter,

spring. Pale green tufts or patches on heaths, moorland and montane

habitats. Cormon in suitable habitats. (59)

cli CY) u (2) .0 u "a r- CA. u =3 t1o > r 4-)

r., S-

Uý 0 t;r- 4- 0E 0 0t; 4- C) N CA

Lf) r u 4) r- .0 ICL CU Ln IT S- u 0 C) CY)

L9 CD C%i Qj 4-) 4-) r_ CU r- Lf) W 0 (a (3) 3: ty) V 4-) (o r- (1) 0 4-3 "a v r- Cý (1) (1) cu C4 u (A ol 41 1 W u 3: W V 1- C) S- (A (U 0 (0 2 C:) (n a) 4-)0) 4-) u m 4-) 1 C c (1) r- tn 4-3 (o C E-= ca. co s- r- r- V) ro to CL 4J CL u CD .0 "Clia) C? (1) -0 >1 >) u 9 r- ch CD r- 0 (tix m r- 4-) 0 Q:t 3: L. (A E x 0 tn "a CL 4.r- ) =3" o- E Ca A 4) (0 r- i%, (v :3 r_ CL I I I: t: n CD ::k Cl (1) 4J 4) a) C =3, CD . co N CD (1) (1) to 8 N (7) c r- +3 C) C2j C2. a L. S- 4-) P cu I- U) co 0 0 tA I oo 4 - aj >1 a) rm "a #-% S- S- Lf) .0 9 N LO r (n r-- S- L. tn c r-_ LO LA cn cn r- 2 5 Cý a) V C; 1- 0 4-J0% r- S- u ml go m 0 3c E r- "0 r- CA. 0 S. c >1 S- r- 0 . I- M 4-3 ea. 0 PCL > tm (1) r- a) a) V) to (0 o>-, 0) 3: S- M S- r- CL) &- 21 4J 4-3 0 . r- > u L) V 0 C n3 4) S.. 4-P cu r- (1) (1) tA Cý. r- ca. Ln r. 0 0 V) W 1. S- :3 tv 0 ýkd u tm (U >ý 10 "0 r- 4-) 4-) r. 1- .0 r- :3 r-W 4-) >a) (1) 0 0 (A (A 0 r- r- 4A cl) CL +3 0. u CL 0) (L) 4J 4-3 C ICL C > > 0 > > C: &- L. 0 mi to M Gj (1) 4) r- V) ca I -. i Q. CA.

C%j cr) q*l (60)

C) 4-) E to 01 42 r 4-

9 4- S- S.: .uu to El L:to

4J M r 4- . r- IA (A A) .S-

4 IS 4-3

..,. % S- .0 4-) Cýj 0) :3 Cl > Its 41 4-) 00 r_ u 0 L. r- L. L. LO 0

+1 (A 0 r- a 0 4-3 V-4 cn S. r- t; Q)-

4- LC)

co :3 r- >1

4. ) C14 u #A

4-3 u 4J 0 cyl u U, 7 Cý 4J 91 4-) 4-J W. (a AS X, a CA L9CD 41 %..0 r- %-0 1 #A LA V7 Q

LO to -61-

I 'CHAPTER6

Variation in'Atr'chum undulatum

6.1 Introduction

The situation in A. undulatum where there are three cytotypes, one haploid, one diploid and the third triploid is of particular interest. They are, however, morphologically indistinguishable

(Smith, 1978b). Cultivation experiments are needed to study the variation between and within the cytotypes and to ascertain whether or not they are morphologically indistinguishable. Similar work has been carried out by Newton (1968) in Tortula muralis with n- 26,27 and 50,52. No differences were found between the two haploid races or between the two diploids. Whilst some populations with n- 50,52 are distinct from haplolds others were indistinguishable from the haploids.

Although the natural chromosome races of Funariaceae are indistinguishable the artificial polyploids produced by Wettestein (1940) had larger cells than plants from-which they were derived. Ramsay (1967) studied Hypopterygium rotulatum with n- 99 18 Ea. 27,36 found few differences between them. Smith and Newton (1968) in studies on

Funaria hygrometrica (in which 11 - 28 and 56), Physcomitriumlpyriforme

QL - 26 and 52), Pohlia nutans QL a 22,33) and As undulatum 70140

21) could find no differences between cytotypes within these species.

As the three cytotypes in As undulatum, are apparently indistinguishable it is necessary to include as many characters as possible in this study to obtain satisfactory results. -62-

6.2 CXtological Investigation ,

The Polytrichales is an order that is particularly well known cytologically. 240 species have been counted; of these 76% are ISome haploid with n-7,17% are. diploid with n- 14 and 7% are of higher ploidy (Smith, 1978b). There are a few examples of chromosome numbers not based on x-7, but it is likely that they are erroneous and they should be discounted. There are a few examples in the genes

PolXtrichum of intraspecific polyploidy (e. g. Polytrichum alvinum var. brevifolium. P. commune. P. 'formosum, P. longisetum and P. 'Piliferam.

(Fritsch, 1972).

The biosystematic situation in A. undulatum is of a particular interest, Three cytotypes are known, one haploid, one diploid and the third triploid. Wilson (1911) reported a count of 16 - 17 for British material. Heitz (1926) reported the chromosome number of this species as "14 - 16" and in (1928) as "(20-)21(-22)". This is the earliest report of intraspecific polyploidy in mosses. Cytological variation appears to occur throughout the range of, &. undulatum (see Table 7).

Haploid and diploid gametophytes have been reported from North

America (Lowry, 1948,1954; Steere, Anderson and Bryan, 1954; Bird,

1962; Khanna, 1967) and both occur in India (Chopra, 1959 (as A. subserratum):

Chopra and Bhandari, 1959)o There have been reports of the three cytotypes from Japan (Tatuno, 1953,1960; Kurita, 1938,1950; Noguchi and

Osada, 1960; Yano, 1957; Tatuno and Kise, 1970). There have been a number of reports of various cytotypes from different parts of Europe, the Ukraine (Lazarenko and Visotskya, 1964; Lazarenko, 1967; Visotskyat 1967;

Fetisova and Visotskya, 1970; LaZ4renko et al., 1971; BArzarina and Solonia, 1972; Lazarenko and Lesnyak, 1977), Austria (Bryant 1973),

Czeckoslovakia (Wight 1972), Denmark (Holment 1958), Latvia (visotskya and -63-

Table 7. Showing the haploid, diploid and triploid counts reported in

the literature for'Atrichum uridulatum.

Author ..... -Source

14-16 Heitz (1926) Europe

20 Heitz (1928) Europe

16-17 Wilson (1911) G, Britain

21 Lewis (1957) G, Britain

14,21 Smith & Newton (1966) G, Britain

7,21 Smith & Newton (1968) G, Britain

21 Holmer (1958) N, Europe

21 Bryan (1973) Austria

21 Wigh & Strandhede (1971) Swedish & Dutch

21 Wigh (1972) Central &. South Europe

14 Lowry (1948) N. America

7,14 Lowry (1954) N. America

7 Steeres Anderson & N. America Bryan (1954)

7 Khanna (1967) N, America

7 Bird (1962) N, America

21 Kurita (1938) Japan

7 Kurita (1950) Japan

7,21 Tatuno, (1953) Japan

7,14,21 Tatuno, and Kise (1970) Japan

7 Yano (1957) Japan

7014921 Noguchi & Osada (1960) Japan

14 Tatuno, (1960) Japan -64-

Table 7 continued: -

n Author Source

14 Chopra & Bhandari India

7 Kumar & Anderson in India Love (1968)

21 Lazarenko & Visotskaya (1964) Ukraine U6S. S. R.

14,21 Lazarenko & Visotskaya (1965) Ukraine U. S*S. R*

21 Festisova & Visotskaya (1970) Estonia U. S. S. R.

21 Visotskaya (1967) Ukraine U, S*S,,R,

l4j2l Lazarenko (1967)

21 Visotskaya (1970) Caucasus U. S. S. R.

7,14 Lazarenko & Lesnyak Kazakhstan and (1977) Tadzhikistan

7 Lazarenko (1967) -65-

Fetisova, 1969), Lithuania (Danilkiv and Visotskya, 1975) and Sweden

(Wigh and Strandhede, 1971). 1966p 1968), counts reported from Britain are n- 14 (Smith and Newton$ 1966,1968). from Ireland n- 21 (Lewis, 1957; Smith and Newton, and 1968). n-7 and 21 (Smith and Newton, (1977) 307 In a large scale study Lazarenko and Lesnyak examined haploids, 6 diploids gatherings from European U. S. S. R., they found no 21. They that with n- 14 and 301 were triploids with n- concluded in A. in spite of morphological similarity the chromosome racesof undulatum Smith Newton they ought to be considered as cryptic sibling species. and differences (1968) also commented that they could find no morphological between different cytotypes. it Clearly A. undulatum has three chromosome races and was studied to investigate the possibility of there being a correlation between between chromosome number and geographical distribution and chromosome n=ber and morphology in the British Isles,

Materials and Methods (Pageig). Methods used are similar to thcEe forH, cupressiform s. l.

Material of A. undulatum was collected from different areas of Britain, for allowed to grow in polythene bags in the laboratory at 10 - 220C at least two weeks, The new shoots that were produced were examined cytologically using aceto-orcein stain. About 0.5 cm of the shoot apex was fixed in acetic acid : absolute alcohol chloroform (I: Jtl) for

2-4 hours (Ramsay, 1969), Fixed material was stained in a saturated solution of aceto-orcein in 45% acetic acid for 12 hours at room temperature. After staining the apex was transferred to a drop of aceto-orcein on a slide, The shoot was macerated witha brass rod and a cover slip added and tapped with the point of a needle to spread the calls. -66-

Pressure was applied to cover slips to spread the chromosomes.

Results and Discussion

Chromosomes of gametophyte mitosis are illustrated in

Fig. (400 co d). The karyotype is but described in terms1of relation lengths (in Jim) as follows:

The haploid n-7

Chromosome 1 (6.34 ± 0.11)

2 (6.34 ± 0.10

3 (6.32 ± 0.90)

4 (5.32 ± 0.12)

5 (5.25 ± 0.11)

6 (4.19 ± 0.10)

7 (3.65 ± 0.13)

The diploid n- 14

Chromosome 1 (6.18 1 0.60)

2 (6.11 ± 0.22)

3 (5.93 ± 0.80)

4 (5.72 ± 0.06)

5 (5.66 ± 0.15)

6 (5.63 ± 0.11)

7 (4.85 ± 0.16)

8 (4.78 ± 0.18)

9 (4.77 t 0.11)

10 (4.74 ± 0.05)

11 (4.70 :t 0.15)

12 (4.67 :t '0.14) 13 (3.2 7 0.0 8)

14 (:3., 30 0.12) I )Nv% (c'. )t /)/) (C%c,

a b

OMM

ojj^ýl 0 4^^? t

( << 4ev T,

Fig. 44. Somatic chromosomes in the gametophyte of Atrichum

(a) A. crispum ; (b) haploid A. undulattim- (c) A. diploid undulatum; (d) triploid A. undulatum,

0 -67-

The triploid n- 21

Chromosome 1 (6.03 t 0.11)

2 (6.13 t 0.15)

3 (6.10 t 0.10)

4 (5.92 ± 0.05)

5 (5.90 ± 0.10)

6 (5.91 i 0.13)

7 (5.80 ± 0.12)

8 (5.70 t 0.21)

9 (5.70 0.15)

10 (4.90 0.04)

11 (4.80 i 0.14)

12 (4.75 ± 0.12)

13 (4.75 t 0.15)

14 (4.81 ± 0.13)

15 (4.75 ± 0.12)

16 (4.40 ± 0.09)

17 (4,30 :t0,05)

18 (4.25 :t0.12)

19 (3.50 ± 0.14)

20 (3.60 ± 0.06)

21 (1.45 ± 0.18)

From Appendix 2 it would appear that the mean length of chromosomes of the haploid, is significantly greater than that Of the diploid and triploid which they do not differ significantly.

From the above data it would appear that the diploid has seven pairs of chromosome, each pair consisting of two chromosomes of almost equal length and sirdlar centromere Position, In the triploid the chromosorms appear to be in threest each consisting of three chromosomes of almost -68-

equal length and similar centromere position.

It seems to be that Ae''Undtilatum is a good example of an

intraspecific polyploid series, with the following karyotype formulae

n-7: V+V+ 2j + 2v

n- 14:, 2V +V+ 4j + 4v

n- 21: 3V + 6V + 6j + 6v

Smith (1978b) comments on using such formulae that the differences

in shape and size implied by the formulae may be misleading as there

is often a complete intergradation in size and position of centromeres between related species. Karyotype formulae may be used for rough

comparisons. There is no significant variation in size and centromere position within cytotypes of populations of different origins

(Fig. 44b, c, d).

Lowry (1954) suggested that reports of haploid and triploid

races in A. undulatum (Figs. 45gp ho 46k, 1) form the most extended natural intraspecific polyploid series known in mosses. Data that have been accumulated since indicate that A. undulatum is not exceptional. Naturally occurring intraspecific poliploidy with three or more cytotypes have been found in Amblystegium riparium (n - 12,24,

36), Funaria hygrometriCa 99 18,27,36) and Pohlia nutans (n

110 220 33) and in the liverwort Dumortiera hirsuta (n - 9j 18,27) (Smith,, 1978b).

In British material of A, undulatum examined by Newton (1968) the meiotic bivalents are very large relative to SMC9., those of the haploid form being largestq and those of the triploid form smallest

(see Fig. 45b, c, d).

Bryan (1973) reported a count of n- 21 from Austrian material where meiosis proceeds normally. The meiotic karyotype is similar to that of triploid British material (see Fig. 45e). sj 46 so r

I %ov to 18;

% 1116 qp *: to qW

IN wä\ ,

,% ir 44 4 Ob %

0?,jo fA.;

Fig. 45. Illustrations of meiotic chromosomes of A. undulatum by:

(a) Smith & Newton (1968); (b-d) Newton (1968);

(e) Bryan (1972) ; (f) Khanna (1967) -,

(g, h) Lawry (1954); (1) Steare, Anderson & Bryan (1954)-,

(j) Lazarenko & Visotska (1964) (k) Lazarenko & Vysotskaya (1965);

(1) Lazarenkoo Visotskaya (m) Visotska (1970).

& Lesnyak (1971): -69-

Russian material with n-7,14 and 21 has been reported. The from diploid and triploid are relatively smaller than those produced

the British material. On the other hand the presence of the largest 45j-m). The bivalent agrees with that of British material (see Figs. (1967) Visotstkya haploid count n-7 has been reported by Lazarenko and

(1965).

(1954) In North America n-7 and 14 have been reported. Lowry (see reported n- 14 and 21. Meiotic bivalents in American material

Figs. 45g, h) of diploid plants are similar to those in British plants.

Steere, Anderson and Bryan (1954) reported n-7 (Fig. 45i) and

Khann (1967) also reported n-7 from North America (Fig. 45f).

The mitotic karyotype has been studied by many authorities.

Wigh and Strandhede (1971) reported n-7 and 21 from Danish and

Swedish material (Fig. 461). He says "it is hardly possible to

distinguish different size classes among the chromosomes though the

longest one is about twice as long as the shortest chromosome". He also

reported (1972) n- 21 from central and southern Europe (Fig. 46j). There

is a difference in size range from British material (see Fig. 44b-d).

Kurita (1938) studied the gametophyte chromosomes of A. undulatum

from Japan, he reported n- 21 (Fig. 46a). Tatuno (1960) reported n- 70 14

and 21 (Fig. 46b, c, d). It would appear that there are some similarities with and differences from triploid British material (see Fig. 44d).

Tatuno and Kise (1970) reported n- 7j 14 and 21 (Figs. 46e-h)

according to the formulae:

n-7: V(Hj X or Y) + 3V + 2J + m(h)

n- 14: V(H, X or Y) + V(H, Y) + 6J + 4J + 2m(h)

n- 21: 2V(HOX) + V(HoY) + 9V +Q+ 3m(h) h

A. 1 4=3P1W400 Xo ry xOry 21 3ý , e4 11e)ý , 0-01

rl<

U's'

0.5*aoý

Fig. 46. Illustrations of mitotic chromosomes of Atrichum. A. undulatum (a-1) and As crispum (mon) byi (a) Kurita (1938); (b-d) Tatuno (1960) (a-h) Tatuno & Kise (1970); (1) Wigh & Strandhade (1971) (j) Wigh (1972); (k-n) Lowry (1954) -70-

The labelling of the largest and smallest chromosomes as heterochromatie

should be discounted. Tatuno (1941) commenced the procedure of

labelling these chromosomes as 111i" and "h" and this has been dogmatically

followed by Japanese authors ever since. This approach is highly

unsatisfactory and may obscure the real situation (Smith, 1978b).

Newton (in Smith, 1978b) says "in so far as quantities of heterochromatic

are concerned the largest chromosome in Pellia epiphZIla and P. neesiana

undoubtedly equate with the H -chromosome of Tatuno (1941) but the

smallest chromosome of the latter species consists of only about 26%

heterochromatin compared with 35% In chromosome 5. In, P. endivitfolia

the largest and smallest chromosomes'contain about 13% and 18% of their

length as heterochromatin, only slightly more than in other chromosomes".

Further, the assumption that particular chromosomes are sex

chromosomes is based on purely circumstantial evidence and in the

absence of proof such chromosomes are best referred to as sex-associated

chromosomes (Smith, 1978b). A more realistic representation of the

karyotype formulae of Tatuno and Kaise (1970) would be:

n-7: il + 32v + 2J +j

n- 14 : 2V + 6V +Q+ 2j

n- 21 : 3V + 9V + 6J + 3.j

Although it is likely that haploid plants are dioecious (R, Es Longton,

pers. comm.), the diploid and triploid are likely to be monoecious and

it is nonsense to designate any chromosomes as sex-associated in mouoecious diploid or triploid plants. But, assuming that there really

are distinguishable morphological differences, then this is of

relevance as will be mentioned later.

A comparison of the karyotype formulae of Tatuno and Kise (1970) with the British material show that there are some similarities and some -71-

differences. These may be summarized as follows:

British n-7: V+2v+ 2j + 2v

Japanese n-7: v+ 3V + 2J +j

British n- 14: 217+V+ 4i + 4v

Japanese n= 14: v+v+ 6v + 4J + 2J

British n- 21: 3V + 617 + 6i + 6v

Japanese n- 21: 2V +V+W+Q+ 3i

Lowry (1954) suggests that diploid A. indulatum arose from haploid

material by apospory and that the triploid arose as the result of hybridity between the haploid and the diploid by apospory. That the

diploid arose from the haploid and the triploid as the result of hybridity between the two is acceptable, but that apospory is involved

is not. It seems more likely that chromosomes doubling results from diplospory (Smith, 1978b), in the first instance a diploid spore

forming by diplospory in a diploid capsule. The triploid is likely to have arisen as the result of the failure of msiosis in a hybrid between a haploid and diploid. Diploid spores have been reported several times from naturally occurring plants, It therefore seems probable that doubling of the chromosome number results from diplospory rather than apospory.

Mehra and Khanna(1961) quote the work of Lowry on A. undulatum as an example of allopolyploidy. Smith (1978b) comments that this is a misinterpretation of the situation. This is a theoretical allopolyploidy but as the three genomes are similar the triploid plants are functionally autopolyploid . -72-

The observations of Tatuno and Kise (1970) provide circumstantial evidence supporting the suggested mode of origin assuming that their

'T' and "Y" chromosomes really are distinguishable, The diploid with

")V' and "Y" would be derived by diplospory in a sporophyte resulting from the fusion of gametes from haploid male and female plants. The triploid, in this instance quoted would arise as the result of diplospory in a sporophyte resulting from the fusion of an "X+Y" diploid however, male gamete and an 'T' haploid female gamete. This does, illustrate the absurdity of labelling chromosomes 'T' and "Y" in polyploids since they clearly have no sex-determining function.

It has been suggested by Rose (1951b) and Nyholm, (1971) that

A. undulatum, var. minus in a hybrid between A. 'undulatum and As' angustatum

In Britain A. undulatum. var. 'minus often occurs outside the range of the former and it seems more likely that var. minus is a hybrid between different cytotypes of A. undulat= (Smith, 1978b).

It would appear from Fig. (44a) that A. crispum, has seven chromosomes. The three largest are metacentrics, two being of almost equal size, then two subterminal and the remaining two metacentrics of almost equal size.

n-7- 2V +V+ 2j + 2v

These somatic chromosomes of the British material of A., crispum

(Fig. 44a) appear not to differ from the north American material

(Lowry, 1954) (see Fig. 46m, n). -73-

CHAPTER 7

Morphological Variation of CvtotZpes

7.1 Materials and Methods

Several approaches were taken in the study of AsI undulat "Is

These were: -

1. Study of field and herbarium material

2. Cultivation experiments

3. Nuclear DNA content

4. The size of heterochromatin bodies

5. Isozymes

6. Distribution of A. undulatum cytotypes.

Methods used are similar to those described for H. cupressiforme s. l. on page Each gathering was divided into two parts, one was cultured, the other preserved as an herbarium specimen. Living material was cultured at Pen-y-Ffridd Field Station, Bangor in an unheated glasshouse in'a mist unit on peat-based potting compost in 25 cm, flower pots. The pots were covered with polythene bags to prevent cross-contamination. Plants were unshaded and watered with tap water

(pH 7.5).. As all cultures were grown in a limited area (300 cm x 150 cm) at the same time, it could be assumed that growth was under uniform conditions, Three months growth was found to give satisfactory results for the purposes of this study. Samples from living material were taken, before and after culture under uniform conditions and preserved for later examination.

Moving means of five shoots, with at least twenty five samples, were found to give satisfactory results. Samples were dissected and the parts mounted in gum chloral. -74-

7.2 'Characters'Sdlddted'for'the'Study'of'A. tindulatum'CytotZpe

1. Plant Morphology

a. Plant height: There is a considerable degree of over-lapping between the three cytotypes (see Table 8). The amount of variation in

in plant height under uniform conditionswas lower than that the wild, suggesting both genetic and environmntal variation.

2. Leaf'Morphology and'Anatomy

a. URper part of stem

(i) Leaf Length: The amount of variation in culture was lower than- that in wild in both haploid and triploid. In the diploid there was a slight increase (see Table 8. There was also a considerable degree of overlap among the cytotypes. A8 with height, the haploids diploid and triploid cytotypes seem to be genetically variable.

(ii) -Leaf Width: There was a remarkable degree of overlapping azwng the cytotypes. The amount of variation decreased with culture in all cytotypes (see Table 8), suggesting that leaf width is affected by environmental factors.

(iii) Cell Width: Like other characterss cell width also showed a considerable degree of overlapping, Thera wasa slight increase in the amount of variation in culture in the triploid, A decrease in haploids and diploids suggest a relatively low environmental and genetical variability.

b. Middle part of stem

(i) Leaf Length: There is a marked degree of overlapping between the cytotypes. There wasa relatively slight decrease in the amount of variation in all plants (see Table 8) after culture,

(ii) Leaf Width: The cytotypes show considerable overlap. The amount of variation was almost the same before and after culture in the triploid -75-

plants; there was a slight increase in the haploid and a decrease in the diploid after culture (see Table 8 ).

c. Acumen shape measured in degrees

This character showeda considerable degree of overlapping between the

There was a decrease in the amount of variation in the triploid cytotypes. I and an increase in the diploids in culture; this amount was almost the same in haploids.

d. Percentage of serration in the leaf

This character showeda. considerable degree of variation between the cytotypes. The amount of variation wasalmost the same under both culture in haploid, with a decrease in both diploid and triploid after culture (see Table 8).

e, The angle between the tooth and leaf margin

There ýasa considerable degree of overlapping between the cytotypeS.

Therewas a marked increase in the amount of variation in culture in the triploid. An increase in. the haploid and almost the same under both conditions in the diploid. This suggests that there is genetic variability in this character in the triploid.

f. Tooth length

There was considerable overlapping between the cytotypes, The amount of variationwaS almost the same in the haploid before and after culture; there was an increase in both diploid and triploid after culture (see Table 8).

S. N=ber of lam llae

The number of lamellae is 2-4, rarely 5 or 6, This character showed some degree of overlapping between the cytotypes. The amount of variation before and after culture in diploid plants was. almost the same (see Table 8). -76-

in diploid On the other hand there was a decrease after-culture the and

the haploid.

h. Lamellae height

The lamellae are 2-4, rarely 5,60 7 cells height. This character

showed some cbgree of overlap between the cytotypes. There was a

decrease in the amount of variation after culture in the cytotypes*

i. Angular cell wall thickness

Three categories were scored: thin, medium and thick. The cytotypes

overlapped and were indistinguishable. Most plants had thin walls and

retained this after cultures sugge'sting some degree of genetical

stability.

j. Leaf margin

Three categories were scored: not detectable, distinct and very

distinct. Most plants had distinct margins, almost all of these

retained it after cultures suggesting some degree of genetical

stability.

k. Nerve strength at apex

Three categories were scored: vanishing, sometimes detectable and

distinct. All plants except a few had a vanishing nerve and retained

this after aulture.

1. Leaf size near the stem base

Three categories were scored: smaller than these above$ equal and

larger. All plants except some had smaller leaves at the stem base and

90% of these retained this character after culture.

m. Number of teeth

Three categories were scored: teeth single, teeth sometimes in in pairs and teeth pairs. Most Plants have teeth in pairs, a few were in the second category.

0 -77-

3. Sporophyte following As sporophytesdid not develop in cultures the characters were studied from wild and herbarium materiali

a. Seta Length is 1.4 3.1 This umsa variable character. The length of the seta - cm they and sometimes reaches 4.2 cm. All plants show an overlap and between were indistinguishable. Discrimination the cytotypes using

this character is not possible.

b. Seta Posture

Three categories were scored: inclineds intermidiate and erect. 5% had All plants except 5% of the gatherings had erect setae, the

intermediate. All plants overlap and are indistinguishable.

c. Capsule Length

The capsule length ranges from 3.4 - 6.2 mm. All plants overlap

and were indistinguishable, Discrimination between the cytotypes

using this character is not possible.

d. Number of peristome teeth

The number of teeth varies from 30 - 34. Most plants have 31 or

32 teeth. The number of peristome teeth of no taxonomic value.

e. Operculum Length

The length of the-operculum in the cytotypes varie. d from 1.4 - 3.1 mm.

The cytotypes overlap and are indistinguishable.

f, Beak Length

Three categories were scored smaller than the capsule, almost equal

to the capsule length and larger than tfiýe capsule. All plants except a

few had a beak of almost equal length to the capsule and were indistinguishable. -78-

Table 8. Showing the Mean and Variability in Cytotypes of A. undulatum

Before and After Culture.

BEFORECULTURE AFTER CULTURE

CYTOTYPE SD SE ýv SD SE cv

n- 21 1Plant height 4.11 1.16 0.212 26.95 4.346 0.75 0.138 18.26 2 Leaf length 7.37 0.62 0.134 9.98 7.62 0.54 0.099 7.15 3 Leaf width 1.72 0.29 0.057 18.04 2.06 0.28 0.052 13,94 4 Call width 22.90 4.06 0.589 14,11 25*03 4,34 0.79 17.35 5 Leaf length 6.35 0., 56 0*113 9.74 6.76 0.49 0.090 7.35 6 Leaf Width 1.36 0.21 0,034 13.92 1.616 0,21 0.040 l3s59 7% of Serrate 79,00 11,66 1.91 13.23 76.33 9.73 1,77 12.75 8 Acumen angle 21.97 6*31 1.16 28.80 31.03 15,11 1.38 2405 9 Teeth angle 46.83 11.24 2.50 29.20 36.76 1.55 2.90 43.29 jo Tooth length 6.613 1942 0,268 22.211 6.07 1.31 0.283 25,53 iiLamalla in no. 3,40 108 0.217 35,05 3.70 1.21 0,24 35.60 12Lamella, cell 3.93 1.09 0.23 31*97 4.1 0.44 0.221 29.60 in height

a- 14 IPlant height 4.84 1.45 0.172 17.75 4.914 0.67 1.034 13.40 2Leaf length 7.20 0.76 0.168 11.68 7.87 0.43 2.57 12.66 3Leaf width 1.69 0.36 0.065 19.36 1.668 0*28 0.045 13.70 4CeI1 width 23.24 4.27 0,. 73 15.77 23.20 2.18 0.420 9.05 5Leaf length 6.25 0.50 0.134 10.74 6.96 0935 01059 4.27 6Leaf width 1.39 0.235 0.046 16.07 1.80 0.23 0.0346 9.62 7% Of Serrate 78.00 11.72 2.516 16; 13 79.24 8.64 2.315 14.60 BAcumen angle 21.12 7.98 1,165 27.58 27.60 8.93 1.917 34.73 qTeeth angle 49.40 12*02 2*57 26.022 49.50 14.13 2.45 24.84 i oTooth length 6.81 1.26 0.28 20.528 6.44 1.27 0.32 25.23 ii Lamella in no, 3.32 1.304 0.250 37.620 3,12 0.75 0.166 26.68 12Lamella cell 3*92 1.34 0.215 27.475 4.16 0.81 0.179 21.59 in height

n-7 IPlant height 4.17 0.88 0.230 26.30 4.38 5.17 0.13 2Leaf length 16.10 7,316 0966 0.164 11.21 7.512 12 89 0.086 5,73 3 Leaf width 1.808 0.23 0.068 18.81 1.70 0,22 0.057 16.89 Cell 4 width 22.48 3.09 0.840 18.70 254,52 2.10 0.436 8,55 5 Leaf length 6.27 0.55 0.118 9.47 6.668 0.29 0,110 8.26 Leaf 6 width 1.40 0.16 0.038 13.55 1.56 0.17' 0.0475 15.21 7Z Serrate of 7,68 12*13 2.49 16.26 83,20 11.57 1,729 16.40 8 Acumen angle 21.72 4.54 1.46 33.79 26.20 9.58 1.786 34,10 9Teeth angle 46.40 8.58 2.37 25.56 46.80 12.27 2.82 30.12 10 Tooth length 6.728 1.10 1 0.278 20.721 6.20 1.62 0.25 20.54 ii L ame l la i n o. n 3.28 1. 19 0.227 34.67 3.36 0.83 0.15 22.53 12Lamella cell , 3.80 1.20 0.223 29.422 2.92 0.89 0.162 27.82 in height

Character 1 in for 2,3.5 mm; & 69 1 unit - 0.43 MM;4, in um-,for 10s I unit -10 um. -79-

g. Calyptra

Three categories were scored: calyptra without teethg sometimes toothed and toothed. All plants except few had toothed calyptras, a few had the second category. This character of no systematic value.

h. Spores 30 This is a variable character with size ranging from 12 - is and the cytotypes were indistinguishable. This character of no systematic value. it As no single character by itself is discriminatory, was clearly necessary to see if all the characters taken in combination would separate these cytotypes from one another.

7.3 Variability Within Cytot)Mes of A. undulatum

1. Plant Height - Increasedin culture in haploids, diploids and triploids

(Figs. 47a - 49a). The variation in height*in culture decreased in the three cytotypes (Table 8).

2. Leaf Length (in upper part of shoot): Leaf length was almost the same in both coDditionsaccompanied by a decrease in leaf length variation in haploids.

There was a slight increase in length, but the variation in length in culture is almost the same in diploids (Figs. 47 - 49b). Triploids increased their leaf length in culture and the variation decreased slightly

(see Table 8).

3. Leaf Width'(in upper part of shoot): Tn haploids there was a slight decrease in width, with a slight decrease in variation in culture.

Diploids showed a slight decrease in width and also a slight decrease in variation in culture. There was a slight increase in width and a decrease in variation in triploids after culture (see Table 8, Figs. 47-49c). Fig. 47. Histograms illustrating variation of haploid A. undulatum

(a) plant height; (b) leaf length (from upper part of stem);

of stem);

(e) leaf length (from mid-stem); (f) leaf width (from mid-stem);

(g) pdrcentage of serration; (h) the angle of the acumen;

(i) tooth angle; W tooth long;

(k) number of lamellar cells; (1) height of lamellar cells.

Note that the Y-axis is not identical in all cases.

Character (a) in mm; for b, c, e&f. I unit - 0.43 MM;d, ln uin for j, unit= Io um. BEFORE CULTURE AFTER CUI TURE

I. 4 a 4

2 4

32 S

is 24 22 to

13 I 10

0 70 is 14 is 10 14 16 12 12 I. 10 I 0 6 4 z

14 14

It 14

12 114 N

I 4

I I

4 4

)

14 22

t4 la 10 I

I .rý

Fig. 48. Histograms illustrating variation of diploid A, undulatum

(a) plant height; (b) leaf length (from upper part of stem);

(e) leaf length (from mid-stem); M leaf width (from mid-stem);

(g) percentage of serration (h) the angle of the acumen

(i) tooth angle ; Q) tooth long ;

W number of lamellar cells (1) height of lamellar cells

Note that the Y-axis is not identical in all cases

Character (a) in for b, 1 t-0 43 mm; c, e&f, uni . MM*s dj n um ; for J, 1 unit = 10 um. BEFORE GULTU RE AFTV,R CULTURE

4.0 a 4.0 - 1.0

Lo F,, 2.4 2.1

1.0

S 0

16 10 14 14 14 t3 Is to

I 'a

'I 4

20 12

S a

4 a

'a 'a

'4 ,0 'I a 4

I S

p 4

at 24 22

14 14

14 Id 4

4 12 io

1 Fig. 49. Histograms illustrating variation of triploid A. undulatum

(a) plant height; (b) leaf length (from upper part of stem)

W leaf width (from upper part W cell width (from upper part of stem); of stem);

(a) leaf length (from mid-stem) M leaf width (from mid-stem);

(g) percentage of serration; (h) the angle of the acumen

(i) tooth angle; Q) tooth long;

W number of lamellar calls (1) height of lamellar cells.

I Note that the Y-axis is not identical in all cases.

Character (a) in m; for b,. c, e&f, 1 unit c 0.43 mm;d, in um. 9 ' for j, 1 unit = 10 um. BE FORE C ULT U RE AFTER CtTLTTIPF

I &A 4 LO

1.4

I 4

20 14 14

to la

10 S

Is U 16

14 II t4 13 I,

*0 a 0 0 3

m $

0 s 14 16

I 12 If

I -80-

4. Cell'Width (in upper part of shoot)% There was an increase in cell width, while the variation in width decreased after culture in haploids.

In diploids cell width is almost the same under both conditions and a decrease in variation is detectable. In contrast triploids increased in cell width and the variation also increased in culture

(Table 8, Figs. 47d- 49d).

S. Leaf Length (from middle part of shoot): The leaf length in both in cultures was almost the sameq and so was the amount of variation haploids after culture. There was a slight increase in length and decreases in variation in diploids and triploids after culture (Table 80

Figs. 479- 49e), suggesting that this character is slightly influenced by environmental factors and the variation is partly genetic in nature.

6. Leaf Width (from middle part of shoot): There was a slight increase in width and a slight increase in variation in haploids after culture.

An increase in width and a decrease in variation is shown in diploids.

There was a slight increase in width, but the variation in width is almost the same under both culture in triploids (see Table 8, Figs. 47f-

49f), suggesting that this character is slightly influenced by environmental factors.

7. Percentage of Teeth: There was an increase in the percentage, of teeth in culture, but the variation is almost the same in haploids.

A similar increase in diploids was found, but there was a slight decrease in variation. There was, a decrease in triploids but the variation is almost the same under both conditiors(Table 89 Figs. 47g-

49g), suggesting some environmental effects.

8. Acumen Angle: This angle-increased in culture in the three cytotypes

(Figs, 47h- 49h), but the variation was increased in diploids, decreased in triploid and almost the same in haploids (Table 8), suggesting some -81-

environmental influence.

9. Tooth Angle,: This angle is almost the same in culture in haploids

and diploids, while it increased in triploids (Figs. 471- 49i), The

amount of variation in culture increased in both haploids and triploids.

In contrast this slightly decreased in diploids after culture (see

Table 8). This character is influenced by some environmental factors.

10. looth Length: Tooth length slightly decreased in culture in the

three cytotypes (see Figs. 47j- 49j). The variation slightly increased

in diploids and haploids while it is almost the same in haploids before

and after culture (see Table 8), suggesting low environmental effects.

11. Number of Lamella Cells: The lamella cells were almost the same

in number in both conditiorsand the variation decreased in culture in haploids diploids. and In contrast the number of lamella calls and in the variation number are almost the same in triploids under both (see cultures Table 8, Figs. 47 - 49k), suggesting that this character is

slightly influenced by environmental factors.

12. Height Lamellae Cells: of There was a decrease in the number of in haploids cells after cultures but the variation was almost the There increases same. are slight in both diploids and triploids after but culture, the variation decreased and almost the same in the two (see cytotypes respectively Table 8, Figs. 471- 491), suggesting that this character was little affected by the environment.

7.4 N=erical Approach_

MIXORD -The same technique as described Qn page ( 30 ) for H. l. . -cuPressiformg, s. was used as follows: - IL

Ln 0 UI0 Fig. 50. Ordination of morphological characters for undulatum _A. herbarium and field material. Key to symbols:

A) Specimens referred to haploid A. undulatum

diploid A. 0) Specimens referred to undulatum

C3 ) Specimens referred to triploid A. undulatum

Note that axis I plotted against II. Fig. 51. Ordination of morphological characters for A. undulat= herbaritm and field material. Key to symbols:

Specimens referred to haploid A. undulatum

Specimens referred to diploid A. undulatum

E3 Specimens referred to triploid A. undulatum

Note that axis I plotted against e

II- co 00 0 S'l

II-III 88

Fig. 52. Ordination of morphological characters for A. undulatum

herbarium and field material. Key to symbols:

Specimens referred to haploid A. undulatum

Specimens referred to diploid A. undulatum

C3 Specimens reffered to triploid A. undulatum

Note that axis II plotted againstAIL' I--- q

80 ý1

ý-20

-40

:z-Z zw - -60 -Z -I 20 Fig. 53. Ordination of morphological characters for A. undulatum

wild material before culture. Key to symbols:

0) Specimens referred to haploid A. undulatum C3 ) Specimens referred to diploid A. undulatum

Specimens referred to triploid A. undUlatum

Note that axis I plotted against II. 'ý

0 120

loo

so r,

20 40 60 80 0i -40 -20

Fig. 54. Ordination of morphological characters for A. undulata=,

wild material before culture. Key to symbols:

Specimens referred to haploid A. undulatum

Specimens referred to diploid A. undulatum

Specimens referred to tri ploid A. undulatum

Note that axis I plotted against Ill. 8

Co k 0

'Fig. 55. Ordination of morphological characters for A. undulatum wild material before culture. Key to symbols:

m) Specimens-referred to haploid A. undulatum

C3 ) Specimens referred to diploid As undulatum

0) Specimens referred to triploid A. undulatum

Note that axis II plotted against III. 80

-60 -40 -20 20 40 60

Fig. 56. Ordination of morphological characters for A. undulatum

after culture. Key to symbols:

Specimens referred to haploid A. undulatum

Specimens referred, to diploid A. undulatum

C3 Specimens referred to t, riploid A# undulatum

Note that axis I plotted against II. 0

8.. I 4 J-J 0

Fig. 57. Ordination of morphological characters-for A. undulatum

after culture. Key to symbolss

specimens referred to haploid At undulatum

0 Specimens referred to diploid A. undulatum

c3 Specimens1referred to, A. undulatum _triploid Note that axis I plotted against III. 8

Fig. 58. Ordination of morphological characters for A. undulatum , after culture. Key to symbols:

Specimens referred to haploid A. undulatum

Specimens referred to, diploid A. undulatum

C3) Specimens referred to triploid A. undulatum,

Note that axis II plotted against-III. -82-

1. To herbarium and wild material

2. To wild material

3. To wild material after culture

From Fig. (50 - 55) it can be seen that the three cytotypes

overlap and are indistinguishable as might be expected from the

discussion of individual characters above. intergradation After culture the three cytotypes showed a similar

in gametophyte characters (see Fig. 56 - 58).

As no differenceswere detectable between haploid, diploid and

triploid races, these cytotypes should be treated as a single taxonomic

species containing three cytotypes, This conclusion is not in line with that

of Lazarenko and Lesnyak (1977) who say "these should be treated as (1978h) %,, beat cryptic sibling species", but is with that of Smith who says

to regard A. undulatum as a single species containing three cytotypes".

It is of interest that in A. undulatum there is no tendency for

diploids and triploids to be larger than haploids. In Tortula Muralis

(Newton, 1968) diploids tend to be larger although they intergrade

completely with haploids. In the case of Tortula murdlis it was suggested

that the new diploids were larger but that there was selection for smaller plants on successive generations. This would be in line with observations of Wettstein (1940) who noted a diminution in size in successive generations in artificial polyploids of Funariaceae. That diploids in T. muralis are often larger than haploid gametophytea'

(Newton, 1968) suggests that some are of recent origin whilst those diploids that are of'si=lar size to the haploids have been selected for over a number of generations. The large diploids correspond to the plant referred to as To'murdlis var. 'rupestris. As an indication of the -83-

relative frequency of what might be new polyploids compared with haplaids

and old polyploids may be obtained from Duncan (1926), Duncan records

T. muralis var. muralis which contains the latter categories of plants

from 112 English and 40 Irish vice-counties whilst varý'tupqstris is

recorded from 63 and 9 vice-counties respectively. That there is not the

slightest indication that polyploids are larger than haploids in

A.. undulatum suggests that either new polyploids are of similar size to parental haploids or that new polyploidsl if larger, are of very rare occurrence and were not encountered during the present investigation.

Rose (1951b) and Nyholm (1971) suggest that A. undulatum var. minus is a hybrid between A. undulatum and A. angustatum. Smith (1978b) commented that in var. minus the sporophyte is stunted and the spores abnormal and apparently sterile and this is indicative of a hybrid nature. The distribution of A. angustatum in Britain precludes itbefMone of 6a parents of

'uiidulatum -var, minus of ten occurs out side the range of the f ormer and it seems likely that var. minus is a hybrid between different cytotypes,

Against the idea that var. minus is a hybrid between different cytotypes are the observations of Dr. M, E, Newton (pers. coam. ) who examined capsules in mixed populations of diploid and triploid plants.

The sporophytes were all normal var. undulatum sporophytes and, whilst there were some irregularities at anaphase-I of meiosis there were always

14 or 21 bivalents. If hybridisation had occurred between diploid and triploid gametophytes then at least some capsules with 14 bivalents and

7 univalents would have been found. Further, in mature sporophytes, the spores were normal; var. 'minus sporophytes contain spores which are very variable in size and with many aborted.

Lowry (1954) suggests that triploid A* undulatum was derived by 94-

apospory from the hybrid baploid X diploid A. 'uridulatum. The suggestion that there is a hybrid between haploid and diploid A. ''undulatum is acceptable, but that by apospory is involved is not. Smith (1978b) commented that it seems more likely that in such a hybrid meiosis is irregular and that occasional spores with a balanced complement of 21 chromosomes occur. Diploid spores have been reported from naturally occurring plants. It therefore seems probable that doubling of the chromosome number results from diplospory rather than apospory. Higher plants frequently produce diploid microspores (Stebbins, 1971) - the situation in mosses is not different from higher plants (Smith, 1978b). -R5-

CHAPTER8

Variation in liowý-morphological charadtars_

8.1 Isozymes in three cytdtypes'of A. undulatum

Electrophoresis of xanthin dehydrogenase (XDH) system was carried is described out on a horizontal slab of poly acrylaMlide gel. The method ill PaW38

Only one (XDH) isozyme phenotype was found (see'Fig. 59) in the three cytotypes of A. undulatum. This consists of two bands one small and the other larger. The smaller band was white while the other was dark, blue. These three cytotypes of A. undulatum have the same number, position, shape and colour of bands, thus indicating a close genetical relationship between them, supporting the suggestion that these plants form an autopolyploid series and are best treated as a single species with three cytotypes.

8.2 DNA,content of nuclei

Iutroduction

Measurement of the relative DNA content of nuclei from gametophytes from different populations may provide evidence of different ploidy levels of these populations and may provide an efficient mean of differentiating populations where karyotype analysis is difficult or impracticable.

The DNA content of nuclei of gametophytes may provide a useful taxonomic criterion and may provide evidence of evolution of one taxon from another.

Table 9 and Fig. 60 show the absolute DNA contents of nuclei from gametophytes of four bryophytes in relation to the contents of nuclei of green algae and annual or ephemeral higher plants. DNA contents of the four bryophytes were estimated from nuclear volumes. These estimated DNA contents per nucleus are relatively low but overlap with the minimum values for vascular plants. The estimated'DNA per chromosome is in the lower range for land abC

1 FI _____

Fig. 59. The isozyme bands of Xanthein Dehydrogenase (XDH) system for

three cytotypes of A. undulat=.

(a) haploid ; (b) diploid ;

I*-- -86-

Table 9. The absolute DNA contents of nuclei from gametophytes of four bryophytes and from nuclei of green algae and higher plants

(Sparrow at al. 1972).

Chromosome "V tcv ONA14611 ortAntsm W Rot. 3) no. Lb Ws (bb set. - (haptoid) Rol. DNA/Chnooome

Oryaphyta'(mossof an4 liverworts) I 0 Karchantia DaIrmer04 (gamecophyte) 9 13 13 13 1.4 13 11.0 a %a III t. 0 a t 1.6 a 10 131 a 101 1 t mrt op. (Smastophyte, shoot seem) ca.? 8.4 62 7.1 a 10 133 a to RiCtia op. (6-tophygO. Coth hoot 63 64 1a 1.0 63 1.6 a 10 132 4pical notch) 0 $Ohio op. (gametophyte. sentrot it 20 61 1.1' 61 2.6 x IQ 132 1.4 IQ Societe" of shoot apex) Poilop$Lds Pollotum fludus (shoot apex. &picat Coll 104 62 3844 62 iM *I :ja toll 131 1.3 to derivatives) it Twelistarts op. (Skoog Spool &pilot 206 47 9366 62 22.1 61 6.1 is 10 132 3.0 a to Coll derivatives) Lycopsids (club wastes) to Isottes fgaltmanail (shoot apex) it 131 498 43 22.6 42 3.3 x 10 133 3.0 a to to tycooodi lucidut (shoot apex) 132 47 196 61 W 63 3.0 a to 133 1.3 it to t jolssinell kratsvolf"f vars brownit to W 17 63 3.1 6: 5.1 is to 131 5.1 a too (shoot apex) sphonapoida (horsetails) to 94misetue j=jjflU (nonspical tells *9 M 41 1114 63 3.2 6: TJ 8 IQ t3a 6.0 a to$

shoot apex) I I '. hVemalf (noft&pioal 86119*49 shoot 100 41 1060 63 4.0 63 7.1 to" 133 6.6 . to, ap*s) ptorepoids (total) Asole"t MLLU' 144 41 Uo 43 1.9 63 3.1 lot* 133 0 is to$ cysto"Bris Irsailis 84 41 "1 61 5.3 6.0 1 f 61 a 1010 13: 7.1 4 to OnOcIsmsaftsibilis 37 42 143 411 23.8 61 1.1 x tall 132 3.0 a lot Ophloslovow (nonspiest pectolet root ca. 310 47 4114 63 4.0 62 3.1 1011 132 1 54 a . to Calls) t Owaynda Sionows U 33 41 114 to I 63 33.9 , 63 946 1110 ; 33 4.4 a to Pteridjus joultin 53 49 316 63 63 1010 t -4.4 a ftort# 10"Ittatis 58 42 451 61 63 6.0 a 1610 Rumohra adt4nciformis 41 4.5 to 1. 1a salvOts op. (Spical calls) 63 63 3.2 63 J. 3 a %*to Ul 4.4 a lot

C-caphyce, 3-call $ease. b

s I goo fli I 1F

I I, 11

. 1

lb it I I 1111IH lb

IQ I*

Fig. 60. The absolute DNA contents of nuclei of bryophyta in'comparison with that in various groups of plants and animals (Sparrow at al. 1972). -87-

plants. The lowest DNA,content of the four bryophytes falls below,

but the highest value exceed', the'upper limit found in green algae.

It is claimed that nuclear volumes or DNA contents of a range of higher

plant species are correlated'with complexity (Sparrow et al. 1972).

There is no evidence that the DNA content in the four bryophytas is

correlated with complexity as it is within the green algae.

The estimation of nucleic acid contents by chemical means requires

large amounts of material and is relatively time consuming. The DNA contents

of single nuclei can be estimated by measuring the density of Feulgen

staining. The light absorbed by the stained nucleus is proportional to the

DNA content. A more sensitive measure can be made using a fluorochrome

which stains DNA in a quantitative manner. Such a flurochrome is DAPI.

Ohher methods depend upon the relationship between nuclear volume and

DNA content. These relationships show that nuclear volume and interphase

chromosome volume are directly proportional to DNA content per cell and

per chromosomet repsectively. Therefore, when the nuclear volume of

meristematic cellsis known, an estimate of DNA content can be made

(Sparrow et al. 1972). This method appears to be inadequate especially when the interphase chromosome volume is obtained by dividing the average

of the interphase nucleus by the somatic chromosome number and represents

the volume occupied by an average chromosome at interphase, neglecting

other nuclear components such as the nucleolus or volume changes during

replication of chromosomes. Clearly the accuracy of measurements with

very small nuclei is questionable. To assess the DNA contents in nuclei, Feulgen

stain was used in the past, but there are many difficultiesfor example

excess or too little stain effects the results as does the irfluence

of the background. To obtain satisfactory results the fluorochrome stain was applied to the three c7totypes of At undulatum -88-

The fluorochrome 4,6-diamidino-2-phenylindole DAPI has been shown to bind specifically to DNA. The DAPI/DNA complex'fluoresces with an intensity of about 15-20 times'that of DAPI alone. The fluorescence of the DAPIIDNA complex has been used as a quantitative estimate of the DNA

(Brunk et al. 1979, Lin et al. 1977). Background fluorescence can be reduced to a negligible amount. DAPI unlike some other stains, such as chromomycin A3, does not display rapid quenching. This allows scanning of preparations under the Ultra Violet (U. V, ) microscope and accurate focussing on the nucleus with little loss of intensity. The simplicity of the staining procedure coupled with the brightness of the DAPIIDNA complex provides a convenient technique for cell cycle studies and comparative estimate of DNA contents of nuclei.

Materials and Methods

Four gametophyte cells from each of twenty five samples of each cytotype were measured. Plant material was fixed in 5% glutaraldehyde EM in Tris buffer pH 7 for 5 minutes. The excess fixative was then removed and replaced with the fluorochrome DAPI.

The stain was made up as a stock solution of 1000 ng/ml in Tris buffer pH 7,100 mM NaCl and 10 mM EDTA, All reagents were started in the dark at 30C. The fixed, tissue was squashed directly on a clean slide.

Staining with DAPI appears almost instantaneously. Stained material was observed under a Leitz Werzlar microscope fitted with an incident U. V. light source and photomultiplier coupled to a pen recorder. Filters used were BC3, UGI, giving a final wave length of 350 =. The DAPI/DNA complex fluoresces. at 450 am. All-nuclear measurements were made from nuclei in cells of young gametophytes near the shoot, apex.

is As the variation proportional to the mean value in the first peak (G 1) of the three cytotypeso transformation of data by, means of loglo was -89-

suggested to overcome this proportional variation. A one way analysis of variance was carried*out. Tukey'stest was suggested to be suitable for this sort of data (see Appendix 3).

Results and Discussion

Bimodality was found in the three cytotypes of A* undulatum

(,Fig. 61 abjc) indicating that some cells had increased DNA contents as they were measured in S, G2 or X phases of cell cycle. In the haploid strains the basal level (mean value of calls in GI) is estimated to be 60 units. The second peak of the distribution would seem to correspond to an expected G2 mean of 130. The G1 level in the diploid cytotype is estimated to be 110 and again the G2 mean is approximately twice this value. The triploid peaks indicate a mean higher than that of the diploid viz 160 for G1 and approximately twice this value for the G2 nuclei

In the haploid stain the moan value of the first peak is estimated 1 to be 55.5 4.2 units. The first peak of the diploid cytotype is t estimated to be 90.80 3.95 units. The mean value of the first peak of 1 the triploid cytotype is estimated to be 134.06 4.89 units. These results show that the mean relative DNA contents increase from the haploid to diploid to triploid as*would be expected from karyoty. Pe. Differencei between diploids and haploids, trip loids and haploids and between triploids and diploids were cofapated using Tukey's test. The Log,, Lo g3 and Log, .5 values are outside the 95% confidence intervals. Therefore the increase in the DNA content is not proportional to increase in chromosome number.

From these results, it would appear that there are differences in

DNA content between the three'ýcytotypes of A. undulatum. Polyplaidy plays a role in the elevation of DNAq but poliploidy is only one of other forces forming the basis of, 'the variability. Stebbins (1950) pointed out some examples where certain temperate genera have larger chromosomes and cm 0-ul Q

1! ! :! V 12 .. a- 10

. 93p4 10

10 0 co,

" r4 0S " .4 f'i C. --

%»0 %0

pt K 4vC% -90-

nuclei than tropical ones. There are significant differences in

chromosome size and DNA content in closely related diploid species

(Stebbins, 1950). These examples suggest the existence of selective

forces influencing the'amount of DNA per nucleus and per chromosome

(Sparrow, 1972).

Clearly, polyploidy has played a main role in generating the three

cytotypes of A. undulatum but there is some evidence from the present

study that loss or gain of DNA has also been involved since the polyploids were formed. Variation in chromosome size is known in tropical

and temeperate numbers of the same genera. Significant differences in

chromosome size-or DNA content is known. to occur in some closely related species of higher plants. (Stebbins, 1950)

8.3 HeterochromAtin. bodidg'iri'A. tindulatum

Introduction

Haterochromatin was first recognized by Heitz in 1928. He found that certain partsof'Pellia chromosomes were more densely staining than most during prophase of mitosis and remained condensed during telophase and the ensuing interphase. ' The ýresence of heterochromatin in other bryophytes has beýnlrepbrted'by many authorities (e. g. Lorbeer, 1934;

Inoue, 1967; Segdwaq ; 971;. Newton, 1977a, b). Heterochromatin bodies have been used toý-identify different levels of polyploidy (Anderson, 1964;

Berrie, 1957; Wigh, 1975)., Anderson (1964) says: "In so-called polyploid species Heitz found more-than one heterochromasoma and consequently more than one heterbpyenotic body in the interphase nuclei of these polypioids.

Heitz concludeds however, that a species is polyploid if it has more than one heterochramosomal". In'BtAchythecium, 'rutabtilumv B. 'rivulare complex

Wigh (1975) found that one large heteropychotic body indicated that a species washaploid and two. large bodies that it was diploid. Further, he -91-

found one body. occurs in dioecious spcies and two in autoecious species.

Some authorities'discuss these bodies in relation to sex chromosomes

in bryophytes (Tatuno, 1960; Mehra and Khana, 1961; Berrie, 1963;

Khana, 1971). Kurita (1938) reported more heterochromatin in the large

X- than in the Y-chromosome of two species of Pogonatum. Anderson (1963)

reported more heterochromatin in female nuclei of Anomodon species than

in male nuclei. Unequal amount of heterochromatin in plant nuclei has been reported by Jackimsky (1935) who found that Pellia epiphylla

(n - 18) had in its complement a pair of chromosomes which differed only in their distirbution of heterochromatin. Diploid and triploid forms of

Dumortiera hirsuta with n- IS and 27, were found to have two or three times the number of interphase heierochromatin bodies than the haploid form (Newton, 1977b). Newton (1971) found that the size of heterochromatin bodies in interphase nuclei offered a means of distinguishing between male and female plants of Plagiomnium undulatum in most instances.

The amount of DNA or its reactivity to Feulgeu stain is reduced by

(Lacour 1956) (1971) low temperature et al., . In contrast, Newton found in studying the size of heterochromatin bodies in, the male and female Plagiomnimillundulatum that temperature had no affect on heterochromatin body size. '

Tatuno (194-1) found a remarkable uniformity in the presence of a large and/or a small heterochromatin body in liverworts, and suggested calling large and small haterochromatic chromosomes, H- and h- chromosomas respectivelys each of which is univeri ally, present in-the haploid gametophytic complement of liverworts. The ektension of these terms H and h to'haterochromatic moss chromosomes was suggested by Yano (1957) but moss heterochromatin is not confined to these two chromosome, *and there are difficulties associated with their designation as R or h-chromosomes (Barrie, 1963). Attempts by -92-

Newton (19774 to identify H- and h-chromosomes in British hepatics proved inconclusive indicating the undesirability of their stereotype recognition in liverworts. The quantities of heterochromatin associated with the longest chromosome in Pellia. dpipylla and Pneesiana undoubtedly equate with H-chromosome of Tatuno (1941), but the samllest chromosome of the latter species consists of only about 26% of heterochromatin compared with 35% in chromosome S. In P. ýdndiviifolia the largest and smallest chromosomes contain 13% and 18% of their length as heterochromatin, only slightly more than in the other chromosomes (Newton, 197u,.

On the basis of this evidenceý there seem no grounds for designating the largest and smallest chromosomes H and h respecitvely and Newton (19774 proposed the abandonment of these terms.

Clearly, with three cytotypes, it would be of interest to investigate the heterochromatin content of interphase nuclei of Atrichum undulatum and to see if temperature had any influence on the size of heterochromatin bodies. The possibility that temperature might: effect the size of heterochromatin bodies of interphase A. undulatum, was therefore considered, but observations indicate that temperature has no affect on the size of heterochromatin bodies of interphase A. undulat=

Materials and Methods

Twenty five haploid, diploid and triploid sampies'were chosen for this study from various localities in England, Wales and Scotland. Material was kept at laboratory temperature from one to six months, further material was cultured in *an unheated'mist unit in a glasshouse at Pan-y-Ff ridd Field Station 0C 0 and a number of samples were kept at (5 to -5 C) in refrigerators in the School of Plant Biology, Bangor. -93-

t Tabl .e 10. Showing the size and the mean value of heterochromatin

bodies in haploid, diploid and triploid'Atrichum, tindulatum.

Cytotype Size of heterochromatin bodies Mean 2 in mm

Haploid 2.88 - 4.80 3.70 1.29

Diploid 2.90 - 4.81 3.94 1.15

Triploid 3-5.80 4.44 1.16

Table 11. Showing the d value of heterochromatin bodies in the

three cytotypes of Atrichum undulatum

Haploid Diploid Haploid

and and and

Diploid Triploid Triploid

-0.552 -0.55 -0.915 -94-

Cytol. ogical preparation technique was similar to that used for

HyRnum cupressiforme and Atrichum'unduldtum (Pagesj9,65),

Drawings of heterochromatin. bodies were made using a camera lucida and the size of drawings calculatecLin' square millimetred.

Results and discussions

The number of nuclei needed to give a suitable sample for each population was determined by calculating the moving means for haploidt diploid and triploid specimens. A sample size of 25 nuclei gave satisfactory results. Table 10 showing the size and the mean value of heterochromatin bodies in the three cytotypes.

From Table 11 it can be seen that the mean size of triploid

A. undulatum is not significantly larger (d - -0.915; P >0.05) than the mean size of the haploid. The mean size of triploid is not significantly larger (d - 0.55; P >0..05) than the mean size of the diploid and the mean size of diploid plants is also not, significantly larger (d - -0.552;

P >0.05) than the mean size of haploid plants. Clearly the size of heterochromatin bodies in interphase nuclei A. undulatum cannot be used to distinguish between haploid, diploid and triploid plants in most instances.

I -95-

CHAPTER 9

Distribution and Conclusion

9.1 Distribution of A. undulatum cy-totypes

A. undulatum. is a very common plant, (Dixon 1924, Rose 1951a,

Watson, 1968i Smith, 1978a). It occurs in a variety of habitats, growing

as yellowish-green to green patches or scattered plants on loose soil in woods, on bankso heaths, in fields. An investigation of the distribution of three cytotypes of A. undulatum in Britain was carried out.

Data for this studywere compiled from field material collected from different localities. Material was determined by Dr. A. J. E. Smith.

Field gatherings were allowed to grow in polythene bags for at least three weeks to enable them to produce young shoots. These were then examined cytologically to determine the chromosome number, using the method described on page Four hundred and seventy two samples were counted from different localities and geographical areas.

For details see Appendix 4. 2 Data were analysed using Chi squared (X ). Whenever there are twomethods of cross-classification, each of which is made up by several more or less qualitative subdivisions, the data can be arranged in the form of a contingency table. X2 is calculated as a measure of deviation between observed and expected. The formula used is: 2 (0 E) E. x2. E where the summation symbol E indicates summation over all the compartments.

The number of degrees of freedom involved is the product of one less than and less the number of rows cne than the number of columns. Probability of a deviation as large as that observed being due to chance is obtained from tables of Chi-squared* -qf; -

The British Isles were divided into four major geographical areas, into The national grid reference was used to divide mainland Britain three (SW). taken areas: North (N), South-east (SE) and South-west Ireland was as a separate area (I). Five contingency tables were examined to show

the most possible associations between chromosome number and geographical distribution of A. undulatum.

Results

There is a significant association between ploidy and geographical distribution, where chi-squared is highly significant at P<0.001

(see Tables 12, l3v

Discussion

These results disagree with the Null h3rpothesis which assumes that there is no'association between numbers of different ploidies in different areas. If this hypothesis is true then the deviation between observed and expected values is due to chance, a situation which is clearly not true.

Inspection of these t ables also shows that this highly significant association is almost mainly due to the distribution of the haploid and partly to the triploid cytotype. In flowering plants it is suggested

(Stebbins, 1971) that the proportion of polyploids is lowest in warm temperature or sub-tropical regions and increases towards both the tropics and the arctic. In flowering plants there is a close correlation between ployploidy and effective vegetative reproduction in colder regions

(Smith, 1978b). Steere (1954) pointed out that there is not this complication in mosses. The results in Fig. 62 and Table 17 show that haploid

A. undulatum is more common in southern areas of Britain which are relatively warmer than in the north which is considered to be relatively colder, This agrees with the hypothesis mentioned by Stebbins (1971). -97-

Table 12. Contingency table relating the four geographical areas and

haploid, diploid & triploid, A. undulatum (observations (0); 2 expectations (E); and value of X corresponding to

probability with 6 degrees of freedom

Geographical areas Cytotypes N S. E S. W Total

0 -0 0 -3 0a 21 0 - 26 50

Haploid E - 2.11 B - 15.25 E- 15.46 E m 17.16

P <0.05 P <0.001 P <0.05 P <0.01

0 -7 0 - 44 0- 47 0 - 58 156

Diploid E - 6.61 E - 47.59 E- 48.25 E - 53.54

P >0.05 P >0.05 P >0.05 P >0.05

0 -U 0 - 97 0- 78 0 - 78 266

Triploid E a 11.271 E - 81.15 E- 82.27 E - 91.29

P >0.05 P <0.05 P >0.05 P <0.05

Total 20 144 146 162 472 J I - 2 24.64

P <0.001 -98-

Table 13. Contin-. ency table relating south and north of Britain-and

haploid, diploid & triplaid A. undulatum. values of,, X2

corresponding to probability with 2 degrees of freedom

Geographical areas cvtntvnp-s

s N Total

0- 47 0-3 50

Haploid B- 34.07 E- 15.92

p <0.05 p <0.01

0- 105 0- 44 149

Diploid E- 101.53 E- 47.46

P >O.05 P >O. 05

0- 156 0- 97 253 rript d ol E- 172.39 E- 80.60

P >O. 05 P <0.05

Total 308 144 452

X2a 20.634

p <0.001 -99-

Table 14. Contingency table relating south east and south west plus

Ireland haploid, diploid and and triploid A. undulatum , values of X2 corresponding to probability with 2 degrees of

freedom

Geographical areas Cytotypes r SE Sw & I. Total

0- 21 0 26 47

Haploid E- 20.92 E 26.07

P >0.05 P >0.05

0- 47 0- 65 112

Diploid E- 49.85 E- 62.14

P >0.05 P >0.05

0- 78 0- 91 169

Triploid E- 75.22 E- 93.77

P >0.05 P >0.05

Total 146 182 328

0.4794

p >O. 05 -100-

Table 15. Contingency table relating the four geographical areas and

diploid, triploid A. undulatum, values of X2 corresponding

to probability with 3 degrees of freedom

Geographical areas Cytotypes SE sw I Total

0- 44 0- 47 0- 58 0-7 156

Diploid E- 52.12 Ea 46.2 E- 50.27 E-7.39

P >0.05 P >0.05 P >0.05 P >0.05

0- 97 0- 78 0- 78 0- 13 266

Triploid E- 88.87 E- 78.79 Ea 85.72 Ea 12.6

P >0.05 P >0.05 P >0.05 P >0.05

Total 141 125 136 20 422

3.917

P >0.05 -101-

Table 16,. Contingency table relating the four geographical areas with

haploid and diploid plus triploid A. undulatum, values of

X2 corresponding probability with 3 degrees of freedom

Geographical areas Cytotypes N SE Sw I Total

0-0 0-3 0- 21 0- 26 50

Haploid E-2.11 E- 15.25 E- 15.46 E- 17.16

P >0.05 P <0,01 P >0.05 P <0.01

0- 20 0w 141 0- 125 0- 136 422 Diploid 'E - 17.88 I E- 128.7' E- 130.53 , E- 144.83 and triploid P >0.05 P >0.05 P >0.05 P >0.05

Total 1 20 1 144 1 146 1 162 1 472

2 x. 20.65

p <0.001 -102-

Table 17. Percentages of haploid, diploid and triploid cytotypes of

A. undulatum in four geographical areas of the British Isles.

Taxon North South South Ireland TOTAL east west

Haploid 2.08% 14.38% 16.05% 0% 10.59%

Diploid 30.55% 32.19% 35.80% 35% 33.05%

Triploid 67.36% 53.42% 48.15% 65% 56.36% Fig. 62. The proportional d. 1stribution of haploidv diploid and triploid A. undulatum in the four geographical areas of the British Isles.

Haploid '(' 'A ), Diploid (A), and Triploid (a

0 -103-

In another example Lazarenko and Lesnjak (1977) found no haploid

A. undulatum in western U. S. S. R. with continental climate with much

colder winters than Britain, thus extrapolating the situation in Britain.

9.2' Conclusion

In Atrichum undulatum there are three cytotypes haploid, diploid and triploid which are indistinguishable morphologically. Morphological variation is both genetical and environmental. Cultivation experiments suggest that plant height is genetically variable and that leaf length is also genetically variable. The angle between teeth and leaf margin shows genetic variability. There is some degree of genetical stability in angular cell wall thickness. Sporophyte characters seta length# sets posture, capsule lengthl number of peristome teeth, operculum length, calyptra and spore are variable and the three cytotypes overlap completely. From this investigation it is clear that there is no correlation between morphology and cytotypeq but that there is a relationship between cytotype and geographical distribution. Inspection of contingency tables shows that this is significant and due to the distribution of the haploid.

Lowry (1954) suggested that n- 21 A. undulatum was derived by apospory from the hybrid n-7A. undulatum xn- 14 A. undulatum.

It seems more likely that in such a hybrid meiosis is irregular and that occasional spores with a balanced complement of 21 chromosomesoccurs

Diploid spores have been reported several times from naturally occuring plants. It thereford, seems more likely that in such a hybrid meiosis is irregular and that occasional spores with a balanced complement of

21 chromosomesoccur - Polyploidy resulting from factors of meiosis - diplospory- has been suggested by Smith (1978b). It therefore seems probable that doubling of the chromosomenumber results from diplospory rather than aposporyo i -104-

the latter only occurring under controlled laboratory conditions

(Smith 1978b). Mehra and Khanna (1961) quote the work of Lowry (1954)

on A. undulatum as an example of allopolyploidy. Smith (1978b) comments

that this is a misinterpretation of the situation. Lowry suggests

that n- 14 A. undulatum is derived from n-7A. undulatum by apospory

and that these two gave rise to n- 21 plants by hybridity followed by apospory. This is a theoretical allopolyploid but as the three genome are similar the a- 21 plants are functionally autopolyploid. -105-

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'APPENDIX I

- Analysis of 'Car6tenoid -Pigments

A. Procedure for-ExtraCtion'df*Car6tertoids, (adapted from Davies, 1965)

1. The plant material was pounded for 1-2 min. with sand and a

small quantity of acetone using a pestle and mortar. The

extract was filtered under reduced pressure and the process

repeated 2-3 times.

2, The extract was concentrated under reduced pressure and

diluted with an equal volume of diethyl ether, then water*

until 2 layers were formed,

3. The lower aqueous phase was run off and re-extracted with

ether to remove any remaining lipid-soltible material and then

discarded.

4. The ether solutions were bulked and washed with water to

remove traces of acetoneg then distilled to a small volume

under reduced pressure and dried by standing over anhydrous

sodium sulphate for about 1 hour.

5. The sodium sulphate was removed by filtering and the extract

was evaporated to dryness on a hot water-bath in a stream of

nitrogen.

B. Sapo ification Procedure

This step was necessary in order to remove unwanted lipid material.

1. Sufficient absolute alcohol was added to dissolve completely

the dry extract, then 60% '(W/V) aqueous potassium hydroxide

was added, I ml to every 10 mlof ethanolic solution.

2. The alkaline mixture was left in the'dark at room temperature

under nitrogen for about 12 hours. -420-

3. The alkaline solution was then diluted with 3 vol=es of

water and freshly distilled diethyl ether was added (I volume

to 3 of the alkaline water (ethanol phase)), The mixture

was shaken firmly in a separating funnel and allowed to stand

until 2 phases appeared, the carotenoids being in the upper

(ether) phase. The lower phase was discarded.

4. The extraction with ether was repeated twice and the three

extracts bulked.

5. The ether solution was washed free of alkali by shaking with

its own volume of water, The washing was repeated several

times, the water being discarded on each occasion,

6. The ether extract was dried by shaking with powdered anhydrous

sodium sulphate (5-10 g per 100 ml of extract) and left to stand

for 1-2 hours*

The sodium sulphate was filtered off and washed with fresh dry

ether, The ether solution was concentrated by distillation in

the dark under reduced pressure and finally blown to dryness

with a steam of nitrogen on a hot water-bath.

8. The residue was stored-under nitrogen at 00C until ready for

use,

C. Thin-LaXer ChromatoEaphZ

The stored extract was re-dissolved in 1-2 ml of fresh, dry ether and loaded on plates coated with Silica Gel GF 254. Effective separation of the carotenoid compounds was achieved using both 20% Ethyl acetate in

Methylene chloride and 15% Methanol in Benzene as developing systems.

Some spots had a spontaneous colouration; others only appeared after spraying with a saturated solution of antimony trichloride in chloroform -121-

and heating at 1100C for 15-20 min. (Davies et al.. 1963), The colours after treatment with the staining reagent are shown on the chromatograms (Figs. 30,31). -122-

. 'APPENDIX 2

-Comparison of Chromosome Length of haploid, clipl6id_and

triRloid Atrichum undulatum. using Tuckey's interval estimate

Tukey's interval estimate was used to calculate the 95% confidence

intervals for the difference between mean value of pairs of cytotypes.

The statistic q' was calculated by:

q+'" qv, r 2n1n2

Where q was derived from tables for v- number of groups (i. e. cytotypes)

and r- number of degree of freedom of error terms in the'analysis

of variance table. 14SE is the mean square error from the analysis of

variance. nl, n2 are the sample size of the two groups.

95% confidence intervals were then calculated as%

(X xj, ) Ui- (X i- -q1<, lui

Where Xi, X3 are the mean values for the two groups.

Pi -Pi is the expected difference between cytotype means.

Chromosome Length (R) in Chromosome um number Haploid Diploid Triploid .

1 6.40 6.14 6.14

2 6.34 5.82 5.9

3 6.32 5.65 5,71

4 5.32 4.81 4.88

5 5.25 4.60 4.80

6 4.19 4.68 4.32

7 3.65 3.19 3.52

Total 5.353 4.984 5.038 -123-

Source S.'s. DF MSE F PROB.

1. Chromosome number 19.10 6 3.00 66.92 01000

2, Cytotypes 18.01 2 0.28 6.18 0.0143

3. Error 0.55 12 0.04

4. Total 0.54 20

As P<0.05t then the differences between groups (ploidy levels or

cytotypes) is significant. The chromosomes in the three cytotypes are not

equal in length, and significant differences have been found between them.

The expected difference between the cytotype means was calculated to

estimate-which cytotype is responsible for this significant difference in

length.

1. Between haploid and diploid cytotypes

(xl X2) ý, Ul (XI - - ql, - 'U2 -<,ql + -

0.369 0.285 0.285 0.369 - 1 -'ýJ2 +

0.084 <<6.654 ' 'Ul - 'U2 ' Assuming that the chromosome length in the haploid and diploid

cytotypes are equal, then P, - P2 - 0, but as 0 is not inside the 95%

confidence intervals, therefore the chromosome length in these

cytotypes are not equal in length and the difference in length is

significant.

2. Between diploid and triploid cytotype

(R X-

0.054 - 0.285 ;U3- P2 0.285 + 0.054

P3- 0.339 -0.231 IU2 -124-

Assuming that the chromosome length in the diploid and triploid inside 95% cytotypes are equals then U3 -U2"0, which is the confidence intervals.

Betweenhaploid add_triploid cXtotype

JJ IJ (X X3) (X, - ,- 3 :5qt+ 1-

0.315 0.285 <<0.285ýl + 0.315 - ' - 'U3 0.03 <<0.600 ý3 lul - As F1 - P3 A E), then there is a significant difference in chromosome length between the two cytotypes.

Conclusion

As the expected difference in means (i, e, Ui - pj - 0) between triploid and diploid cytotypest which is inside the 95% confiden6e intervals, therefore the total chromosome length is not significantly different in these cytotypes. In the haploid and triploid and haploid and diploid cytotypes the difference in means is outside the 95% confidence intervals. Therefore there are significant differences between each group of cytotypes. It would appear that the chromosomes in the haploid cytotype are responsible for the significant difference found. The diploid and triploid chromosome lengths are similar, but both are different from the haploid chromosomes. The total chromosome 3 length of the triploid is /2 times that of the diploid, but the total length in the diploid and triploid are not twice and three times that in the haploid -125-

'APPENDTX comparisýoýn'of'DNA'Contdrit'6f, 'Thrd#-'5Zt6tyoed! 6f, 'Atrithum'undulatum 'Usirig Mikeyd Interval'EAtimate

Tukeyts interval estimate was used to calculate the 95% confidence intervals for the differences between means of pairs of groups of samples. The statistic q was calculated by:

MSE qv -q + V"; 2n1n2

qv, was derived from. tables for v- number of groups (iseo where r in cytotypes) and r- number of degrees of freedom of error term the analysis of variance.

MSE is Mean Square Error from the Analysis of Variance. n,,, n2 are sample sizes of the two groups.

95% confidence intervals were then calculated as: -

(x - x, )-qI<, (; i - )ý )

in The DNA content haploid, diploid and triploid. A. ' undulatum was assumed to be in the ratio of 1: 2: 3:, A loglo transformation was applied to the data to give it a normal distribution and equal variance for the three cytotypes as follows: -

Cytotype Size (n) Mean'; St. Dev.

Haploid 45 1.78 0,117747

Diploid 58 1.80 0,112555

Triploid 65 2.10 0,125273 -126-

Source Sum' 6f , SQ. D. F. M. S. F

Between 4.110306 2 2.055 146.00

Within 2.322456 165 0.01408

Values of (ý12- ý11) under the above assumptiom was taken as follows: - log 2 0.30102 1. 'Between diploid and hapl6id'dy totjýes_ 10 - -- qI < (, ý11) <, q' + ýX2 - XI) 21 ' u2 -

0.02 - 0,0555 <' (P2 - pl) 0.0555 + 0.02

- 0.0355 <' (ý12 - Pl) 0.0755

2. Between triEloid'arid'hapl6id eVtotypes loglo 3-0.47712

(X3 (113 (X3 xl) - XI) qI

0.34 - 0.054 ýU3 -ful ) <, 0.054 + 0.34

(F3 0.394 0.286 - 111) '<

log 1.5 0.17609 3. ' Betwgen'trißloid'and'diplöid' Lytotypes 10 -

(x < (93 < + (ý3 ; 3-x2)- qI - ;12) qt - 2)

(; 0.0504 0.3 0.3 - 0.0504 < 13 - P2) e< +

0.2496 (ý13 0.3504 <' - P2) ý<

As none of the three confidence intervals contain the expected

loglo 20 log 3 loglo 1.5 values of 10 and the assuraption that the ratio of DNA content in haploid: diploid: triploid is 1: 2: 3 is therefore rejected by the data. -127-

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