Himantoglossum Adriaticum</Emphasis>

Acta Botanica Hungarica 50(3–4), pp. 257–274, 2008 DOI: 10.1556/ABot.50.2008.3–4.3

GROWTH OF HIMANTOGLOSSUM ADRIATICUM AND H. CAPRINUM INDIVIDUALS, AND RELATIONSHIP BETWEEN SIZES AND FLOWERING

J. BÓDIS1 and Z. BOTTA-DUKÁT2 1Georgikon Agricultural Faculty, University of Pannonia H-8360 Keszthely, Deák F. u. 16, Hungary; E-mail: [email protected] 2Institute of Ecology and Botany, Hungarian Academy of Sciences H-2163 Vácrátót, Alkotmány u. 2–4, Hungary; E-mail: [email protected]

(Received 14 January, 2008; Accepted 17 April, 2008)

The long-term protection of plant species is impossible without a comprehensive and thorough knowledge of their biology. This paper deals with some of the important aspects of the biology of two strictly protected species, Himantoglossum adriaticum and H. caprinum. The changes of the leaf area of plants were measured with a non-destructive method and the growth pattern was investigated during the vegetation period. It was also studied how the plant size of individuals changed from year to year, and what kind of connection there was between the plant condition and the flowering. Large size plants of both species were characterised by intensive growth rates in autumn and spring, their growth stopped in winter. Often they lost some of their leaf areas because of damage from insects (mainly in the case of H. caprinum), and frost (almost exclusively in the case of H. adriaticum). In some years the medium and small size plants were characterised by similar growth pattern to those of large size ones, but more often their growth did not stop in winter, though their growth was not so fast. The medium and small size H. caprinum individuals showed an intense autumn growth only in one vegetation period, which characterised both large size individuals of H. caprinum and all individuals of H. adriaticum. The constant annual rate of growth, which may have been slowed meaning that the leaf number did not grow, but it did not stop either, was detected in hardly more than 10% of individuals with both species. In the case of H. adriaticum the critical size for flowering seemed to be 50 cm2, which was usually reached in the four-leaf stage of the rosettes. The same value was 100 cm2 in the case of H. caprinum and it needed at least a six-leaf basal rosette. The leaf number and the leaf area of reproductive plants had been larger for already two years before flowering took place, than those of the plants that remained vegetative.

Key words: critical rosette size, flowering, growth pattern, Himantoglossum adriaticum, H. caprinum, leaf area

INTRODUCTION

Because of their beauty and special life history orchids have been quite frequently studied and are well-known, but most of the knowledge is reduced only for their presence data and general characteristics. However, hardly any- thing is known about the biology of Hungarian orchids, even that of strictly protected species. The long-term protection of plant species is impossible without a comprehensive and thorough knowledge of their biology. Because a lot of plant characteristics are size-dependent, the size and its changes is very important in the case of many species. The facts, that many monocarpic perennials have a critical plant size for flowering (Harper 1977) and the length of the vegetative growth period depends on the environmental conditions have been well-known for decades (Hirose and Kachi 1982). Lots of characteristics have been used as a measure of size: plant height, rosette diameter, the number of leaves in the rosette or in the whole plant, dry mass, etc. Relationships were established in the case of many monocarpic plant species between rosette sizes and the probability of dying, remaining vegetative or flowering. The fate of a rosette appeared to be inde- pendent of its age. The probability of flowering increased with increasing size, growth over the period immediately prior to bolting, and age (in order of importance is variabled) (Gross 1981, Eliaš 1985, Klinkhamer and Jong 1987, Klink- hamer et al. 1987). There are related results in connection with endangered Hungarian native plants, too. During the demography study of Digitalis lanata, strictly protected species in Hungary, Pozsonyi (2000) found positive correlation between rosette diameter and number of flowers. Vegetative plants of Ferula sadleriana, the interglacial relict endemism of the Carpathian Basin, showed two size classes: one with 1–2 leaves and one with 3 or more leaves. The rate of those plants that belong to the larger groups showed a similar pattern with that of the reproductive plants (Kalapos 1998). Not only the monocarpic perennials, but polycarpic species also have a critical plant size for flowering. The plant size is very important for orchids, too. In the case of Cypripedium acaule the probability of remaining dormant below ground in a given year was strongly dependent on plant size in the previous year. Furthermore, the length of the dormancy period (one to several years) was a significant and inverse function of plant size just prior to dormancy (Primack and Stacy 1998). Those studies were more important for us, which dealt with orchids which have tubers, similarly the Himantoglossums. Wells and Cox (1989) dem- onstrated that Ophrys apifera has a minimum rosette size, required for flowering, the probability of flowering increases with increasing rosette size and

Acta Bot. Hung. 50, 2008 GROWTH OF LIZARD ORCHIDS 259 number of leaves. Kindlmann and Balounová (1999, 2001) summarised and critically reviewed the hypotheses that had been put forward as explanations of the irregular flowering phenomenon of orchids: irregular flowering was at- tributed to costs associated with sexual reproduction, to herbivory or to the chaotic behaviour of the system represented by difference equations described growth of the vegetative and reproductive organs. None of these seemed to fully explain the events of a transition from flowering one year to sterility or failure the next year. Data on seasonal growth of leaves and inflorescence of two terrestrial orchid species Epipactis albensis and Dactylorrhiza fuchsii were reported, too. The influence of age, size, reproductive effort and climatic conditions on flowering variability of Himantoglossum hircinum were examined using data collected in a long-term project (1976–2001) in Germany. Future size after flowering to quantify costs of reproduction was also studied. The probability of flowering was strongly determined by plant size, while there was no significant influence of age class on flowering probability of the population (Pfeifer et al. 2006). This paper deals with some important aspects of the biology of two strictly protected species, Himantoglossum adriaticum and H. caprinum, the Adriatic and the Purple Lizard orchids, respectively. Our questions were: – 1. How do the leaf number, the leaf area, and the growth rate of H. adriaticum and H. caprinum individuals change within one year and from year to year? – 2. Is there any difference between the growth patterns of the species? – 3. Is there any relationship between the general condition of the plant and its flowering? Is there a critical size for flowering and if there is, what is it?

MATERIAL AND METHODS

Species characteristics

Because of the uncertain taxonomic situation of the Himantoglossum genus, all Himantoglossum presences in West and Central Europe were believed to be those of H. hircinum, which might have taken differences in subspecies only. There was a very important event in taxonomy of the genus when H. Baumann found a population on the Istrian peninsula in 1973 and declared that it was a new species, which was accepted and its name became H. adriaticum, which refers to its locus classicus. According to Baumann, this new

Acta Bot. Hung. 50, 2008 260 BÓDIS, J. and BOTTA-DUKÁT, Z. species occurs in Central and North Italy, Croatia, Slovenia, South and West Austria, West Hungary and Slovakia (Baumann 1978). What is more, the presence of other species, the H. caprinum was also reported by Baumann in the area of Hungary, which is almost the northwest boundary of H. caprinum’s distribution area (Baumann and Künkele 1982). Füller (1981) and Buttler (1986) agree with Baumann about the occurrence of H. adriaticum and H. caprinum in Hungary. Transdanubia (western part of Hungary) is the territory where the eastern margin of distribution of H. adriaticum and the western boundary of distribution of H. caprinum overlap. H. hircinum occurs only in western and southern Europe according to the opin- ions of the authors mentioned above. Until the 1980s all the Himantoglossum specimens found in Hungary had been determined as H. hircinum. Since that time the taxonomic situation of every population has been identified (Dénes et al. 1993, Molnár et al. 1995, Bódis and Almádi 1998). Although the two above-mentioned species are close to each other, it is not difficult to identify the differences. Both are CITES species and strictly protected plants in Hungary. Himantoglossum is a perennial genus that survives from one year to the next via tuber. Its flowering time is in mid-summer and its leaves disappear during the dry and hot months. Unlike the majority of the Hungarian native species, its leaves appear in early autumn (depending on the rainfall) and they have green leaves during the winter. Their vegetation period lasts from Sep- tember until June–July that is their plant year. Himantoglossum species have basal rosettes. The size of the leaves be- comes smaller and smaller upwards from below. H. adriaticum is the smaller, more graceful species (Molnár 1999a). We found that the highest leaf number was 7, the large size individuals (with at least 4 leaves) have 40–110 cm2 as characteristic leaf area. The highest reported leaf number of H. caprinum was 9, the typical leaf areas of large size individuals (having at least 6 leaves) were 100–160 cm2. Because of the varieties of the basal rosette sizes it was supposed, that the different size meant different growth patterns, so the plants were di- vided into 3 groups on the basis of the number of basal leaves (Table 1), and the groups were analysed separately.

Table 1 Size classes of the species Species Small size plants Medium size plants Large size plants H. adriaticum 2 leaves 3 leaves 4 or more leaves H. caprinum 2 leaves 3–5 leaves 6 or more leaves

Acta Bot. Hung. 50, 2008 GROWTH OF LIZARD ORCHIDS 261

Localities

The locality of H. adriaticum studied is situated in the western part of Hungary, in the Keszthely Hills, at the boundary of the village Gyenesdiás and Keszthely town. The orchid population was found by I. Szabó, on both sides of the Pilikán-Szoroshad minor road, in dolomite grassland, on the edge of the forest (Szabó 1987). The study site of H. caprinum is situated near the village Kisharsány, in the southern part of Hungary. The site is situated on Fekete Hill, which is the part of the Villány Hills. This locality used to be a grazing area but 20–30 years ago it was abandoned. The former grazing area has changed considerably due to encroachment by scrubs and growth of ash trees, so the largest part of the orchid population live in the shade after the development of tree canopy (Csere et al. 2004).

Methods

This paper describes 3 year-observations, from 1999 to 2002, made on the behaviour (vegetative or reproductive) and performance of two Himantoglos- sum species, including measurements of basal rosettes leaf number and area during their vegetation period, 8–12 times yearly. There is no commonly accepted method for the measurements of the orchid’s leaf area. Investigations based on the direct measurements used destructive method (Wells and Cox 1989), which are to be avoided because the Himantoglossums are strictly protected species and their population sizes are just too low to apply destructive methods. The “Montgomery-formula” is known as one of the plant-friendly non- destructive methods (Willems 1982, Willems and Ellers 1996), which is the product of the leaf length multiplied by the leaf width at its broadest part and a constant. The determination of the constant is a special problem for each species, and its accuracy is even more doubtful, particularly in those cases when the shapes of the leaves are as varied as in the case of Himantoglossums (Molnár 1999b). During the preliminary studies, we tried several non-destructive methods to measure the leaf growth. The number of the leaves of the basal rosettes was a useful but not a sufficient indicator. The length and width of leaves were difficult to measure first of all because many of them were damaged anyway (insect bites, frost spots, etc.), secondly because of their considerable bend, when we tried to lay them on an even surface to be able to measure them, they simply were broken down.

Acta Bot. Hung. 50, 2008 262 BÓDIS, J. and BOTTA-DUKÁT, Z.

Finally the method used for determination of the photosynthetically active leaf area was the following: after the appearance of leaves in September paper sheets were put under the leaves of the basal rosettes of the individually marked plants and the leaves were outlined. The damages were indicated, too. This was repeated each 3–4 weeks (depending on snow conditions in winter). The “drawings” were cut out and the actual leaf areas of samples were measured in the laboratory using a Li-Cor leaf area meter. Unfortunately, this method was not good enough to measure the assimilating area of the flowering stalk or the upper leaves, embracing the stem. The measurements of the upper leaves proved to be so unreliable that we decided to neglect these data. Our data refer to basal rosettes in all cases.

Data analyses

Because of the above-mentioned sampling problems there was no possibility to apply such growth characters which consider the changes in biomass, so Leaf Growth Rate (LGR) was calculated, which is analogous with Crop Growth Rate (CGR). We calculated the average values of growth speed between two data points and plotted it against the medium of the time interval. The leaf number, the maximum leaf area and the maximum value of Leaf Area Duration (LAD) (at the end of vegetation period) were used to character- ise the condition of the plants. The LAD (day × cm2) is the time integration of the leaf area (i.e. how large leaf area was active for how long time) up to the given point of time, and according to the references it is a very suitable tool for the measurement of the vigour of individuals (Hunt 1978). Statistical analyses of the morphometric data of flowering and non-flowering (vegetative) individuals were possible only in the case of H. adriaticum and only in 2002. There were not enough flowering plants in the other years or of the other species. A Mann–Whitney U-test was applied for comparison (Sprent and Smeeton 2001).

RESULTS

Annual growth of individuals

Himantoglossum adriaticum – The leaves of the Lizard orchids appear after the late August, early September rainfall, usually in September (that is why the 1st September is the 1st day in all figures). There was an intensive growth period after the autumn appearance in all individuals. The intensive growth lasted until November, and then the growth of individuals in different size classes became different. The large size plants showed stagnation or only a

Acta Bot. Hung. 50, 2008 GROWTH OF LIZARD ORCHIDS 263 slight growth until the end of March in all three years. During this time, the as- similation area often was reduced because of the damages. The large size plants presented very powerful leaf growth in all of the three years since the end of March until the arrival of the warm period in May, the end of the growing period of orchids (Figs 1–2). The large size plants in all three years fol- lowed the next growth pattern: intensive growth period in September–Octo- ber and in April, stagnating or slight growth in winter. There was a stagnation period in the case of the medium and small size plants only in the first and third vegetation periods after the intensive growth phase of autumn. In the second sampling year, the growth was characterised by an almost constant rate in both size classes, there were no considerable differences between the autumn and spring or the winter phases. In the subse- quent year, their intensive growing and stagnation periods were the same as in the case of large plants. The medium size plants grew nearly as fast as the large ones, only the small size plants grew more slowly (Figs 1–2). The intensive growth period of the autumn is caused by the sudden leaf appearance. The fast growing period of the first leaves lasted until November, after that slower pace or rather significant leaf area decrease was measured. Although the first leaves often got damaged, their areas remained the largest during the whole vegetation period. During further rosette development, the leaves of the upper layers overshadow the first leaves, which induced their se- nescence. The second leaves were smaller and their autumn growth rate was as fast as that of the first ones. Their leaf area reached only half or two-thirds of the first leaves by winter and later. At the end of the vegetation period, they had nearly the same size, because the first leaves had lost some of their area but the second leaves retained it. The second leaves appeared together with the first and retained their area during the whole vegetation period. The decrease of their area was not typical, moreover in the second spring there was a significant improvement. A definite spring growth characterised the third and the upper leaves causing a very strong increase in the photosynthetically active leaf area. The appearance of the third leaf happened right after the emergence of the first two in autumn, the fourth was found the soonest in November, the fifth was likely to emerge any time from November until March. Under favourable weather conditions, the basal leaf rosette of H. adriaticum can be possessed of seven leaves. The sixth and the seventh leaves appeared in late spring (Fig. 3). Himantoglossum caprinum – We measured only medium and small size plants in the first year. They grew at a constant speed during their vegetation period. In this year their growth rate did not decrease in winter. The growth was only a bit less intensive than in autumn or in April (Figs 1–2).

Acta Bot. Hung. 50, 2008 264 BÓDIS, J. and BOTTA-DUKÁT, Z.

a 80

2 40 cm

0 28.08.1999 15.03.2000 01.10.2000 19.04.2001 05.11.2001 24.05.2002

b 160

140

120

100

2 80 cm

0 28.08.1999 15.03.2000 01.10.2000 19.04.2001 05.11.2001 24.05.2002 10.12.2002

Fig. 1. Temporal changes of the leaf area (LA) in the three size classes: a = H. adriaticum, b = H. caprinum

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a 2

1.6

1.2

0.8

0.4 1 - 0 .day 2 -0.4 cm

-0.8

-1.2

-1.6

-2

-2.4 01.09.1999 19.03.2000 05.10.2000 23.04.2001 09.11.2001 28.05.2002

b 3

1 -1

.day 0 2 cm

-1

-2

-3 01.09.1999 19.03.2000 05.10.2000 23.04.2001 09.11.2001 28.05.2002

Fig. 2. Temporal changes of the relative leaf growth rate (LGR) in the three size classes: a = H. adriaticum, b = H. caprinum

Acta Bot. Hung. 50, 2008 266 BÓDIS, J. and BOTTA-DUKÁT, Z.

The second and third years there were characterised by intensive growth periods in autumn and in spring in the case of H. caprinum, too. The large size plants grew more slowly in the second vegetation period, their area decreased a little during wintertime in the third year. The intensive spring growth re- sulted in a considerable expansion of the area that was reached by the begin- ning of winter (Figs 1–2). The small and medium size plants did not manage to increase their leaf area so considerably by early winter. Their growth was constant but very slow during the second winter. In the third year the plants suffered large damage in winter and the spring growth was able to compensate only for that. According to LGR – which measures the absolute growth rate – the autumn and spring growth period was not so intensive as in the case of the large plants. The small and medium size plants were characterised by a rather constant growth rate (Fig. 2). The reason for this was that the leaves’ appearance had a special rhythm. The first three, or rather four leaves emerged still in autumn, and the medium and small size plants did not develop more leaves in spring, so they had no intensive spring growth period, which, however, was present in the case of large ones (and caused another intensive growth period). The leaf area of the first leaves grew continuously only in the first year, while in the second and third years the leaf area decreased from December because of the damage. That was true for the first three leaves in the third year. The fourth and fifth leaves appearing still in autumn and the further leaves emerging in spring compensated for the damage (Fig. 3). In that way, large plants were able to increase their total leaf area because the new leaves compensated for the damage suffered by the first leaves. It was not possible for smaller plants because of their lower leaf number. That is why the damage of the first leaves in the case of medium and small size plants had such a crucial effect in their lives.

Year to year size changes of the individuals

Himantoglossum adriaticum – It is obvious that the leaf number of the large size plants cannot increase year by year; nevertheless, we were surprised to find that in most cases the leaf number decreased. The individuals of H. adriaticum did not retain their leaf number that they had reached in the previous years (Table 2). On the other hand, a dramatic decrease was observed in only one case: after having a 5-leaf basal rosette in one year the same plant managed to develop only a 2-leaf one. In this case, we may not ignore the possibility that another neighbouring tuber sprouted instead of the marked plant having turned into dormant in the next year. The changes of the leaf number were most balanced in the case of the medium size plants. We observed that the leaf number decrease was rare, gener-

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1. leaf 4. leaf 7. leaf 2. leaf 5. leaf 8. leaf a 3. leaf 6. leaf 9. leaf

20 18 16 14 12

2 10 cm 8 6 4 2 0 28.08.1999 15.03.2000 01.10.2000 19.04.2001 05.11.2001 24.05.2002 b 25

15 2 cm 10

0 28.08.1999 15.03.2000 01.10.2000 19.04.2001 05.11.2001 24.05.2002

Fig. 3. Temporal changes of the area of the leaves: a = H. adriaticum, b = H. caprinum

Acta Bot. Hung. 50, 2008 268 BÓDIS, J. and BOTTA-DUKÁT, Z.

Table 2

Changes of the leaf number (Lno) and the maximum leaf area (LAmax) in the case of H. adriaticum, in the percentage of the individuals investigated Size classes Increase Constant Decrease

Lno Lmax Lno Lmax Lno Lmax Large size plants 38 63 19 0 44 37 Medium size plants 50 93 43 0 7 7 Small size plants 30 60 50 30 20 10 All plants 40 73 35 7 25 20 ally it did not change or it rather increased. The decrease of leaf number was more often detected among the small plants, probably because of their easier vulnerability. Regarding all H. adriaticum plants, in every fourth case there was a decrease of leaf number, which means that growth is not an “easy task” for Liz- ard orchids. The question arose whether the tendency observed in leaf number change would be similar or the same as that of the leaf area in three years of study. The answer is a definite “yes”, the trend proves to be similar in the changes of leaf area too, in every size category (Table 2). Medium size plants were able to increase their leaf areas in almost every case, but the large and small size plants often remained below the leaf area that they had reached the year before. The leaf area sizes were influenced by the plant reproduction, too. Only in one case did it happen that after flowering the leaf number and the leaf area increased in the next year, but there was a decrease in four cases. Unfortunately, the sampling number was too small to draw more general conclusions. Himantoglossum caprinum – The proportion of year-by-year increase of leaf number was more or less the same in every size class characterising one third of the individuals (Table 3). There was no decrease in the case of large size orchids, their leaf number was invariable. As the plants marked individu-

Table 3

Changes of the leaf number (Lno) and the maximum leaf area (LAmax) in the case of H. caprinum, in the percentage of the individuals investigated Size classes Increase Constant Decrease

Lno Lmax Lno Lmax Lno Lmax Large size plants 29 80 71 0 0 20 Medium size plants 33 23 28 0 39 77 Small size plants 29 45 42 0 29 55 All plants 31 38 41 0 28 62

Acta Bot. Hung. 50, 2008 GROWTH OF LIZARD ORCHIDS 269 ally were not too large and/or old to have no chance to increase their leaf number, the unfavourable weather and deteriorating habitat conditions may have induced to the cessation of the growth. These reasons might also contribute to the lack or conspicuous scarcity of reproduction in all three years (there were only three flowering plants and only in the third year). The leaf numbers were decreased in 40% of medium size plants, while about 30–30% of them increased or stopped growing. In the case of small size plants, the changes of the leaf number were similar to medium ones, though the cessation of the growth was observed the most frequently. The large size plants were more generally (80% of sampling number) characterised by the growth of the leaf area, even if the leaf number did not increase (Table 3). In the case of the medium size plants, however, only 23% of them enlarged their leaf area. The rate of increases and decreases was nearly the same among the small size plants. Among the large plants, the constant leaf number meant increasing leaf area while in the case of medium size plants it referred to leaf area decrease.

The comparison of flowering and non-flowering plants

With all the size fluctuations of large size H. adriaticum plants detailed above, we found that the individuals flowering in the year 2002 had been sig- nificantly larger in the previous two years as well (Table 4). The correlation between the size of the leaf area and the leaf number influenced the reproduction ability of individuals. Based on the investigations of H. adriaticum individuals, it was clear that the medium size plants which had only 3 leaves at the begin- ning of our study developed no flowers in their 4-leaf stage either. On the

Table 4 Results of the statistical comparisons of the flowering and non-flowering H. adriaticum individuals Growth period Characteristic Mann–Whitney U-test Exact probability of Type I errors (p-values) for one-side test 1999/2000 leaf number 8.00 0.000532 2000/2001 leaf number 12.50 0.002048 2001/2002 leaf number 1.50 0.000016 1999/2000 max. leaf area 4.00 0.000095 2000/2001 max. leaf area 12.00 0.002048 2001/2002 max. leaf area 4.00 0.000095 1999/2000 max. LAD 2.00 0.000032 2000/2001 max. LAD 10.00 0.001080 2001/2002 max. LAD 5.00 0.000151

Acta Bot. Hung. 50, 2008 270 BÓDIS, J. and BOTTA-DUKÁT, Z. other hand, one-third of the plants that had 4-leaves and retained that number produced flowers in that second 4-leaf period. It seems that the 3–4 leaf size is a special “border-line” phase, that individuals may be young adults or subsenile matured plants. The minimum size of rosette for flowering is very small in the case of H. adriaticum. Even an individual of 29 cm2 diameter was able to flower. How- ever, it was the only individual under 50 cm2 that produced a flower. The critical size for flowering seems to be 50 cm2, which is usually made up by at least 4 leaves. One-third (29%) of even those above that critical size (50 cm2) remained vegetative in that plant year. When we investigated the size classes separately (those above 50 cm2), we found that one quarter of the large size plants and half of the medium size plants did not develop flowers in that plant year, respectively. There were flowering H. caprinum individuals only in the third year, the smallest size rosette was 108 cm2, the largest one was 248 cm2. The size of the basal rosette was in 10 cases larger than 100 cm2, but there were flowers only in four cases. The flowering is expected above 100 cm2 (at least with a 6-leaf basal rosette), its probability was 40% during the investigation period and in this size.

DISCUSSION

The results suggest that the growth differ in the case of large size as well as medium and small size plants in both Himantoglossum species. Large size plants were characterised by intensive growth rates in autumn and spring, their growth stopped in winter, and often they lost some of their leaf area because of damage from insects, molluscs and frost. In some years the medium and small size plants were characterised by similar growth pattern to those of large size ones, but more often their growth did not stop in winter, though the growth intensity was slowed down. In the cases of the medium and small size H. caprinum individuals, there was an intense autumn growth only in one vegetation period, which characterised by both large size individuals of H. caprinum and all individuals of H. adriaticum. The area of leaves developed in autumn often was reduced in winter because of the frost and animal nibbling. The whole deficit was larger in the case of H. adriaticum. H. caprinum was more often damaged by nibbling, while H. adriaticum showed more frost damage. The difference in the size of frost damage may not be really explained by the differences of their habitats, but rather by the differences between the species (a lot of H. caprinum individuals grew intensively in extremely cold weather conditions; the mean temperature in December 2001 was –4 °C). The winter growth and almost the total lack of frost

Acta Bot. Hung. 50, 2008 GROWTH OF LIZARD ORCHIDS 271 damage are in connection with its distribution area. H. caprinum is an East Bal- kan floral element, and therefore it is better adapted to harder winters, than H. adriaticum, distributed from the medium of the Apennine peninsula to Slovakia. This hypothesis is supported by the frequent occurrence of frost damage of the leaves of the H. hircinum, which is native to the Atlantic Mediterranean area (Good 1935, Füller 1981). The nibbling damage of H. caprinum was especially large in early spring and when the winter was mild, with the majority of the perennials and the trees having no leaves for the phytophags to feed on. Wells and Cox (1991) states that larger leaves of Ophrys apifera suffer nibbling damage more often and more severely, which proved very true for H. caprinum as well – what is more the larger basal rosettes seemed to be more threatened, then the smaller ones. The large size plants were able to compensate for their large leaf area damage in spring, in their intense growth period, when they developed new leaves. All leaves of the medium and small size plants emerged in autumn, so they had no chance to compensate for their damage. The medium size plants suffered the largest nibbling damage, because they were large enough to at- tract the herbivores, but they did not develop larger size leaves in spring. Species that produce leaves in autumn and remain green do not suffer from summer droughts because they have no above-ground organs at that time (Wells and Cox 1989). But as we saw above, in winter and springtime the orchids may suffer serious damage, so these seasons are critical periods for the individuals. The constant annual rate of growth, which may have been slowed meaning that the leaf number did not grow, but it did not stop either, was detected in hardly more than 10% of individuals with both species. That means: one group grew their leaf number year-by-year, while the other group may have failed to bring out new leaves in one year, but then resumed development without loss. The importance of the leaf number decrease or increase is that it determines whether the plant remains in its size category or changes towards the larger or the smaller size categories. For H. adriaticum the most interesting status is that of the 4-leaf plant, which is the starting point of the large size in this species. The leaf number of the largest plants (with 6–8 leaves) was reduced by the end of the study, but 35% of them remained in the large size category. The starting point of the large size in the case of H. caprinum is the 6-leaved status, which only half of the largest individuals were able to retain. Similarly to other orchids with tubers (Willems 1982, Wells and Cox 1991), on the basis of our study we may conclude that both Himantoglossum species need to reach a critical size to be able to flower. However, we have not been able to verify the statement, that the given year reproduction can be surely estimated on the basis of the number, size and the shape of the basal

Acta Bot. Hung. 50, 2008 272 BÓDIS, J. and BOTTA-DUKÁT, Z. leaves (Molnár 1999b) because we found that only 71% (H. adriaticum) and 40% (H. caprinum) of those individuals flowered which were above the critical size for flowering. It is worth mentioning that we did not take the shape of the leaves into consideration. The critical size for flowering is different for the two species. In the case of H. adriaticum the individuals with more than 50 cm2, which usually reach this stage with 4 basal leaves can be realistically expected to flower. The critical size of H. caprinum is 100 cm2, however, it needs at least 6 basal leaves. The habitat of H. caprinum is a mostly shady area because of the dense bush canopy that may contribute to leaf number increase. An Orchis simia population (in the Netherlands) proved that the plants, living under a dense, higher vegetation, need larger basal rosettes to be able to flower than those, which live in a sunny habitat, because the same photosynthetic output of carbohydrates can be made with larger leaf areas as a consequence of the weaker light conditions (Willems and Ellers 1996). The difference between the Himantoglossum species is caused by not only the characteristics of the species but the differences in the light conditions of their habitats as well. To distinguish the two effects a com- mon garden experiment would be needed, which is impossible in the case of that strictly protected species that are difficult to propagate. The size of the basal rosette after flowering helps to quantify the “cost” of reproduction. In the case of H. adriaticum only once did it happen that there was an increase in the leaf number and leaf area in the next year, after flowering, but there were decreases on four occasions. The tendency is similar in the case of H. hircinum, which, after flowering, also decreased its size and reduced the probability of flowering again (Pfeifer et al. 2006). Otherwise, neither a significant decrease in leaf area, nor a reduced likelihood of flowering was observed following 1 or 2 years of inflorescence production in the case of another orchid species, Spiranthes cernua (Antlfinger and Wendel 1997). The current year reproductive efficiency depends on primarily the amount of carbohydrates in the tuber stored during in the previous vegetation period. The amount of accumulated carbohydrates is determined by the size of the cu- mulative leaf area, which the plants produce before April (Wells and Cox 1989). We may conclude that the flowering plants had larger leaf numbers and area throughout two years before reproduction, than the vegetative. It means despite the twin-tuber life form, there is a possibility to accumulate carbohydrates, probably in a way that from a larger tuber a larger plant develops, which again can produce even larger tubers. On this basis, the strategy of re- source management of Himantoglossums does not differ from that of other perennials, which are able to accumulate carbohydrates, if they need carbohydrates even for many years before the flowering. It was previously proved, that the probability of flowering is influenced by the plant sizes in the given

Acta Bot. Hung. 50, 2008 GROWTH OF LIZARD ORCHIDS 273 and the previous year (Carey and Farrell 2002, Pfeifer et al. 2006), but the (possible) effects of the year prior to these two last years have not been published yet.

* Acknowledgements – We are pleased to acknowledge the help in the field and during the measurement given by Szilvia Csere and Ildikó Szalóky. We would like to thank Edit Molnár for her valuable comments on an earlier draft on this paper. The study was supported by the OTKA F029484.

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