OBSERVATIONS ON NATURAL CLONES IN HOLCUS MOLLIS

Transcription

1 New Phytol. (1967) 66, OBSERVATIONS ON NATURAL CLONES IN HOLCUS MOLLIS BY D. J. HARBERD Department of Agriculture, The University, Leeds 2 {Received 26 September 1966) SUMMARY Morphological examination of a group of fifty-eight isolates of Holctis mollls from natural hill vegetation suggested that the collection was composed of four plant-types only. Further experiments proved that there were major discontinuities between the plant-types, and relatively little heterogeneity within them. It is argued that each plant-type corresponds to a single colonizing seedling. Acceptance of this interpretation implies that natural stands of H. mollis contain very few genotypes, some of them replicated very many times, and this fits in with the presumed cytological history of the species. One of the genotypes observed was found in an area over half a mile across, in an ecological non-uniform terrain, so that the actual migration route might have been much longer. INTRODUCTION During the summer of 1955 vegetation samples were taken from eighty sites oi Agrostis- Festtica grassland in the Pentland Hills, south of Edinburgh, for genecologieal investigation. The following year isolates of thirteen taxa from these samples were planted out in randomized trials. In several of the taxa it seemed likely from the morphological observations recorded that some of the individual genotypes had been collected several times in the samples and further work supplied corroborative evidence for this interpretation (Harberd, 1961). The present paper is concerned with the evidence for clonal spread of this kind in Holctts mollis. MATERIALS AND METHODS Plant materials The original eighty collecting sites were grouped in the hills in three areas: sixty-tw o of the sites were clustered together near Loganlee, eight sites were half a mile away at Grain Burn on the other side of the hill, and the remaining ten sites were four miles distant at Boghall (Fig. i). Five small turves were cut at each of the eighty sites and from each of these 400 turves; when Holcus mollis was present, a tiller of it was isolated and grown on. This yielded fifty-eight isolates and these were planted out as randomized spaced plants in the experimental ground the following year. It soon became apparent that there were four distinct plant-types present in the collection, all isolates being referrable to one of the four whose distinguishing characteristics were: (i) Late flowering; long, broad, drooping leaves; big pale inflorescences with a drooping habit at the pre-flowering stage; sheaths of flag leaves somewhat inflated; 401

2 402 D. J. HARBERD abundant long fat rhizomes; shoot bases red between darker veins; rust susceptible; overwintering plants fawn-brown. (2) Earlier flowering and smaller in all parts; more erect in habit, leaves narrower; with less drooping of leaves and inflorescences; inflorescence darker; less inflated sheaths; fewer and shorter rhizomes; shoot bases white between the red veins; rust susceptible, fawn-brown in winter. (3) Very similar to (2), and not easily distinguished from it, but more erect and spindly and of a slightly paler colour. (4) Differed essentially from the rest of the material in being both immune to rust and winter-green. Otherwise very similar to (2) but leaves softer in colour and to the touch, also slightly earlier and smaller in all parts with very little rhizome development, slightly more erect in habit. Fig. I. Sketch map of the northern part of the Pentland Hills showing the collecting areas in relation to the main topographical features. Contours at 250-ft intervals; hatched over 1250 ft. The distribution of these four plant-types between the collecting sites was particularly interesting (Table i). Types 3 and 4 were the only types collected from Boghall to which they were restricted. All isolates from Grain Burn were type 2, and isolates from Loganlee belonged either to type i or 2, type i being restricted to Loganlee. Thus it appears that the different plant-types were limited to relatively small parts of the whole collecting area. With such marked discontinuities in plant-type it seemed likely that the trial was not composed of a single taxon. As it was known (Beddows and Jones, 1953) that H. mollis exists in several chromosome races, representative material was sent to Dr Jones for examination. It transpired (Jones, 1958) that types i, 2 and 3 belonged to the commonest

3 Genotype distribution in Holcus mollis 403 cytological race, with thirty-five chromosomes, and that type 4 was of the twenty-onechromosome race. According to Jones's interpretation (1958) the twenty-one-chromosome race is a hybrid between a twenty-eight-chromosome H. mollis and the fourtcen-chromosome H. lanatus. This hybrid is apparently produced experimentally with ease but it had not previously been recorded in the wild yet thirteen of the sixteen plants from Boghall were of this type. Such a concentration of a rare type in one locality invites comment. The immediate reaction is that ecological conditions at Boghall are conducive both to its formation and survival. But while Boghall must differ in many small respects from other localities it does not appear to be in any way ecologically unique. There is, however, an alternative interpretation that there is only one genotype of the twenty-one-chromosome race at Boghall, that this has spread vegetatively over an area of at least 100 yd across and that it was collected there many times. This hypothesis seemed worth testing and the investigations that ensued form part of the present paper. Jones interprets the thirty-five-chromosome race of H. mollis as being a backcross between the twenty-one-chromosome hybrid (an unreduced gamete) and the twentyeight-chromosome H. mollis. Presumably plants would have arisen in this way relatively rarely so that this race could only have become the commonest form of H. mollis by virtue of its very vigorous vegetative spreading growth. We should then expect to find that this race consists of relatively few distinct genotypes, some of them dispersed over Table i. Numbers of isolates of each of the four plant types from the different collecting areas Boghall Grain Burn Longalee i8 I Plant types considerable distances. The preliminary observations, both morphological and distributional, suggested that this had in fact been found. Type 3 was only collected from a very small area of Boghall and might easily be a single genotype. Type i had an observed spread of 300 yd but its very vigorous vegetative reproduction could account for this given adequate time. The most dubious part of the evidence, as it stood, was the suggestion that type 2 with a spread of over half a mile which straddled two sides of a hill mass could be the vegetative products of a single seedling. It was necessary, therefore, that the collection should be examined in more detail. Methods In investigations of clonal spread with other species two methods have been used (Harberd, 1961). The more reliable of these has been found to be the one involving the self sterility mechanism but, in view of the cytological background of H. mollis, this was judged to be inappropriate in the present case. Accordingly, all further investigations were based on 'cloned clone' trials and two were laid out, one of twenty-one-chromosome plants and the second of thirty-five-chromosome plants. In a trial of this type a group of isolates is cloned to several ramets each and grown in randomized trial, the object being to see whether the original isolates can be differentiated by any recorded characters. Holcus proved to be different from many other grass materials in that it grows so fast that it flowers profusely in the year of planting. It is possible in the ist year to get a

4 404 D. J. HARBERD continuous crop of flowering stems by cutting the older ones to the ground as they mature, and thus to record characters many times during a season. (In the 2nd year the much larger number of flowering stems all mature at approximately the same time). The stems are easily measured for a large number of characters, and in most cases a set of eight standard characters was recorded: stem length, panicle length, internode length for both the ultimate and the penultimate internodes, and sheath length and lamina length for both the ultimate (flag) and penultimate leaves. RESULTS The twenty-one-chromosome material The six isolates used as the basis of this trial had all been examined cytologically hy Jones and each came from a separate site at Boghall so that the fullest known distribution of the race was represented. Each isolate was cloned to eight pieces and the forty-eight plants were laid out as eight randomized blocks in the spring of Throughout the period of trial the forty-eight plants always appeared to be replicas of one another. There were, of course, small differences between them but it always seemed that these were no more than the slight cultural modifications that inevitably occur in spaced plant trials. This was substantiated by the repeated observations that the isolates could not he differentiated in the replications by matching these slight morphological variants. The trial was recorded for ear emergence in the spring of 1957 and for each of the eight standard characters ten times during the growing season. Stem length was measured once during There are thus eighty-two records, all of which have been analysed for differences between isolates. If the isolates were identical we should expect four of these analyses by chance to be statistically significant at the 5 ^ level; in fact sixteen of the analyses passed the 5% significance level three of them being significant at the 1% level. Thus there is some evidence that the six isolates were not, in fact, all of one common genotype. However, the evidence is not very weighty, especially when looked at in more detail, as the different scores tended to conflict with each other. This can be illustrated well by the character 'Penultimate internode' which was significantly different between isolates in four of the ten dated records, passing the i % significance level on two dates. However, no one isolate can be picked out as extreme in this character, the ranking order changing erratically from date to date. In fact, summing the scores over dates, there is no significant difference between the isolates in this character. (Had the scores reinforced one another their significance would have gone up following this practice.) When the other seven characters were treated in the same way only one just passes the 5% significance level. The thirty-five-chromosome material Twenty-four isolates were chosen for this experiment; each was cloned to four pieces and the trial was laid out as four randomized blocks. All of the isolates chosen had been examined cytologically. Twenty-two of them originated in Loganlee, eleven each of the late flowering type i and the early flowering type 2, representing for each the widest known distribution of the type in that part of the hills. There was also one isolate of type 3 from Boghall (site 4) and one of type 2 from Grain Burn (site 80). By contrast with the adjacent trial of twenty-one-chromosome isolates, the heterogeneity between isolates in this trial was especially obvious at all times. In each replication the eleven isolates of the later flowering type i could be picked out with the greatest of

5 Genotype distribution in Holcus mollis 405 ease for most of the season. The difference vi^as particularly marked at the time of ear emergence. All ramets of the early flowering plants had inflorescences showing for the first time during the last 12 days of June. The later plants reached the same stage between the dates 5 July and 18 July. The single Boghall isolate in each replication was also identified with relative ease. The variability in earliness of development in the trial was so great that it was possible to record the standard set of eight characters on the early plants three times before the later plants could be measured. The trial was then recorded as a whole on three more dates and, at a final scoring, only the late plants were recorded. Looking flrst at the results of analyses of the records for the late plants, we have thirtythree records to consider the single score of the date of first ear emergence and the four dated records for each of the eight standard characters. One of these analyses was significant at the 1% level and a further three at the 5 ^ level, so that again, as in the twenty-one-chromosome material, there is some evidence for heterogeneity present and, although the evidence exceeds the levels expected by chance alone, it is not very substantial. There is, however, a difference from the comparable results for the twenty-onechromosome material because in two ofthe characters the lamina lengths ofthe ultimate and penultimate leaves the ranking orders of the different dates of recording show reasonable agreement. Three of the isolates always occur together at the top of the list. In the four separate dated analyses for these characters each attains significance once, one at the i % level and one at the 5 % level, and by summing over dates these significances are retained, though not enhanced. None of the other characters (two of which were significant at the 5 ^ level in one analysis each) are significant in their summed analyses. These results suggest that the collection of eleven isolates could be divided into two groups, viz. the eight shorter-leaved ones and the three longer-leaved ones. If this is done none of the possible analyses suggest any heterogeneity within either group. There is, however, no corroborative evidence for this division into two groups. In the trial the plants all appeared to be replicas of one another and they could not be classified on any qualitative character. Even in the leaf lengths there was no evidence of discontinuity in the character so that a division into the six shorter-leaved and the five longer-leaved is just as effective in eliminating significance and there is nothing to choose between these two, or any other equally efficient classification. The early plants in the trial were recorded for date of first ear emergence and for the standard set of eight characters on six separate occasions, a total of forty-nine recordings. As has been noted the isolate from Boghall was morphologically distinct from the rest of the early plants. Analyses of the forty-nine records for differences between the Boghall isolate and the other early plants show significant differences in twenty-two records, five of them reaching the 0.1% level of significance. This substantial body of evidence supports the hypothesis that there is a genetical difference between the Boghall isolate and the others. The remaining twelve early isolates appeared to be uniform in the trials but on analysis there are significant differences between the isolates in thirteen of the forty-nine records, one being significant at the 0.1% level. Once again, as in the cases of both the twentyone-chromosome plants and the late flowering thirty-five-chromosome plants, there are more differences here than would be expected by chance in a population of identical isolates. Examined in more detail the evidence parallels that for the late flowering plants in that three isolates are frequently at one extreme of the ranking order, this time at the bottom, and particularly so in the significant analyses. Once more there is no evidence of N.P.

6 D. J. HARBERD discontinuity in the characters and so no logical justification for dividing the isolates into the three smaller and the nine larger but, as before, if this is done there is virtually no evidence of differentiation within either group. It is of some interest to note that the isolate from Grain Burn (^ mile from the collecting area at Loganlee) is one of the three smaller isolates. Omitting this one and analysing for differences between the eleven Loganlee isolates there are still significant differences in nine of the forty-nine characters. Actually the Grain Burn isolate is not the extreme one at the small end of the character ranges the extreme isolate being from site 71 at Loganlee, closely followed by Grain Burn (site 80) and then the Loganlee site 72. We have seen earlier that there are no significant differences within this group of three isolates so that there appears to be no evidence for suggesting that the Grain Burn isolate should be treated as separate from at least some members of the Loganlee collection. It will be recalled that both the early and the late plants of the thirty-fiive-chromosome trial were recorded for the eight standard characters on three of the recording dates. There is also a complete record of the date of first ear emergence for the whole trial, so that there are twenty-five analyses possible for differences between the two plant types (omitting the Boghall isolate). There was apparently no difference in the lengths of the flag leaves, but nearly all of the other characters were very differently differentiated, twenty of the analyses being significant at the 0.1% level. Thus once again there is a very substantial body of evidence in favour of genetic difference between types that had already been recognized. INTERPRETATION AND DISCUSSION Interpretation The interpretation of 'cloned clone' trials has been discussed in an earlier paper (Harberd, 1961), when it was pointed out that significant differences did not necessarily accompany genetic diversity; nor can one assume that genetic uniformity automatically leads to trials showing no significant differences. We would, however, expect trials to tend in these directions. There were four distinct plant types among the Holcits mollis plants collected and these have been shown to be differentiated from each other in a highly significant manner in respect of various characters recorded. There seems no reason to doubt that this division refiects genetical differences. It is further believed that each of these plant-types is a single genotype but, before we can accept this interpretation, it is necesssary to consider the origin of the frequently recorded differences within the plant types. In the earlier paper it was pointed out that environmentally induced differences between parent plants of the same clone might be expected to persist in their vegetatively derived progeny for a short period of time, just how short a time being unknown. Any 'cloned clone' trial which contained only a single genotype might reveal significant differences between parent plants from different environmental pre-treatments if it was recorded too soon after establishment. Libby and Jund (1962) publish figures illustrating this 'hang over' from an earlier environment in 'cloned clone' trials, which they term 'c effects' variation. At the start of the present experiments the rapid rate of growth of H. mollis was seen as a distinct advantage in an experimental material. However, as a result of this rapid growth nearly all of the recording w as done in the year of planting and not, as in other grass materials, in the season following the first winter. Possibly then the Holcus was recorded too soon after the start of the experiment and 'c effects' were present. If

7 Genotype distribution in Holcus mollis 407 this were so, then, following Libby and Jund's argument, the variabihty between the ramets of each isolate was too small because, up to the time of division, the ramets had a uniform environment instead of a random assortment of the available environments. Consequently the error items in the analyses would be too small; had more realistic estimates been available fewer significant differences would have been recorded. It is interesting to note that if the errors of the analyses are increased by an arbitrary 50% (Table 2) the results conform closely to those that had been anticipated: the different 'genotypes' are still highly differentiated from each other, whereas within 'genotype' about 5% of the analyses between isolates are significant at the 5% level. More permanent intraclonal variability can arise in several ways. It can, for instance, be traced to differences of systemic infections (Harberd, 1962). This possibility was considered carefully in the present materials but it did not appear to be a likely cause of the differentiation observed. Mutations are relatively rare events, so that presumably they could not account for the variability within all three 'genotypes'. Finally there has been Table 2. Effect of increasing the size of the error items of the analyses by 50% Type of analyses No. of analyses Statistical significance I. Within'genotype': Differences between twenty-one- 82 Three significant chronnosome isolates Differences between early 49 Four significant, one thirty-five-chromosome isolates, at the i % level omitting Boghall Differences between late 33 None significant thirty-five-chromosome isolates II. Between 'genotypes'; Differences between Boghall 49 Seventeen significant, ten and other early thirty-five- at the i % level chromosome materials Differences between early 25 Twenty-one significant, eighteen and late thirty-five-chromosome at the o. i % level materials, omitting Boghall a considerable increase in interest lately in the possibility of intraclonal somatic segregation in higher plants (Breese, Hayward and Thomas, 1965; Simmonds, 1965). This was given no serious thought at the time and, even with the present improvement in knowledge, it would be very difficult to design an experiment to check whether it had taken place. From this discussion of the evidence it appears that the data presented contains no proof that there was genetic variability within the four plant types recognized; each might in fact consist of the vegetative progeny of a single seedling. No direct evidence can be produced for this contention it is most unfortunate for instance that the crosssterility test is inapplicable with this material. However, it is noteworthy that plants which could be seen to be different are much more significantly different in several of their measured characters than are plants which appeared alike. In fact if we reject the hypothesis that there are only four genotypes present in the collection and assume that there are several more, the evidence would still be difficult to interpret. We should then have to explain how the variability is distributed at two levels an obvious and highly significant level dividing the plants into four types, and a much less significant level which does not clearly subdivide the types.

8 4o8 D. J. HARBERD While the evidence is admittedly far from unamhiguous it does appear to be most likely that the four types recognized were the vegetative products of four colonizing seedlings. Discussion If the interpretation be accepted that only four genotypes were present in the collection there are two points that call for discussion, both arising from a comparison of the present findings with those of the earlier publications. The first of these concerns the apparent lack of genotypes with greatly restricted areal spread. It appeared from the earlier work, particularly on Festuca rubra (1961), that relatively few genotypes spread any great distance; most were greatly restricted in their spread. It seemed likely that these genotypes of smaller spread were those of recent origin. E. rubra has, of course, normal sexual reproduction so that new seed is produced every year. It would appear that very little of the new seed can successfully result in new genotypes in the sward. By comparison, new seed of thirty-five-chromosome Holcus mollis must be rare, since the twentyone-chromosome parent is itself rare, and if the chances of survival are as small, very few new seedlings can arise nowadays. Consequently nearly all genotypes of the thirty-fivechromosome race should be very old ones and we should expect each of them to cover a great area, just as appears to have been found. The second point for discussion concerns the sizes of the clonal patches. Each of the clones is at least 100 yd across, and one was isolated from points upwards of-^ mile apart. Even if we cannot accept that all of the early thirty-five-chromosome plants (excepting those from Boghall) were of the same genotype, it will be remembered that there was no evidence of any difference at all between the Grain Burn isolate and some of those from Loganlee and these alone constituted sites ^ mile apart. With its much greater vegetative spreading ability this distance for H. mollis is not unlike the spread of 240 yd published for Eestuca rtibra. The two cases are also alike in that in both species the territories concerned are obviously not ecologically uniform. There is probably a continuous cover of Holctts tnollis if one includes the very sparse occurrence of the species in some of the vegetations. Alternatively, if we assume that the species would have spread most rapidly in the vegetation where it is most abundant, that is in the flushed grassland, its route from one side of the hill to the other would be down the slope and then up the valley leading to the pass a total distance of possibly well over a mile. REFERENCES BEDDOWS, A. R. & JONES, K. (1953). Chromosome numbers in Holcus mollis. Nature, Lond., 171, 938. BREESE, E. L., HAYWARD, M. D. & I'HOM.AS, A. C. (1965). Somatic selection in perennial rye grass. Heredity, Lond., 20, 367. HARBERD, D. J. (1961). Observations on population structure and lonkevits' of Festuca rubra L. Nero Phytol., 60, 184. H.WBERD, D. J. (1962). Some observations on natural clones in Festuca ovina. Neio Phytol., 61, 85. JONES, K. (1958). Cytotaxonomic studies in Holcus. I. The chromosome complex in Holcus mollis L. New Phytol., 57, 191. LiBBY, W. J. & JuND, E. (1962). Variance associated with cloning. Heredity, Lond., 17, 533. SiMMONDS, N. W. (1965). Somatic segregation of the spectacle pattern on potato tubers. Heredity, Lond., 20, 277.

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