Received 19 March 2014; revised 19 April 2014; accepted 25 April 2014

ABSTRACT

A strong effect of climate on phenological events in conifers has been documented by several stu- dies. To study adaptation of reproductive processes in Abies sibirica Ledeb. to changing environ- ment, the phenology of the development of the species male reproductive cones at introduction was studied. Phenological shift in Abies sibirica meiosis and pollination was observed. An earlier start of male bud reproductive development is founded in V.N. Sukachev Institute of Forest Arboretum resulted in increasing meiosis and pollen irregularities. Insufficient high quality pollen in the species at its pollination stage may be a major factor responsible for the incapability to produce the viable seeds in quantities sufficient for pollination in seed gardens. Responses of the fir male cone development to the current environmental conditions at the Arboretum may be considered as a model of adaptation of the species to climatic changes.

Keywords:

Abies sibirica Ledeb., Forest Arboretum, Male Buds Development, Phenological Shifts

1. Introduction

Several studies have documented the strong effect of climate on phenological events of conifers, such as cone initiation, meiosis, pollination and seed development [1] - [6] . Due to their longevity, multiple reproductive cycles and exposure to large year-to-year environmental heterogeneity within their prolonged lifespan, conifers have improved their phenotypes to tolerate changes in climatic conditions [7] and formed a number of climo- types. Ecological conditions experienced by parents could directly influence the progenies in succeeding genera- tions [8] - [13] . The temperature regime which prevailed during megagametogenesis, zygotic embryogenesis and seed maturation induced shifts in the embryo development program to result in long-term phenotypic changes affecting the growth cycle of the progeny [14] . Provenance trials have shown that southern and western climo- types of conifers begin to grow at lower air temperature and their seasonal developmental stages are shifted to an earlier period of time and to lower accumulated temperature as compared to northern and eastern climotypes [15] - [18] . Even slight changes of air temperature and relative humidity can have marked effects on climotypes growth, ontogenesis, and reproductive system. High sensitivity to new conditions is characteristic of the juvenile plants and of the beginning of the reproductive period during sporogenesis and gametogenesis adaptation to new temperature and light regimes [19] [20] .

Studying the adaptation of reproductive processes in plants to changing environmental conditions is crucially important. It has been shown [21] [22] that insufficient high quality pollen in the species at its pollination time may be a major factor responsible for the incapability to produce the viable seeds in quantities sufficient for pol- lination in seed gardens. Because male reproductive organs are very sensitive to small variations in climate, es- pecially to temperature, phenological records can be a useful proxy for temperature in historical climatology, especially in the studying climate change and global warming.

The aim of the work is to study phenology of Abies sibirica Ledeb. male reproductive cones development at introduction.

2. Material and Methods

The study was conducted in 2002-2004 and in 2011. Two study plots were laid out in fir stands growing in the East Sayan Mountains and one plot in the arboretum of the V.N. Sukachev Institute of Forest. The study sites were found in the extremely continental climate having an average annual temperature of 0.8˚C. The weather information provided by Krasnoyarsk Weather Service showed that April and May air temperatures in 2002 and 2003 were within their average multi-year range, whereas the 2004 spring came 7 - 12 days later than usual and in 2011 late frosts occurred [23] .

The sampled fir trees grew in the low-mountain zone, more specifically in the Laletina River valley (450 - 520 m a.s.l.), in the middle mountains (the Upper Kaltat, 640 - 720 m a.s.l.) and in V.N. Sukachev Institute of Forest arboretum which located at the wildland/Krasnoyarsk town interface, on a terrace ( 275 m a.s.l.) on the left bank of Yenisei River. Notwithstanding its proximity to Krasnoyarsk, the arboretum is not affected by in- dustrial pollution, as it is situated outside the prevailing industrial emission transfer [24] . Siberian fir saplings were transferred to the arboretum in 1977 from the forest nursery of Siberian Institute of Fruit Growing (Barnaul, Altai, Altai Region, Russia) found in a moderately continental climate with the average annual temperature be- ing 4.0˚C). They were grown from seeds collected from Siberian fir stands naturally occurring in Altai region.

Pollen cones of Siberian fir were collected at the dormancy stage (in October), and at 1 - 3 days intervals during the pollen development period (from mid-April to mid-May), and at the time of pollination (late May). Samples were fixed in alcohol mixed with acetic acid at a ratio of 3:1 for one day after their appropriate trimming or dissection. Then they were dehydrated in a xylol alcogol series [25] , embedded in paraffin tissueprep, sectioned at 6 - 12 μm and stained in iron-hemotoxylin. A number of samples were transferred to 70% alcohol, and stained with acetohematoxylin. More than thirteen thousands of developing pollen grains were tested using Micromed-2 microscope. On each plot, the pollen cones were collected from 10 - 15 sample trees and the pollen was ana- lyzed for viability (in vitro % germination and tube length, in μm) and size (two perpendicular diameters of grain body, and length and height of air sacs, μm). Up to 300 pollen grains were analyzed per sample tree. STATISTICA 7.0 [26] was used to calculate descriptive statistics including the standard deviations and the con- fidence levels of the differences.

3. Results

3.1. Pollen Bud Development

Siberian fir pollen cones develop during 12 month, from late June till the end of May. Pollen-cone buds usually differentiate from axillary buds that have formed in axils of leaves on the sides and lower surfaces of the shoots (Figure 1). The pollen cones of sampled trees were clustered (up to 35 cones in a cluster). Each pollen cone in a cluster occurred in a at leaf base and was sessile. The pollen buds became conspicuous at the end of June-be- ginning of July and developed until early October, since when they became totally enclosed in imbricate scales. They were made up by large numbers of spirally arranged bisporangiate microsporophylls. All microsporophylls were initiated during the first month of pollen bud development. Sporogenous tissue in microsporangia was

formed during August. In late August, microsporangia in sample fir trees increased to be 212 × 147 mkm in size. They stayed dormant at the premeiotic stage of the pollen mother cells (PMC), with tapetal cells remaining un- inucleate (Figure 2(a)).

3.2. Time of Meiosis and Pollen Development

Pollen buds in sample trees enlarged in spring and developed during May (Table 1). The rate of development of the post-dormancy pollen cones strongly depended on site conditions and air temperature; it increased in warm weather and decreased when it became cooler.

In the East Sayan study populations meiotic division usually starts in the pollen mother cells (PMCs) at ac- cumulated temperatures ranging 40.8˚C to 47.8˚C during the first ten days (in low mountains) and the 10th through the 20th (middle mountains) of May (Figure 2(b)). In 2002, 2003 and 2011 in the low mountains mei- osis was observed to begin on the 5th and 7th of May, and lasted one - two days (Figure 3). In 2004, meiosis started as late as May 13-15. This delay was presumably due to the cold spring of 2004. Meiotic division lasted for 2 - 3 days (from diakinesis through the tetrad stage). The time of meiosis varied no more than one day among the sample trees. Meiosis occurred at a generally higher rate in microsporangia situated in the lower parts of the strobile as compared to those found in the upper parts. At the end of meiosis, microspore tetrads separated quickly. Early microspores were initially angular and gradually became orbicular. During in about two weeks following meiosis, pollen grains were forming air sacs. Haploid microspore cells then experienced three unequal divisions: the first two divisions resulted in production of two small lens-shaped prothallial cells on the micro- spore proximal side and a small generative and large tube cells resulted from the third division.

At the Arboretum, the pollen cones collected in April of 2002, 2003 and 2011 contained PMC at early meiosis stages and first meiotic divisions were observed on the 23rd April, at an accumulated temperature of 11.2˚C - 23.7˚C in while this process only started on the 5th May in the cold spring of 2004, with the accumulated tem- perature being 57.5˚C. Meiotic division lasted for 13 - 17 days; microspore cells experienced divisions and in- creased in size during ten days.

3.3. Meiosis and Pollen Irregularities

The fir trees moved to the arboretum had species-specific features, such as rapid progression through telophases I and II, and conspicious prophase II, elongated chromosomes (Figure 2(c)), and asynchrony (Figure 2(d)) characteristic of where the species occurs naturally. Although meiosis proceeded in a fairly regular way in most cells, irregularities of general and specific types were found nearly throughout meiosis and pollen formation (the irregularities occured in 11.7%, 13.3%, and 17.9% of the cells in low mountains, middle mountains and in the arboretum (Figures 2(e)-(f)). In the Arboretum, irregularities frequency of occurence increased at all meiotic stages and reached 48.2% at anaphase I [27] . Regular Siberian fir pollen grains have, as a rule, two large sym- metrical air sacs. However, our study identified five-cell pollen grains containing a stalk cell adjacent to two prothallial cells, a tube cell, and a body cell. We also found grains that had either no, or one, three, or four air sacs (Figure 2(g) and Figure 2(h)). Irregular pollen grains were less than 1% of all pollen produced annually by each tree.

3.4. Pollen Size

The pollen grain size varied considerably among sample trees (Table 2). Mature pollen grain variability in diameter was 5% - 12% among the years of observation, 12% - 21% among individual trees, with the respective ranges being 4.0% - 9.0% and 2.6% - 9.3% for air sac size. It should be noted that the mature pollen grains from the arboretum were the same size as in low-mountain Siberian fir populations and 17.1% - 46.6% larger than in middle-mountain populations found at elevation 640 - 720 m a.s.l.

3.5. Time of Pollination

We presume there was a relationship between the climatic conditions and the pollen dispersal period. The air temperature-caused differences in rate of development of the post-dormancy pollen cones resulted in differences in pollination time (Figure 3). In the naturally growing populations pollination was occurred between May 23 and June 5 at accumulated temperature 243˚C - 267˚C; earlier in warm weather and later when it became cooler. In the trees sampled in arboretum, the pollen was dispersed between 21st and 31st of May in different years of observation, with the accumulated temperature ranging 155.6˚C to 292.2˚C. In the natural populations, wind pollination lasted for 3 - 5 days, as opposed to 3 day at the arboretum. Pollen viability at the arboretum was low: in 2002 and 2003, pollen germination varied among sample trees from none to 89.2% and none to 20%, respec- tively [28] . In 2004 and 2011, pollen germination was 34.4% - 96.0% almost three times more that in the former two years.

4. Discussion

Plant responses to the environment are controlled by naturally evolving mechanisms of adaptation responsible for plant environmental tolerance, including tolerance of environmental constraints, and resistance external in- fluences [29] [30] . A. sibirica belongs to a group of species within the genus Abies in which buds start to enlarge earlier in spring than in nearly all other conifers [31] . In this group, microsporogenesis and pollen development start in early spring, immediately after winter dormancy, when the probability of damaged by low air tempera- ture is very high. Extreme temperatures occurring during pollen development and flowering, with former being the stage at which meiosis is most temperature-sensitive, have a negative impact on meiotic cell division pat- terns, including a slowdown of meiosis. The slowdown is manifested the structural and physiological distur- bances, which strongly affect gamete formation and embryo development [22] [32] - [36] . It was established that firs are affected by spring frosts when buds are in the burst phase [37] . The risk of the low temperature-induced development slowdown is reduced only provided that period of dormancy is sufficiently long [38] . For western fir climotypes transferred from where the climate is less severe, development of male buds clearly tended to be- gin earlier (and at lower air temperatures) and to occur at a lower rate compared to native trees. A phenotypic shift was presumably a cause of the decrease in the moved plant homeostasis and developmental disturbances. It is likely that dormancy terminated too early and late frosts resulted in increasing number of irregular cells oc- curring during meiosis, like in 2004. Moreover, PMC damage caused by low air temperatures at the premeiotic phase took a long time to manifest [39] .

The lower amount of pollen in the air and the lower pollen viability found in the arboretum compared to the mountain study plots could be factor limiting A. sibirica trees pollination in the arboretum. Sampled trees grow- ing in the arboretum were characterized by high pollen sterility [28] . Climatic differences between high-eleva-

tion location of natural Siberian fir stands and low-elevation locations of the arboretum might be a major factor accounting for low Siberian fir seed viability in the arboretum. While pollination periods recorded for Abies sp. take long, from 18 days in A. homolepis [40] to as long as one month in A. pinsapo [41] in A. sibirica it usually lasts only 3 - 5 days. The earlier start of male cone reproductive development observed for sampled trees from arboretum might prevent cross-pollination with individuals of natural population. In the arboretum, the probabil- ity of sample fir trees self-pollination was high, because natural populations are situated more than 10 km away from arboretum and fir pollen is usually dispersed to fairly short distances. Fir pollen is relatively heavy, for which reason pollination distances greater than 60 m may be another factor limiting viable seed production [42] . In some conifers, self-pollination is an important cause of empty seed formation [43] [44] . It is generally ac- cepted that isolated tree populations may have low genetic diversity which may be responsible for the lower species performance of such populations [45] [46] .

Woody plants have developed adaptation mechanisms to modify their phenotype to tolerate changes in cli- matic conditions. It have been recognized that the phenology vary within species at different elevations and lati- tudes [47] - [51] . To remain alive in changing climate, plant species have to be flexible in their responses by changing their phenology or physiology, adapting to new climate condition via selection, migration to suitable climates, otherwise they will become extinct. Recent studies have shown that different environmental factors may induce epigenetic changes in the mother trees, which changes are through the seeds to offspring and, can alter offspring gene activity by modifying its phenotype [7] [11] [52] [55] . This phenomenon may be due to ge- netic features of the trees, since pollen development is genetically controlled and since epigenetic mechanisms are also genetically determined [56] - [58] .

Result of phenological shift research studies at introduction can be a useful proxy for studying climate change and global warming. Responses of ontogenetic development of the male reproductive buds of the Siberian fir sampled in the arboretum to environmental conditions may be considered as a model of species adaptation to climate change.

Acknowledgements

Author thanks Dr. Olga Kvitko for conducting of some cytological investigations as well as Prof. Elena N. Mu- ratova for supporting the researches.

The work was supported by Russian Foundation of Basic Research and Krasnoyarsk Foundation, projects 11-04-00281 and r_sibir_a 13-04-98045.

References