CYTOTAXONOMIC STUDIES IN HOLCUS. II. MORPHOLOGICAL RELATIONSHIPS IN HOLCUS MOLLIS L

GYTOTAXONOMIG STUDIES IN IIOLCUS

IL MORPHOLOGICAL RELATIONSHIPS IN HOLCUS MOLLIS L.

BY KEITH JONES* AND CHRISTOPHER P. CARROLL

Welsh Plant Breeding Station, Aberystwyth

{Received 2'i May 1961)

(With Plate i)

SUMMARY

A randomized block experiment was laid down to compare the morphology of the tetraploid,pentaploid and hexaploid races of Holcus mollis. No qualitative differences have been found todistinguish the races.

Quantitative differences exist, but are not sufficiently large to allow a taxonomic separation.The morphology of the pentaploid mollis is discussed in relation to its possible hybrid origin

involving H. lanatus.

INTRODUCTION

In the first paper of this series (Jones, 1958) it was shown that in Britain H. mollis L.occurs as a complex of polyploids {zn = 28, 35, 42, 49) of which the pentaploid {zn = 35)is the most abundant. Evidence given in that paper suggests that this latter form is infact a hybrid derivative of H. mollis {zn = 28) and H. lanatus {2n = 14). In an attemptto substantiate this idea studies have been undertaken of the comparative morphologyof the chromosome races using the randomized block technique. The results of thisexperiment are reported here. They are relevant to the possibility of recognizing thepolyploids in nature, their origin and to the general effects of increase in chromosomenumber.

In the experimental lay-out only the tetraploids, hexaploids and pentaploids have beenincluded. The omission of the heptaploid has been made necessary because only twospecimens have yet been found. However, although this race has had to be omitted fromthe statistical treatment the two plants were cloned and grown as a guard row around theblocks, and from these some observations on morphology were made.

Preliminary measurements were made on a large number of characters but only thosewhich afford some possibility of separating the chromosome races, or of showing re-lationships, were selected for the present experiment. Statistical treatment was givenonly to macroscopic characters likely to be of service to the taxonomist.

MATERIAL AND METHODS

Tillers were collected from established plants in their natural habitat. To reduce thechance of replicating genotypes no two plants came from sites less than 70 yd apart, andmost were much more widely separated. During the collection of data it became apparent

* Present address: Royal Botanic Gardens, Kew, Richmond, Surrey.

63

64 K. JONES AND C . P. CARROLL

that three of the hexaploids (H II, H III, H IV) closely resembled one anotlier althoughthere were small differences in the numbers of spikelets on a homologous branch of theinflorescence. It is possible, despite the precautions taken during collection, that thesethree hexaploids could be parts of the same clone, and a certain amount of caution shouldbe exercised in drawing conclusions from the statistical comparisons involving hexaploids.The origin of the plants is as follows:

Race W.P.B.S. No. Location

TetraploidT I Bs 1903(2) Penglais Hill, Aberystwyth, Cards.^T II Bs 1905(1) Penglais Hill, Aberystwyth, Cards.*T HI Bs 1912(1) Frongoch, Aberystwyth, Cards.T IV Bs 1913(1) Cwm Woods, Aberystwyth, Cards.T V Bs 1913(2) Cwm Woods, Aberystwyth, Cards.*T VI Bs 1932(1) Durham Woods, Durham.

PentaploidPI Bs 1915(1) Clarach, Aberystw^rtb, Cards.P II Bs 1928(1) Pencaerau Woods, Neath, Glam.P HI Bs 1929(1) Gnoll Woods, Neatb, Glam.P IV Bs 1931(1) Malvern, Worcs.P V Bs 1932(2) Durham Woods, Durham.P VI Bs 1939(1) Exmoor, Devon.

HexaploidHI Bs 1903(14) Penglais Hill, Aberystwyth, Cards.H II Bs 1911(12) Frongoch, Alserystwyth, Cards.H HI Bs 1912(4) Frongoch, Aberystwyth, Cards.*H IV Bs 1912(6) Frongoch, Aberystwyth, Cards.*H V Bs 1917(10) Parson's Bridge, nr. Aberystwyth, Cards.H VI Es 1926(5) Glaspwll, Cwm Llyfnant, Aberystwyth, Cards.

HeptaploidG.R. I Bs 1917(13) Parson's Bridge, nr. Aberystwyth, Cards.G.R. II Bs 1918(1) Devil's Bridge, nr. AberysUvyth, Cards.

* Different site.

In order that the plants should initially be as identical as possible, they were preparedas equal-sized bunches of trimmed tillers. The plants were potted individually and keptin randomized order from the start. After establishment in a warm glass-house they werehardened-off and transferred to the experimental field on 5 May 1958. A 3 ft spacing wasallowed between plants to obviate competitive effects, and a guard row at the samedistance around the perimeter consisted of alternate clonal plants of the two heptaploidsso that it could also serve to determine whether they were separate genotypes.

Six randomized blocks, made up by clonal replication, formed the experimental layout.Each block contained eighteen genotypes, six from each chromosome race. The chromo-some number of each genotype was checked from counts of root-tip mitosis just beforeplanting in the field.

Data were collected mainly from the flowering tiller. To reduce within-plant variabilitya homotype (in the sense of Gregor, Davey and Lang, 1936) was selected; measurementswere made on the three best developed tillers from each plant. The spikelets from aselected branch of the three inflorescences chosen were pooled and twelve taken atrandom to provide a plant mean. The actual identity of the plants was not known to theobserver when measurements were taken. The method of statistical analysis is given inthe Appendix.

Holcus mollis 65

RESULTS

Habit of young plants. Standardized photographs were taken of the plants in oneblock, because it was found to be easier to compare growth habit in this way (Anderson,1949). The tetraploids had a more tufted appearance than the other races, due probablyto the more rapid emergence of new tillers from axillary buds (Plate i, Figs. 5-7). Theyalso gave the impression of having much narrower leaves but this was caused mainly bythe presence at that stage of a large number of immature leaf blades. It was not possibleto distinguish pentaploids and hexaploids. The two heptaploids were noticeably differentin habit from the rest of the polyploids, being stiffly erect with broad, short leaves of abright green colour (Plate i, Fig. 8).

Radius of the plant. Because the radius of the plant before flowering has proved auseful indicator of growth habit in the examination of the primary hybrid between H.lanatus and H. ?7iollis (Carroll and Jones, 1962) the measurement was taken on the threepolyploid races also. The overall radius was measured in three specified directions, usinga rule clamped to a spiked pivot which was inserted into the centre of each plant in turn.The mean values are:

Tetraploid Pentaploid HexaploidNo. of genotypes 6 6 6Mean radius (in.) 9.22 11.68 9.27

The actual mean value for the pentaploid is larger, but there were no significant differ-ences between races at P = 0.05.

Date of flowering. Data collected in previous seasous have indicated that the date ofpanicle emergence is generally earlier for H. lanatus than for the various races of H.mollis which differ little among themselves. The date of emergence of the third in-florescence was recorded throughout the experiment and subjected to statistical analysis:numerical values were obtained by counting from the earliest date as i. The differencesbetween races fell well below the 5 % significance level.

The flowering tiller. Measurements were taken of the length and greatest width of theblade, also of the greatest width of the sheath when unrolled. The length of the bladeshowed no differences between races, and was so variable within plants as to be uselessunder ordinary experimental conditions. The width of the sheath proved to be one of themost useful measurements, there being no overlap in range between tetraploid andhexaploid values. The within-plant variation was low compared with many other measure-ments. The statistical analysis is shown in Table i.

The hexaploids and pentaploids were signiflcantly larger (P = o.oi) than the tetra-ploids, and the flgures strongly suggest that the pentaploids may in fact exceed thehexaploids, as the 5 % level is nearly reached in that comparison.

The greatest width of the flag-leaf blade gave very similar results, though the signi-flcance levels attained were not quite as high, probably because the within-plantvariability was greater.

Panicle. Typical 4X, 5X, 6x and 7X panicles are illustrated in Plate 1, Figs. 1-4.The length of the whole inflorescence was measured from its lowest node to the extremetip, including spikelets (Table 2).

These results are again like those for flag-leaf sheath width: 5X and 6x races havesimilar means and both exceed the 4X race.

Two internodes, the basal and the third from the base of the inflorescence, wereselected for measurement as there might be differences in proportions between the

£ N.P.


Number of genotypesMean width (mm)

(i) General analysisItem

ReplicatesChromosome races 2Genotypes within races 15

Genotypes overallErrorTotal

Table i. Width of the sheath of the flag-leafPentaploid Hexaploid

6Tetraploid

611.6s 17-03

Analysis of variance

DF

5

1785

107

Variance

1.83264.89

22.2050-75

O.Q8

614.94

Varianceratio

1.8711.93**

51-79**

DFinF test

S/852/15

17/85

(ii) Comparisons of range of variationItem DF

Within-4X varianceWithin-5x variance"Within-6x variance

Variance

11.2549-75

5-61

Varianceratio

8.87*t

DF inFtest

5/5 (5x/6x)

t In view of the significant ratio, a Bartlett's x'̂ test was carried out to see whether the variancescould be regarded as forming one population. Significance at P = 0.05 was not quite reached;X2 = 5-35; D F = 2.

(iii) t-test comparisons of chromosome races (see mean values above)For 4X with 6x** L.S.D. (P = o.oi) 3.27 mmFor 6x with 5x L.S.D. (P = 0.05) 2.37 mm

* Values significant at P = 0.05** Values significant at P = 0.01

Table 2. Length of the inflorescence

Number of genotypesMean length in cm

i) General analysisItem

ReplicatesChromosome racesGenotypes within races

Genotypes overallError

2

15

Analysis

D F

5

1785

Tetraploid6

9.89

of variance

Variance

0.9543-9911.8915.660.64

Pentaploid6

11.91

Varianceratio1.483-70*

24.47**

Hexaploid6

11.68

DFinF test

5/852/iS

17/85

Total 107

(ii) Comparisons of range of variationWithin-4x variance 5Within-5x variance 5Within-6x variance 5

10.5820.684.40

4.70

(iii) Z-test comparisons of chromosome races (see mean values above)For 4x with 6x* L.S.D. (P = 0.05) 1.73 cmFor 5x with 6x L.S.D. (P = 0.05) 1.73 cm

* Values significant at P = 0.05** Values significant at P = o.oi

5/5 (5x/6x)

Holcus moUis 67panicles of different races. In neither instance did difl'erences reach the 5 % level ofsignificance, but a selected branch (the longest at the second node from the base) gavesignificant differences between race means, and also between the variabilities of the races(Table 3).

Table 3. Length of the longest branch at the second panicle node

Number of genotypesMean length in mm


ReplicatesChroniosome races 2Genotypes within races 15


Total

ii) Comparisons of range of variation

Item

Within-4x varianceWithin-5x varianceWithin-6x variance

Analysis

DF

5

1785

107

D F

555

Tetraploid6

40.69

of variance

Variance

10-331388.91

190.55331-53

18.62

-

Variance

62.00471-49

43-07

Pentaploid6

52.86

Varianceratio

0-557.29**

_17.81**

-

-

Varianceratio7.60*

10.95*-

Hexaploid6

48.69

DF inF test

5/852/1S

17/85

-

DFinFtest

5/5 (5X/4X)5/5 (5x/6x)

In view of the significant variance ratios shown above, a Barlett's x̂ test was carried out toascertain whether the variances could be regarded as forming one population. The result wassignificant at P<o .5 ; x^ = 7-4i; DF = 2.

(iii) Comparisons of chromosome races (cf. mean values above)

As the variances for the races could not be regarded as equal the f-test could not be applied. Thecomparisons were made according to the method due to Behrens which gives an appropriateweighting for the differences in variance.

For 4X with 5xt d — 3.46 (6 = 7o°O5')For 4X with 6x** d = 5.18 (6 = 5i"oo')For 5x with 6x d = 1.21 (6 = 73^39')


t Values significant at P = 0.02 (Behrens's test only)

The results so far as mean values are concerned agree with those for the charactersalready mentioned. As the length of the branch varies in the same way as the total lengthof the inflorescence there is reason to suppose that the races differ only in size and not inshape. This was borne out by observation of the plants in the field. Calculations of the

total lengthratio gave similar results for all three races.

length of branch

H. lanatus has considerably more spikelets per unit of the inflorescence than has the28-chromosome H. mollis (Carroll and Jones, 1962), and the effect of this is apparentin the primary interspecific hybrid. A count was made of the average number of spikeletson the selected branch for each of the three polyploid races. No significant differenceswere obtained. The race means are:

Tetraploid Pentaploid HexaploidNumber of genotypes 6 6 6Mean number of spikelets 27.15 39.00 26.25


The high, but non-significant, value for the pentaploid race was due almost entirelyto the influence of one genotype, P IV, which had more than twice as many spikelets asany other pentaploid.

The two heptaploids differed in spikelet number. No overlap in range occurred be-tween the replicate values for the two plants, and it is reasonable to suppose that twodifferent genotypes are involved. The means and ranges are:

Mean spikelet number RangeG.R. I 15.7 14.3-18.8G.R. II 27.8 22.3-33.7

Spikelet. The lengths of the large (inner) glume and of the awn were measured. Soalso was the length of the lower floret, and this provided more precise data than thoseobtained for any other character. Variability was very low and the effect of polyploidygreatly exceeded that due to genotypic differences. The results followed the pattern forthe characters mentioned so far: pentaploid and hexaploid were effectively the same andthe tetraploid was very signiflcantly smaller (P == o.ooi) (Table 4).

Table 4. Length of the lower floret

Number of genotypesMean length in mm


ReplicatesChromosonne racesGenotypes within races


215

Analysis

DF

5

1785

Tetraploid

62.36

of variance

Variance

0.0163-IO0-150.50O.OIO

Pentaploid

62.86

Varianceratio

1.6020.7**

50.0

Hexaploid

62.87

DFinF test

5/852/15

17/85

Total 107

(iij Comparisons of range of variationItem DF


Variance

0.140.16

ratio

1.10

DFinFtest

5/5

(iii) «-test comparisons of chromosome races (see mean values above)For 4x with sx*** L.S.D. (P = 0.001) 0.37 mmFor 6x with 5x L.S.D. (P = 0.05) 0.19 mm


*** Values significant at P = o.ooi (f-test only)

The results for length of the larger glume were the same as those for the length ofthelower floret, apart from a greater amount of variation between replicates. The most note-worthy of the characters examined was the length of the awn because the pentaploid inthis mstance resembled the tetraploid and was smaller than the hexaploid (P = o.ooi)(Table 5).

Number of genotypesMean length (mm)


ReplicatesChromosome racesGenotypes within races

Genotypes overallFrror

Total

(ii) Comparisons of range ofItem


Holcus mollisTable 5. Length of the

Tetraploid6

4-54

Analysis of variance

D F

52

15

1785

1 0 7

variationD F

555

Variance

0.067.19O-571-350.02

-

Variance

0.690.810.21

awn

Pentaploid6

4-56

Varianceratio3.00*

12.61**

67.50**

-

Varianceratio

1.174

Hexaploid6

5-32

D F inF" test

5/852/15

17/85

-

DF inF test

5/5 (5x/6x)

69

(iii) ?-test comparisons of chromosome races (see mean values above)For 4X with 6x*** L.S.D. (P = o.ooi) 0.72 mmFor 4x with 5x L.S.D. (P = 0.05) 0.38 mm


*** Values significant at P = o.ooi (i-test only)

DISCUSSION

Early observations on the chromosome races of H. mollis did not suggest that there wasany obvious character which could be used to separate them. All were very similar intheir inflorescence structure and character whilst in general habit, apart from an appar-ently greater vigour of the pentaploid, they were indistinguishable. The results of ourmore detailed examination are in general a confirmation of the previous studies. It isapparent that the polyploids do not form a series in which size increases with chromo-some number. Most frequently the pentaploid characters are as great or even greater indimension than those of the hexaploid which in turn is usually larger than the tetraploid.However, there is no qualitative difference in morphology and one must turn to those of aquantitative nature. Here again, however, we find that no single dimension provides arange which is sufficiently distinct to be of value. If we consider the ratios between

awn lengthcharacters the most sensitive of these is which gives a ratio of less

lower floret lengththan 1.7 units for the pentaploid and between 1.7 and 2.1 for the tetraploid and hexa-ploid. Once more, therefore, there is little to distinguish the races and there is nocertainty that the pentaploids can always be identified. Even a non-morphologicalcharacteristic, such as date of flowering, offers no opportunity of separating the races.

Is there any morphological evidence which points to the hybrid nature of the penta-ploid? In looking for this one has to bear in mind several points. Firstly, if the previoushypothesis of origin (Jones, 1958) is correct only one-fifth of the genetic material of thisrace has been derived from H. lanatus. As we shall show in a later paper (Carroll andJones, 1962) triploid hybrids having one-third of their chromosomes from this source

yo K. JONES AND C . P. CARROLL

show a strong resemblance to H. ?nollis and it would in consequence be anticipated thatin the pentaploid the greater predominance of the mollis chromosomes would be reflectedin morphology. Secondly, of the two species, H. lanatus is larger in most of its organs; itis likely, therefore, that its effect on the hybrid would be to increase the size of the polyploidover and above that due to pure chromosome reduplication. Our data show that sueh anincrease can indeed be found in the pentaploids but this could as well be attributed to anoptimum chromosome number as to hybridity. One factor which may argue againstpentaploidy being the ideal state is that the heptaploids are not lacking in size or vigourin comparison.

The case for the participation of H. lanatus in the origin of the pentaploid receives somesupport from the results obtained for the flag-leaf sheath and awn. H. lanatus has a hulkyinflorescence and a correspondingly broad flag-leaf sheath. Tetraploid mollis has a paniclewith fewer spikelets per unit length and the flag-leaf sheaths are narrower. Now penta-ploid mollis does not significantly differ from the tetraploid in its number of spikeletsbut the flag-leaf sheath is larger even than that of the hexaploid. Again the awn of lanatusis about half the length of that of tetraploid mollis when straightened. The awns of thepentaploid are no greater in length than those of the tetraploid — a result which is incontrast to all other measured characters. This may be a demonstration of the geneticeffect of lanatus. A flnal point in favour of the theory of hybridity is the greater variabilit)'in many of the characters of the pentaploid when compared with the other races. Thepresence of a 'new' chromosome set would make this situation more easily understood.

Clearly our experiments have not proved the hybrid nature of the pentaploid butneither have they detracted from the hypothesis. When taken in conjunction with thecytological evidence, and with morphological data published in Part III, they makehybridity the most likely explanation for the vigour and adaptability (and consequentlythe success) of the pentaploid.

If we are right in our presumption it becomes evident that neither polyploidy norhybridity need necessarily produce morphological changes which are adequate for thepurposes of taxonomy. Even with the randomized block technique we cannot make anypositive distinction between the races. We would hardly consider it wise, therefore, tosuggest that the chromosome races should receive separate taxonomic recognition. Wehave recorded, and called attention to, the complex nature of H. mollis. Herbarium sheetsof each of the races will be deposited in the herbaria at Kew and the Welsh Plant BreedingStation and the final judgment on taxonomic status of the entities will be left to theexperienced taxonomist.

ACKNOWLEDGMENTS

We wish to thank Mr. A. R. Beddows for his interest and comments, and for supplyingmaterial for this study; also Dr. A. Durrant, Department Agric. Botany, U.C.W,,Aberystwyth, who gave considerable assistance with our statistical analyses. We aregrateful to Miss S. Davies and Miss M. LI. Jones, B.Sc, who assisted in collecting thedata, and finally to Miss Irene D. Rees for editing the manuscript.

REFERENCES

ANDERSON, E. (1949). Introgressive hybridisation. Chapman & Hall Ltd., London.CARROLL, C. P. & JONES, KEITH (1962). Cytotaxonomic studies in Holcus. III. A morphological study of

the triploid Fi hybrid between Holcus lanatus L. and H. mollis L. New PhytoL, 61, 73.

THE NEW PHYTOLOGIST, 6i, PLATE

JONES .\ND CARROLL -HOLCUS MOLLIS {facing p . 7')

Holcus mollis 71FISHER, R. A. & YATES, F . (1957). Statistical Tables for Biological, Agricultural and Medical Research. 5th

ed. Oliver & Boyd, London.GREGOR, J. W., DAVEY, V. McR'I. & LANG, J. M. S. (1936). Experimental taxonomy. I. Experiment garden

technique in relation to the recognition of small taxonomic units. Nezo PhytoL, 35, 323.JONES, KEITH (1958). Cytotaxonomic studies in Holcus mollis L. I. The chromosome complex in Holcus

mollis L. New PhytoL, 57, 191.SNEDECOR, G. W . (1946). Statistical Methods. 4th ed. Iowa State College Press. Ames.

APPENDIX

SCHEME OF STATISTICAL ANALYSIS

I. General analysis of variance (using Snedecor's F test)I if significant

at P = 0.05

2. Comparison of within-race variances (greatest v least)

3. Test population of within-ivariances for homogeneity (usingBartlett's x̂ test)

if significantat P = 0.05

5. Comparison of race-means (using Behrens'method for means with differing variances).

not

significant

4. Comparison of race means(using the f-test for leastsignificant differences)

In order to provide a sensitive test for significance when chromosome race comparisonswere being made the total sum of squares due to genotype differences in the generalanalysis was split into two items: (i) chromosome races S.S. and (ii) genotypes withinraces S.S. Thereafter, the variance obtained from the latter was used as the error term,because if real differences are to be detected between races, they must exceed thevariation due to genotypes as such.

EXPLANATION OF PLATE i

1. Inflorescence of tetraploid Holcus mollis2. Inflorescence of pentaploid Holcus mollis3. Inflorescence of hexaploid Holcus mollis4. Inflorescence of heptaploid Holcus mollis(The size range cannot be shown as only two genotypes have been found).5. Young plant of tetraploid H. mollis, showing growth habit.6. Young plant of pentaploid H. mollis, showing growth habit.7. Young plant of hexaploid H. mollis, showing growth habit.8. Young plant of heptaploid H. mollis, showing growth habit.In 1-3 the white bars represent the average lengths ofthe shortest and longest genotypes ofthat ploidy.

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CYTOTAXONOMIC STUDIES IN HOLCUS. II. MORPHOLOGICAL RELATIONSHIPS IN HOLCUS MOLLIS L