Polien viability in three Juniperus taxa

Polien viability in three Juniperus taxa | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Polien viability in three Juniperus taxa Martin Galgoci, Andrej Kormutak, Miroslav Klobucnik, Dusan Gomory, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3748501/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Feb, 2025 Read the published version in Biologia → Version 1 posted 5 You are reading this latest preprint version Abstract Pollen viability in three Juniperus taxa native to Slovakia was investigated using in vitro germination test. The highest germination percentage and longest pollen tubes were surprisingly found in J. communis var. intermedia which is believed to be a putative hybrid between J. communis var. communis and J. sibirica . Germination rate of the hybrid averaged at 78.70% as compared with 64.96% and 60.83% of the pollen germination in the corresponding parental species. The same tendency holds for the pollen tube length reaching 38.68 µm in the putative hybrids, 31.39 µm in J. communis var. communis and 28.08 µm in J. sibirica . At the taxa level, the above given data were statistically highly significant in both pollen viability characteristics. Highest viability of J. communis var. intermedia pollen does not corroborate the widespread notion on reduced fertility of the plant interspecific hybrids. Additional tests are suggested to verify the hybrid nature of the putative hybrid population using DNA based population study and artificial crossing experiments involving the putative parental species J. communis var. communis and J. sibirica . Juniperus species Putative hybrid Pollen viability Figures Figure 1 Introduction In his treatment on „Junipers of the World“, Adams ( 2014 ) listed 76 species, 32 varieties and 8 forms which are recognized within the genus Juniperus . Being a prominent representative of the genus in Europe, J. communis L. was included into the section Juniperus by its typical variety communis , along with additional 8 varieties distributed in the eastern and western hemispheres. In Slovakia, the species' richness is restricted to the three taxa only which are ingenious to the country (Futak et al. 1966 ). The species J. communis var. communis may be found from the hills till the alpine zone on the entire territory of Slovakia reaching its maximal altitude at 1495 m (Blatny and Stastny 1959 ). J. sibirica .is a second representative of the junipers that is native to Slovakia and which is less numerous than J. communis var. communis. It occurs in a subalpine zone of the Carpaticum at the altitude range 1090–2140 m. In literature, it is also known under the synonymous names J. communis var. montana , J. communis subsp. alpina , J. communis subsp. nana , and J. communis subsp. sibirica. The last taxon which is necessary to mention in this context was described under the name J. communis var. intermedia . According to Futak et al. ( 1966 ), the taxon is of hybrid origin which has emerged as a product of spontaneous hybridization between J. communis var. communis and J. nana . It is distributed on the clearings and pasture lands of the mountains, preferably on the localities with common occurrence of J. communis var. communis and J. sibirica . There is an urgent claim for detailed taxonomic and phytogeographic descriptions of the hybrids (Futak et al. 1966 ). Owing to the meiotic irregularities which are very common in the interspecific hybrids, the pollen size and morphology were recently used as indirect criteria in assessing the regularity of meiosis in the putative hybrids between the tetraploid J. thurifera and the diploid J. sabina and in their parental species in the French Alps (Farhat et al. 2020 ). Of the three putative hybrid individuals analyzed so far, one individual displayed a large variance in pollen size while the remaining two hybrids possessed 35.3% and 32.5% of abnormal pollen grains as compared with only 0.9% of morphological abnormalities revealed in the pure species J. thurifera . The authors Farhat et al. ( 2020 ) are of the opinion that these data reflect the irregular meiosis in the respective hybrids. In order to evaluate the utility of pollen analysis in screening interspecific hybrids of junipers in the field, the authors recommend the assessment of the pollen viability in both the hybrids and the corresponding parental species. In present study, we have applied this approach in pollen viability analysis in all the three domestic taxa of junipers, aiming in obtaining the indications which may provide a support or deny the postulated hybrid natuire of the J. communis var. intermedia taxon. Materials and Methods Three Juniperus species were subjected to pollen viability analysis all of which are naturally distributed in Middle Slovakia. Pollen samples of J. communis L. var. communis were harvested on the locality Sumiac (1000 m a. s. l.), those of J. sibirica Lodd. in Burgsd. on the locality Kralova hola (1846 m a. s. l.) and J. communis var. intermedia (Schur) Sanio on the locality Kralova studna (1284 m a. s. l.). Pollen collection was done during anthesis when a massive release of pollen from microsporangia was taking place on the respective localities. Owing to the profound differences in flowering phenology of these species, the collection dates extended from June 5, 2023 on Kralova studna Mtn., via June 13, 2023 in Sumiac to June 21, 2023 on Kralova hola Mtn. All the three localities under studyare integral parts of the National Park „Low Tatras“. As a consequence, the collection of plant material on these localities was carried out with the permission issued by the Administration Office in Revuca under no. NAPANT/551-001/2022 on May 6th 2022 which was subsequently confirmed by the District Office in Banska Bystrica, Department of Environmental Care on May 13, 2022. In total, 10 shrubs of each species were used in pollen quality estimation. The twigs of individual shrubs with shedding pollen were cut and transferred in the paper bags to the laboratory. Next day, the twigs were sieved and obtained pollen was left on a sheet additional 1–2 hours to dry. Extracted pollen was stored in glass tubes that were plugged loosely with cotton-wool caps and placed into a desiccator with silica gel. The desiccator was kept in a refrigerator until the germination test. The period between storing of fresh pollen in refrigerator and its use in germination test has not exceeded 2 days. In vitro germination of juniper pollen was carried out at 25 ºC in the dark with a separate evaluation of pollen viability in each shrub under study. The cultivation medium consisted of 1.5% agar and 5% sucrose (Brewbaker and Kwack 1963 ), the latter has been reported to be optimal for common juniper pollen germination in culture (Duhoux 1972 ; Surso 2018 ). The pollen was evenly dusted over the gel surface in small Petri dishes (47 mm diameter) by blowing it from a soft hair brush. Each dish was placed in a larger Petri dish (70 mm) containing 2 ml H 2 O and covered with a lid. Each sample was tested in triplicate. Plates were incubated for 6 days which was necessary for juniper pollen to fully express the growth potential of pollen tubes. During the second half of the culture period, contamination of the cultivation media occurred by fungal spores and bacteria on the pollen surface and from the air. The number of germinated pollen grains was recorded from a random sample of 100 pollen grains, and the pollen tube length was measured on a sample of 30 pollen grains on each Petri dish (total of 300 pollen grains and 90 pollen tubes per shrub). The evaluation was made with a NU 2 microscope (Carl Zeiss Jena) using a 10x eyepiece with a scale and 25x objective. A pollen was considered viable when pollen tube reached a length comparable to the diameter of the pollen grain body (Lindgren and Lindgren 1996 ).The presence of clearly differentiated pollen tubes in individual pollen grains served as a main criterion of pollen viability. Another viability criterion was mitotic division of the microspore nucleus, giving rise to the vegetative and generative cells, both of which were visualized in growing pollen tubes by Feulgen staining. When measuring the length of a pollen tube, the distance from a zone where the round shape of the pollen grain body extended into the pollen tube was considered to the tip of the pollen tube. Pollen from each of the shrubs was evaluated separately. Differences in both viability traits were tested statistically using two-way analysis of variance (ANOVA). Germination percentages were arcsine transformed prior to analysis. Calculations were performed using the GLM (General linear model) procedure in the statistical package SAS, version 9.4. Pairwise differences between individual shrubs were tested by Duncan's test. Results Pollen germination rate in studied Juniperus species was relatively low, not exceeding 78.7%. This amount of germinating pollen was registered in the putative hybrid population J. communis var. intermedia only. The corresponding characteristics of the pollen in the putatively parental populations J. communis var. communis and J. sibirica were incomparably lower averaging at 64.96% and 60.83%, respectively (Table 1 ). ANOVA confirmed significant difference in pollen germination percentage between all the three taxa investigated (DF = 2, F = 57.75 *** ) and between individual shrubs of a given species (DF = 27, F = 9.97 *** ). The outlined variation in pollen germination rate has as a rule been paralleled by the pollen tube length with longest pollen tubes being revealed in J. communis var. intermedia (38.68 µm) and shortest tubes in J. communis var. communis (31.39 µm) and J. sibirica (28.05 µm). Like in germination percentage, the differences in pollen tube length were statistically significant between species (DF = 2, F = 283.58 *** ) and between individuals of a given species (DF = 27, F = 42.42 *** ). Table 1 Pollen viability in three Juniperus taxa Species Locality Germination [%] Pollen tube length [µm] Mean ± s.d. Duncan`s gr. Mean ± s.d. Duncan`s gr. J. communis var. communis Sumiac 64.96 ± 16.24 B 31.39 ± 17.89 B J. sibirica Kralova hola 60.83 ± 13.00 C 28.05 ± 7.06 C J. communis var. intermedia Kralova studna 78.70 ± 10.36 A 38.68 ± 13.39 A Different letters of Duncan's grouping indicate significant differences in pollen viability parameters Except for pollen viability parameters, some peculiarities of germinating pollen grains were taken into account contributing to a better understanding of the pollen germination in this group of conifers. Immediately after sowing of pollen grains on cultivation medium, an intensive penetration of water through exine aperture into intine takes place resulting in hydration of the intine and subsequent bursting of the exine which is detached from the pollen grain surface and left in the medium (Figs. 1–2). The duration of the process is about 60–90 min. and is accompanied by the hydrophilic capsule formation (Fig. 2). Germination of microspores into microgametophyte initiates pollen tube formation which acquires an oval and subsequently „shoe-like“ shape (Fig. 3). The generative cell moves to the distal part of the prolonging pollen tube, the vegetative cell remains in the central zone (Fig. 4). Discussion Most studies on variation within the genus Juniperus refer to the morphological features of leaves, seeds and cones (Klimko et al. 2007 ). In particular, it is true of the Mediterranean species J. deltoides , J. communis subsp. communis , J. communis subsp. intermedia and J. communis subsp. alpina the needle and stem anatomy of which was found to be of taxonomic importance at the species and interspecies levels (Lakušiċ and Lakušiċ 2011). Among the species J. drupacea , J. communis var. communis , J. communis var. saxalis and J. oxycedrus subsp. oxycedrus native to Turkey, the differences were made between three species based on the palisade parenchyma arrangement and location of the secretory canals (Güvenç et al. 2011 ). In five Juniperus taxa of the same country, the species was found to miss double stomatal band on the inner surface of needles. Also, the absence of resin pits on the seed surface was registered in J. drupacea (Köroglu et al. 2018 ). In North America, no differentiation was found in terpenoid content of the J. ashei central Texas populations (Adams 1977 ). However, the profound intraspecific and interspecific differences were reported to exist between J. scopulorum , J. phoenica , J. polycarpos , J. thurifera and some other juniper species at the DNA level (Adams 2014 ). Pollen quality seems to be neglected completely in this respect.There is only one attempt known so far which deals with the interaction between nutritional status of J. communis shrubs and quality of their pollen. Accordingly, the shrubs with applied nitrogen fertilizer were foud to be negatively affected by the nitrogen in the soil. The germination percentage of their pollen averaged at 3.1% only as compared with 37.11% germination rate of the pollen in non-fertilized control (Pers-Kamczyc et al. 2020 ). The last mentioned pollen characteristic is much lower than those presented in this study (60.83% and 78.7%). Most probably, the reason for this difference is due to the different methodology used in the respective studies. In study by Pers-Kamczyc et al. ( 2020 ), Brewbaker and Kwack`s liquid medium was used for in vitro germination test, whereas in our study a solid medium of the same sucrose content was applied. Also, the duration of the test was restricted by the above authors to 4 days, while in our study the characteristics of germinating pollen were checked on the 6th day. Obviously, the germination potential of J. communis pollen is even higher than that given in present study (64.9%). In our previous experiment, it has reached the magnitude range of 67.3–88.6% what indicates considerable annual variation (Kormutak et al. 2021). Except for pollen viability characteristics, the attempt by Bortenschlager ( 1990 ) has also been made to utilize pllen morphology for taxonomy and systematics of the Cupresaceae whcih resulted in conclusion that uniformity of pollen morphology supports the systematics unity of the family. A single pore with an annulus and the sexinous elements (orbicules) are considered here as the characteristic traits of the pollen sculpture. A further subdivision at the generic or sectional level, based on pollen morphology, is impossible. A key role of porus in pollen hydration and its subsequent germination behaviour was convincingly demonstrated in Cupressus arizonica and J. communis by Duhoux ( 1975 ) and Surso ( 2018 ). In an aqueos medium, the dehydrated and contracted pollen grains become spherical due to hydration within a few seconds (Duhoux 1982 ). On a solid medium, used in this study, the process is more prolonged lasting 60–90 min. Surso ( 2018 ) recommends higher temperature (26 ºC) for in vitro cultivation of juniper pollen as well as a longer cultivation period. According to the author, a 6–7 day period is necessary to reveal fully the germination potential of the species` pollen under in vitro. As far as the basic aspect of this study is concerned, i. e. the hybrid or pure species status of the J. communis var. intermedia population, the question has not been resolved satisfactorily. In spite of postulated hybrid nature of the population based on needle characters (Futak et al. 1966 ), we have not found convincing indicators which would support its hybrid origin. Exhibiting the highest pollen viability parameters, the population does not satisfy the requirement for the lowered pollen viability in the interspecific hybrids of plants. Instead, presented data suggest a pure species nature of the population. Isolated location of the J. communis var. intermedia on the locality Kralova studna without admixture of neither the putatively parental species J. communis var. communis nor J. sibirica supports this conclusion. It is of interest to note in this connection that Hall ( 1952a ) considers the presence of the parental species on a common locality for the basic prerequisity of hybrid swarm formation in junipers, along with the experimental evidence that spcies in question share the potential to hybridize mutually. The last mentioned aspect of the reproductive behaviour of junepers remains to be verified in species J. communis var. communis and J. sibirica . Formely, the opinion has prevailed that hybrid swarms of junipers are very common in the genus covering extensive areas in the USA. Especially it was true for the interspecific combinations of J. virginiana with J.ashei , J. horizontalis and J. scopulorum as well as for the interspecific hybrids of J. scopulorum with J. horizontalis and J. pachyphloea as evidenced by morphological assessment of the respective populations (Hall 1952b ). However, repeated verification of these hybrids has not substatiated their hybrid origin as evidenced by their needle terpenoids (Flake et al. 1969 ; Adams 1975 , 1977 ). Instead, based on needle terpenoids and to some degree on DNA markers, Adams ( 2014 ) has provided in his monograph a list of six interspecific hybrids occurring naturally in USA. On the contrary, the studies involving 22 Juniperus taxa in Mexico and Quatemala failed to show any evidence of hybridization except for the hybrids between intraspecific taxa or between the most closely related species (Zanoni and Adams 1975 , 1976 ). In spite of well documented areas of hybridization in Juniperus in North America, ageneral opinion prevails that widespread hybridization does not appear to be the general rule in Juniperus (Adams 2014 ). Conclusions Contrary expectations, the in vitro germination test has not proved the reduced viability of pollen in the putative hybrid population of J. communis var. intermadia. Instead, the population has surpassed in this respect the putative parental species J. communis var. communis and J. sibirica in both the germination rate and pollen tube length. Based on this finding, the putative hybrid population may be taken for either the subspecies that has evolved from J. communis var. communis or independent species which has emerged during the evolution from the common predecenssor. Additional tests are necessary to specify the taxonomic status of the putative hybrid population on the locality Kralova studna. Highly desirable are the population studies of all the three taxa under question using DNA markers which aim in description the genetic structure of individual populations. The artificial crossing experiments which are at present in progress should answer the question on relationship between J. communis var. communis and J. sibirica to confirm either their mutual hybridization or reproductive isolation in the nature. Declarations Acknowledgements This work was supported financially by the VEGA Grant Agency (project no. 2/0005/23) and COST CA 22136. Competing Interests The authors declare, they have no conflicts of interest. Authors ' Contributions Statement All authors contributed to the study conception and design. Martin Galgóci, Andrej Kormuťák, Miroslav Klobučník and Peter Boleček harvested pollen in the field. Martin Galgóci and Andrej Kormuťák extracted pollen in the lab and carried out germination test. Andrej Kormuťák wrote the first version of the manuscript. Peter Boleček made figure graphics (MS Word, DPI Convertor, MS 3D Skicar). Dušan Gömöry processed experimental data statistically. All authors read and aproved the manuscript. References Adams RP (1975) Gene flow versus selection pressure and ancestral differentiation in the composition of species: analysis of populational variation in Juniperus ashei Buch. using terpenoid data. J Mol Evol 5:177–185 Adams RP (1977) Chemosystematics – Analyses of populational differentiation and variability of ancestral and recent populations of Juniperus ashei . Ann Mo Bot Gard 64:184–209 Adams RP (2014) Junipers of the world: The genus Juniperus. 4th edition. Trafford Publishing Co., Bloomington Blatny T, Stastny T (1959) Natural distribution of forest trees in Slovakia. Slovak Publisher of Agriculture Literature, Bratislava Bortenschlager S (1990) Aspects of pollen morphology in the Cupressaceae. Grana 29:129–138. https://doi.org/10.1080/00173139009427743 Brewbaker JL, Kwack BH (1963) The essential role of calcium ions in pollen germination and pollen tube growth. Am J Bot 50:859–865. https//doi.org/10.2307/2439772 Duhoux E (1972) Evolution structurale de la paroi du grain de pollen du Juniperus communis L. (Cupressacées), cultivé in vitro au cours de la phase d`hydratation. C R Acad Sci 274:2767–2770 Duhoux E (1975) L`aperture dans l`exine et dans l`intine externe du pollen du Juniperus chinensis L., et de Juniperus communis L. Pollen Spores 17:191–201 Duhoux E (1982) Mechanism of exine rupture in hydrated taxoid type of pollen. Grana 21:1–7. https://doi.org/10.1080/00173138209427673 Farhat P, Takvorian N, Avramidou M, Garaud L, Adams RP et al (2020) First evidence for allotriploid hybrids between Juniperus thurifera and J. sabina in a sympatric area in the French Alps. Ann For Sci 77:93. 10.1007/s13595-020-00969-7 , hal-03354950 Flake RH, von Rudloff E, Turner BL (1969) Quantitative study of clinal variation in Juniperus virginiana using terpenoid data. Proc Natl Acad Sci (USA) 62:487–494 Futak J, Jasicova M, Schidlay E (1966) Flora of Slovakia. Slovak Academy of Sciences Publisher, Bratislava Güvenç A, Hürkul M, Erdem A (2011) The leaf anatomy of naturally distributed Juniperus L. (Cupressaceae) species in Turkey. Turk J Bot 35:251–260. https://doi.org/10.3906/bot-0904-1 Hall MT (1952a) Variation and hybridization in Juniperus. Ann Mo Bot Gard 39:1–64. https://www.jstor.org/stable/2394490 Hall MT (1952b) A hybrid swarm in Juniperus. Evol VI : 347–366. https://www.jstor.org/stable/2405698 Klimko M, Boratyńska K, Montserrat JM et al (2007) Morphological variation of Juniperus oxycedrus subsp. oxycedrus (Cupressaceae) in the Mediterranean region. Flora 202:133–147. DOI10.1016/j.flora.2006.03.006 Kormuťák A, Bolecek P, Galgóci M, Gömöry D (2021) Longevity and germination of Juniperus communis L. pollen after storage. Sci Rep 11:12755. https://doi.org/10.1038/s41598-é21-90942-9 Köroglu A, Kendir G, Şimşek D, Miceli N, Pinar NM (2018) The morphological properties of leaves, cones, seeds of some Juniperus species native to Turkey. Commun Fac Sci Univ Ank Ser C 27:61–84. https://communications.science.ankara.edu.tr./index.php?series=C Lakušić B, Lakušić D (2011) Anatomy of four taxa of the genus Juniperus sect. Juniperus (Cupressaceae) from the Balcan peninsula. Bot SERBICA 35:145–156. https://botanicaserbica.bio.bg.ac.rs/2011 35 2.html Lindgren K, Lindgren D (1996) Germinability of Norway spruce and Scots pine pollen exposed to open air. Silva Fenn 30:3–9. https://doi.org/10.14214/sf.a9216 Pers-Kamczyc E, Tyrala-Wierucka Ż, Rabska M, Wrońska-Pilarek D, Kamczyc J (2020) The higher availability of nutrients increases the production but decreases the quality of pollen grains in Juniperus communis L. J Plant Physiol 248:153–156. https://doi.org/10.1016/jplph.2020.153156 Surso MV (2018) Pollination and pollen germination in common juniper ( Juniperus communis : Cupressaceae). Arct Environ Res 18:162–174. https://doi.org/10.3897/issn2541-8416.2018.18.4.162 Zanoni TA, Adams RP (1975) The genus Juniperus (Cupressaceae) in Mexico and Quatemala: numerical and morphological analysis. Bull Bot Soc Mexico 35:69–92 Zanoni TA, Adams RP (1976) The genus Juniperus (Cupressaceae) in Mexico and Quatemala: numerical and chemosystematic analysis. Biochem Syst Ecol 4:147–158 Cite Share Download PDF Status: Published Journal Publication published 05 Feb, 2025 Read the published version in Biologia → Version 1 posted Reviewers agreed at journal 14 Jan, 2024 Reviewers invited by journal 19 Dec, 2023 Editor invited by journal 15 Dec, 2023 Editor assigned by journal 13 Dec, 2023 First submitted to journal 11 Dec, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3748501","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":261534551,"identity":"be6937d2-9309-45bc-a988-0f99fab4f029","order_by":0,"name":"Martin 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13:04:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3748501/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3748501/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11756-024-01795-2","type":"published","date":"2025-02-05T15:58:08+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":48620111,"identity":"c55d3c08-b3f0-411e-8957-a2d002093adb","added_by":"auto","created_at":"2023-12-21 15:55:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1037617,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigs. 1-4 \u003c/strong\u003eDynamics of juniper pollen germination showing dormant pollen grains with shrivelled pollen body surface after sowing on medium (Fig. 1), followed by hydrophilic capsule formation and exine release (Fig. 2) and pollen tube growth (Fig. 3) with vegetative and generative cells inside pollen tube (Fig. 4). hc-hydrophilic capsule, cyt-cytoplasm, in-intine, ex-exine, pt-pollen tube, dp-dormant pollen grain, vc-vegetative cell, gc-generative cell\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3748501/v1/9f76ffcbf1ddaa627cc016d8.png"},{"id":75931197,"identity":"5ba284b3-303e-4f01-aa36-f4961ab5560c","added_by":"auto","created_at":"2025-02-10 16:14:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1309472,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3748501/v1/77d5272e-ea12-4260-b3e4-2f541c5a750c.pdf"}],"financialInterests":"","formattedTitle":"Polien viability in three Juniperus taxa","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn his treatment on \u0026bdquo;Junipers of the World\u0026ldquo;, Adams (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) listed 76 species, 32 varieties and 8 forms which are recognized within the genus \u003cem\u003eJuniperus\u003c/em\u003e. Being a prominent representative of the genus in Europe, \u003cem\u003eJ. communis\u003c/em\u003e L. was included into the section Juniperus by its typical variety \u003cem\u003ecommunis\u003c/em\u003e, along with additional 8 varieties distributed in the eastern and western hemispheres. In Slovakia, the species' richness is restricted to the three taxa only which are ingenious to the country (Futak et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1966\u003c/span\u003e). The species \u003cem\u003eJ. communis\u003c/em\u003e var. \u003cem\u003ecommunis\u003c/em\u003e may be found from the hills till the alpine zone on the entire territory of Slovakia reaching its maximal altitude at 1495 m (Blatny and Stastny \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1959\u003c/span\u003e). \u003cem\u003eJ. sibirica\u003c/em\u003e .is a second representative of the junipers that is native to Slovakia and which is less numerous than \u003cem\u003eJ. communis\u003c/em\u003e var. \u003cem\u003ecommunis.\u003c/em\u003e It occurs in a subalpine zone of the Carpaticum at the altitude range 1090\u0026ndash;2140 m. In literature, it is also known under the synonymous names \u003cem\u003eJ. communis\u003c/em\u003e var. \u003cem\u003emontana\u003c/em\u003e, \u003cem\u003eJ. communis\u003c/em\u003e subsp. \u003cem\u003ealpina\u003c/em\u003e, \u003cem\u003eJ. communis\u003c/em\u003e subsp. \u003cem\u003enana\u003c/em\u003e, and \u003cem\u003eJ. communis\u003c/em\u003e subsp. \u003cem\u003esibirica.\u003c/em\u003e The last taxon which is necessary to mention in this context was described under the name \u003cem\u003eJ. communis\u003c/em\u003e var. \u003cem\u003eintermedia\u003c/em\u003e. According to Futak et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1966\u003c/span\u003e), the taxon is of hybrid origin which has emerged as a product of spontaneous hybridization between \u003cem\u003eJ. communis\u003c/em\u003e var. \u003cem\u003ecommunis\u003c/em\u003e and \u003cem\u003eJ. nana\u003c/em\u003e. It is distributed on the clearings and pasture lands of the mountains, preferably on the localities with common occurrence of \u003cem\u003eJ. communis\u003c/em\u003e var. \u003cem\u003ecommunis\u003c/em\u003e and \u003cem\u003eJ. sibirica\u003c/em\u003e. There is an urgent claim for detailed taxonomic and phytogeographic descriptions of the hybrids (Futak et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1966\u003c/span\u003e). Owing to the meiotic irregularities which are very common in the interspecific hybrids, the pollen size and morphology were recently used as indirect criteria in assessing the regularity of meiosis in the putative hybrids between the tetraploid \u003cem\u003eJ. thurifera\u003c/em\u003e and the diploid \u003cem\u003eJ. sabina\u003c/em\u003e and in their parental species in the French Alps (Farhat et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Of the three putative hybrid individuals analyzed so far, one individual displayed a large variance in pollen size while the remaining two hybrids possessed 35.3% and 32.5% of abnormal pollen grains as compared with only 0.9% of morphological abnormalities revealed in the pure species \u003cem\u003eJ. thurifera\u003c/em\u003e. The authors Farhat et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) are of the opinion that these data reflect the irregular meiosis in the respective hybrids. In order to evaluate the utility of pollen analysis in screening interspecific hybrids of junipers in the field, the authors recommend the assessment of the pollen viability in both the hybrids and the corresponding parental species. In present study, we have applied this approach in pollen viability analysis in all the three domestic taxa of junipers, aiming in obtaining the indications which may provide a support or deny the postulated hybrid natuire of the \u003cem\u003eJ. communis\u003c/em\u003e var. \u003cem\u003eintermedia\u003c/em\u003e taxon.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eThree \u003cem\u003eJuniperus\u003c/em\u003e species were subjected to pollen viability analysis all of which are naturally distributed in Middle Slovakia. Pollen samples of \u003cem\u003eJ. communis\u003c/em\u003e L. var. \u003cem\u003ecommunis\u003c/em\u003e were harvested on the locality Sumiac (1000 m a. s. l.), those of \u003cem\u003eJ. sibirica\u003c/em\u003e Lodd. in Burgsd. on the locality Kralova hola (1846 m a. s. l.) and \u003cem\u003eJ. communis\u003c/em\u003e var. \u003cem\u003eintermedia\u003c/em\u003e (Schur) Sanio on the locality Kralova studna (1284 m a. s. l.). Pollen collection was done during anthesis when a massive release of pollen from microsporangia was taking place on the respective localities. Owing to the profound differences in flowering phenology of these species, the collection dates extended from June 5, 2023 on Kralova studna Mtn., via June 13, 2023 in Sumiac to June 21, 2023 on Kralova hola Mtn. All the three localities under studyare integral parts of the National Park \u0026bdquo;Low Tatras\u0026ldquo;. As a consequence, the collection of plant material on these localities was carried out with the permission issued by the Administration Office in Revuca under no. NAPANT/551-001/2022 on May 6th 2022 which was subsequently confirmed by the District Office in Banska Bystrica, Department of Environmental Care on May 13, 2022. In total, 10 shrubs of each species were used in pollen quality estimation. The twigs of individual shrubs with shedding pollen were cut and transferred in the paper bags to the laboratory. Next day, the twigs were sieved and obtained pollen was left on a sheet additional 1\u0026ndash;2 hours to dry. Extracted pollen was stored in glass tubes that were plugged loosely with cotton-wool caps and placed into a desiccator with silica gel. The desiccator was kept in a refrigerator until the germination test. The period between storing of fresh pollen in refrigerator and its use in germination test has not exceeded 2 days.\u003c/p\u003e \u003cp\u003eIn vitro germination of juniper pollen was carried out at 25 \u0026ordm;C in the dark with a separate evaluation of pollen viability in each shrub under study. The cultivation medium consisted of 1.5% agar and 5% sucrose (Brewbaker and Kwack \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1963\u003c/span\u003e), the latter has been reported to be optimal for common juniper pollen germination in culture (Duhoux \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1972\u003c/span\u003e; Surso \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The pollen was evenly dusted over the gel surface in small Petri dishes (47 mm diameter) by blowing it from a soft hair brush. Each dish was placed in a larger Petri dish (70 mm) containing 2 ml H\u003csub\u003e2\u003c/sub\u003eO and covered with a lid. Each sample was tested in triplicate. Plates were incubated for 6 days which was necessary for juniper pollen to fully express the growth potential of pollen tubes. During the second half of the culture period, contamination of the cultivation media occurred by fungal spores and bacteria on the pollen surface and from the air. The number of germinated pollen grains was recorded from a random sample of 100 pollen grains, and the pollen tube length was measured on a sample of 30 pollen grains on each Petri dish (total of 300 pollen grains and 90 pollen tubes per shrub). The evaluation was made with a NU 2 microscope (Carl Zeiss Jena) using a 10x eyepiece with a scale and 25x objective. A pollen was considered viable when pollen tube reached a length comparable to the diameter of the pollen grain body (Lindgren and Lindgren \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1996\u003c/span\u003e).The presence of clearly differentiated pollen tubes in individual pollen grains served as a main criterion of pollen viability. Another viability criterion was mitotic division of the microspore nucleus, giving rise to the vegetative and generative cells, both of which were visualized in growing pollen tubes by Feulgen staining. When measuring the length of a pollen tube, the distance from a zone where the round shape of the pollen grain body extended into the pollen tube was considered to the tip of the pollen tube. Pollen from each of the shrubs was evaluated separately. Differences in both viability traits were tested statistically using two-way analysis of variance (ANOVA). Germination percentages were arcsine transformed prior to analysis. Calculations were performed using the GLM (General linear model) procedure in the statistical package SAS, version 9.4. Pairwise differences between individual shrubs were tested by Duncan's test.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003ePollen germination rate in studied \u003cem\u003eJuniperus\u003c/em\u003e species was relatively low, not exceeding 78.7%. This amount of germinating pollen was registered in the putative hybrid population \u003cem\u003eJ. communis\u003c/em\u003e var. \u003cem\u003eintermedia\u003c/em\u003e only. The corresponding characteristics of the pollen in the putatively parental populations \u003cem\u003eJ. communis\u003c/em\u003e var. \u003cem\u003ecommunis\u003c/em\u003e and \u003cem\u003eJ. sibirica\u003c/em\u003e were incomparably lower averaging at 64.96% and 60.83%, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). ANOVA confirmed significant difference in pollen germination percentage between all the three taxa investigated (DF\u0026thinsp;=\u0026thinsp;2, F\u0026thinsp;=\u0026thinsp;57.75\u003csup\u003e***\u003c/sup\u003e) and between individual shrubs of a given species (DF\u0026thinsp;=\u0026thinsp;27, F\u0026thinsp;=\u0026thinsp;9.97\u003csup\u003e***\u003c/sup\u003e). The outlined variation in pollen germination rate has as a rule been paralleled by the pollen tube length with longest pollen tubes being revealed in \u003cem\u003eJ. communis\u003c/em\u003e var. \u003cem\u003eintermedia\u003c/em\u003e (38.68 \u0026micro;m) and shortest tubes in \u003cem\u003eJ. communis\u003c/em\u003e var. \u003cem\u003ecommunis\u003c/em\u003e (31.39 \u0026micro;m) and \u003cem\u003eJ. sibirica\u003c/em\u003e (28.05 \u0026micro;m). Like in germination percentage, the differences in pollen tube length were statistically significant between species (DF\u0026thinsp;=\u0026thinsp;2, F\u0026thinsp;=\u0026thinsp;283.58\u003csup\u003e***\u003c/sup\u003e) and between individuals of a given species (DF\u0026thinsp;=\u0026thinsp;27, F\u0026thinsp;=\u0026thinsp;42.42\u003csup\u003e***\u003c/sup\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePollen viability in three \u003cem\u003eJuniperus taxa\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eLocality\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eGermination [%]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003ePollen tube length [\u0026micro;m]\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;s.d.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDuncan`s gr.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;s.d.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eDuncan`s gr.\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eJ. communis\u003c/em\u003e var. \u003cem\u003ecommunis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSumiac\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e64.96\u0026thinsp;\u0026plusmn;\u0026thinsp;16.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e31.39\u0026thinsp;\u0026plusmn;\u0026thinsp;17.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eJ. sibirica\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKralova hola\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e60.83\u0026thinsp;\u0026plusmn;\u0026thinsp;13.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e28.05\u0026thinsp;\u0026plusmn;\u0026thinsp;7.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eJ. communis\u003c/em\u003e var. \u003cem\u003eintermedia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKralova studna\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e78.70\u0026thinsp;\u0026plusmn;\u0026thinsp;10.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e38.68\u0026thinsp;\u0026plusmn;\u0026thinsp;13.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eDifferent letters of Duncan's grouping indicate significant differences in pollen viability parameters\u003c/p\u003e \u003cp\u003eExcept for pollen viability parameters, some peculiarities of germinating pollen grains were taken into account contributing to a better understanding of the pollen germination in this group of conifers. Immediately after sowing of pollen grains on cultivation medium, an intensive penetration of water through exine aperture into intine takes place resulting in hydration of the intine and subsequent bursting of the exine which is detached from the pollen grain surface and left in the medium (Figs.\u0026nbsp;1\u0026ndash;2).\u003c/p\u003e \u003cp\u003eThe duration of the process is about 60\u0026ndash;90 min. and is accompanied by the hydrophilic capsule formation (Fig.\u0026nbsp;2). Germination of microspores into microgametophyte initiates pollen tube formation which acquires an oval and subsequently \u0026bdquo;shoe-like\u0026ldquo; shape (Fig.\u0026nbsp;3). The generative cell moves to the distal part of the prolonging pollen tube, the vegetative cell remains in the central zone (Fig.\u0026nbsp;4).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eMost studies on variation within the genus \u003cem\u003eJuniperus\u003c/em\u003e refer to the morphological features of leaves, seeds and cones (Klimko et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). In particular, it is true of the Mediterranean species \u003cem\u003eJ. deltoides\u003c/em\u003e, \u003cem\u003eJ. communis\u003c/em\u003e subsp. \u003cem\u003ecommunis\u003c/em\u003e, \u003cem\u003eJ. communis\u003c/em\u003e subsp. \u003cem\u003eintermedia\u003c/em\u003e and \u003cem\u003eJ. communis\u003c/em\u003e subsp. \u003cem\u003ealpina\u003c/em\u003e the needle and stem anatomy of which was found to be of taxonomic importance at the species and interspecies levels (Lakušiċ and Lakušiċ 2011). Among the species \u003cem\u003eJ. drupacea\u003c/em\u003e, \u003cem\u003eJ. communis\u003c/em\u003e var. \u003cem\u003ecommunis\u003c/em\u003e, \u003cem\u003eJ. communis\u003c/em\u003e var. \u003cem\u003esaxalis\u003c/em\u003e and \u003cem\u003eJ. oxycedrus\u003c/em\u003e subsp. \u003cem\u003eoxycedrus\u003c/em\u003e native to Turkey, the differences were made between three species based on the palisade parenchyma arrangement and location of the secretory canals (G\u0026uuml;ven\u0026ccedil; et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In five \u003cem\u003eJuniperus\u003c/em\u003e taxa of the same country, the species was found to miss double stomatal band on the inner surface of needles. Also, the absence of resin pits on the seed surface was registered in \u003cem\u003eJ. drupacea\u003c/em\u003e (K\u0026ouml;roglu et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In North America, no differentiation was found in terpenoid content of the \u003cem\u003eJ. ashei\u003c/em\u003e central Texas populations (Adams \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1977\u003c/span\u003e). However, the profound intraspecific and interspecific differences were reported to exist between \u003cem\u003eJ. scopulorum\u003c/em\u003e, \u003cem\u003eJ. phoenica\u003c/em\u003e, \u003cem\u003eJ. polycarpos\u003c/em\u003e, \u003cem\u003eJ. thurifera\u003c/em\u003e and some other juniper species at the DNA level (Adams \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Pollen quality seems to be neglected completely in this respect.There is only one attempt known so far which deals with the interaction between nutritional status of \u003cem\u003eJ. communis\u003c/em\u003e shrubs and quality of their pollen. Accordingly, the shrubs with applied nitrogen fertilizer were foud to be negatively affected by the nitrogen in the soil. The germination percentage of their pollen averaged at 3.1% only as compared with 37.11% germination rate of the pollen in non-fertilized control (Pers-Kamczyc et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The last mentioned pollen characteristic is much lower than those presented in this study (60.83% and 78.7%). Most probably, the reason for this difference is due to the different methodology used in the respective studies. In study by Pers-Kamczyc et al. (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), Brewbaker and Kwack`s liquid medium was used for in vitro germination test, whereas in our study a solid medium of the same sucrose content was applied. Also, the duration of the test was restricted by the above authors to 4 days, while in our study the characteristics of germinating pollen were checked on the 6th day. Obviously, the germination potential of \u003cem\u003eJ. communis\u003c/em\u003e pollen is even higher than that given in present study (64.9%). In our previous experiment, it has reached the magnitude range of 67.3\u0026ndash;88.6% what indicates considerable annual variation (Kormutak et al. 2021). Except for pollen viability characteristics, the attempt by Bortenschlager (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1990\u003c/span\u003e) has also been made to utilize pllen morphology for taxonomy and systematics of the Cupresaceae whcih resulted in conclusion that uniformity of pollen morphology supports the systematics unity of the family. A single pore with an annulus and the sexinous elements (orbicules) are considered here as the characteristic traits of the pollen sculpture. A further subdivision at the generic or sectional level, based on pollen morphology, is impossible. A key role of porus in pollen hydration and its subsequent germination behaviour was convincingly demonstrated in \u003cem\u003eCupressus arizonica\u003c/em\u003e and \u003cem\u003eJ. communis\u003c/em\u003e by Duhoux (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1975\u003c/span\u003e) and Surso (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In an aqueos medium, the dehydrated and contracted pollen grains become spherical due to hydration within a few seconds (Duhoux \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1982\u003c/span\u003e). On a solid medium, used in this study, the process is more prolonged lasting 60\u0026ndash;90 min. Surso (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) recommends higher temperature (26 \u0026ordm;C) for in vitro cultivation of juniper pollen as well as a longer cultivation period. According to the author, a 6\u0026ndash;7 day period is necessary to reveal fully the germination potential of the species` pollen under in vitro. As far as the basic aspect of this study is concerned, i. e. the hybrid or pure species status of the \u003cem\u003eJ. communis\u003c/em\u003e var. \u003cem\u003eintermedia\u003c/em\u003e population, the question has not been resolved satisfactorily. In spite of postulated hybrid nature of the population based on needle characters (Futak et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1966\u003c/span\u003e), we have not found convincing indicators which would support its hybrid origin. Exhibiting the highest pollen viability parameters, the population does not satisfy the requirement for the lowered pollen viability in the interspecific hybrids of plants. Instead, presented data suggest a pure species nature of the population. Isolated location of the \u003cem\u003eJ. communis\u003c/em\u003e var. \u003cem\u003eintermedia\u003c/em\u003e on the locality Kralova studna without admixture of neither the putatively parental species \u003cem\u003eJ. communis\u003c/em\u003e var. \u003cem\u003ecommunis\u003c/em\u003e nor \u003cem\u003eJ. sibirica\u003c/em\u003e supports this conclusion. It is of interest to note in this connection that Hall (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1952a\u003c/span\u003e) considers the presence of the parental species on a common locality for the basic prerequisity of hybrid swarm formation in junipers, along with the experimental evidence that spcies in question share the potential to hybridize mutually. The last mentioned aspect of the reproductive behaviour of junepers remains to be verified in species \u003cem\u003eJ. communis\u003c/em\u003e var. \u003cem\u003ecommunis\u003c/em\u003e and \u003cem\u003eJ. sibirica\u003c/em\u003e. Formely, the opinion has prevailed that hybrid swarms of junipers are very common in the genus covering extensive areas in the USA. Especially it was true for the interspecific combinations of \u003cem\u003eJ. virginiana\u003c/em\u003e with \u003cem\u003eJ.ashei\u003c/em\u003e, \u003cem\u003eJ. horizontalis\u003c/em\u003e and \u003cem\u003eJ. scopulorum\u003c/em\u003e as well as for the interspecific hybrids of \u003cem\u003eJ. scopulorum\u003c/em\u003e with \u003cem\u003eJ. horizontalis\u003c/em\u003e and \u003cem\u003eJ. pachyphloea\u003c/em\u003e as evidenced by morphological assessment of the respective populations (Hall \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1952b\u003c/span\u003e). However, repeated verification of these hybrids has not substatiated their hybrid origin as evidenced by their needle terpenoids (Flake et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1969\u003c/span\u003e; Adams \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1975\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1977\u003c/span\u003e). Instead, based on needle terpenoids and to some degree on DNA markers, Adams (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) has provided in his monograph a list of six interspecific hybrids occurring naturally in USA. On the contrary, the studies involving 22 \u003cem\u003eJuniperus\u003c/em\u003e taxa in Mexico and Quatemala failed to show any evidence of hybridization except for the hybrids between intraspecific taxa or between the most closely related species (Zanoni and Adams \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1975\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1976\u003c/span\u003e). In spite of well documented areas of hybridization in \u003cem\u003eJuniperus\u003c/em\u003e in North America, ageneral opinion prevails that widespread hybridization does not appear to be the general rule in \u003cem\u003eJuniperus\u003c/em\u003e (Adams \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eContrary expectations, the in vitro germination test has not proved the reduced viability of pollen in the putative hybrid population of \u003cem\u003eJ. communis\u003c/em\u003e var. \u003cem\u003eintermadia.\u003c/em\u003e Instead, the population has surpassed in this respect the putative parental species \u003cem\u003eJ. communis\u003c/em\u003e var. \u003cem\u003ecommunis\u003c/em\u003e and \u003cem\u003eJ. sibirica\u003c/em\u003e in both the germination rate and pollen tube length. Based on this finding, the putative hybrid population may be taken for either the subspecies that has evolved from \u003cem\u003eJ. communis\u003c/em\u003e var. \u003cem\u003ecommunis\u003c/em\u003e or independent species which has emerged during the evolution from the common predecenssor. Additional tests are necessary to specify the taxonomic status of the putative hybrid population on the locality Kralova studna. Highly desirable are the population studies of all the three taxa under question using DNA markers which aim in description the genetic structure of individual populations. The artificial crossing experiments which are at present in progress should answer the question on relationship between \u003cem\u003eJ. communis\u003c/em\u003e var. \u003cem\u003ecommunis\u003c/em\u003e and \u003cem\u003eJ. sibirica\u003c/em\u003e to confirm either their mutual hybridization or reproductive isolation in the nature.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported financially by the VEGA Grant Agency (project no. 2/0005/23) and COST CA 22136.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare, they have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u003csup\u003e\u0026apos;\u0026nbsp;\u003c/sup\u003eContributions Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Martin Galg\u0026oacute;ci, Andrej Kormuť\u0026aacute;k, Miroslav Klobučn\u0026iacute;k and Peter Boleček harvested pollen in the field. Martin Galg\u0026oacute;ci and Andrej Kormuť\u0026aacute;k extracted pollen in the lab and carried out germination test. Andrej Kormuť\u0026aacute;k wrote the first version of the manuscript. Peter Boleček made figure graphics (MS Word, DPI Convertor, MS 3D Skicar). Du\u0026scaron;an G\u0026ouml;m\u0026ouml;ry processed experimental data statistically. All authors read and aproved the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAdams RP (1975) Gene flow versus selection pressure and ancestral differentiation in the composition of species: analysis of populational variation in \u003cem\u003eJuniperus ashei\u003c/em\u003e Buch. using terpenoid data. 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Biochem Syst Ecol 4:147\u0026ndash;158\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"biologia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"biol","sideBox":"Learn more about [Biologia](http://link.springer.com/journal/11756)","snPcode":"11756","submissionUrl":"https://www.editorialmanager.com/biol/default2.aspx","title":"Biologia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Juniperus species, Putative hybrid, Pollen viability","lastPublishedDoi":"10.21203/rs.3.rs-3748501/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3748501/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePollen viability in three \u003cem\u003eJuniperus\u003c/em\u003e taxa native to Slovakia was investigated using in vitro germination test. The highest germination percentage and longest pollen tubes were surprisingly found in \u003cem\u003eJ. communis\u003c/em\u003e var.\u003cem\u003eintermedia\u003c/em\u003e which is believed to be a putative hybrid between \u003cem\u003eJ. communis\u003c/em\u003e var. \u003cem\u003ecommunis\u003c/em\u003e and \u003cem\u003eJ. sibirica\u003c/em\u003e. Germination rate of the hybrid averaged at 78.70% as compared with 64.96% and 60.83% of the pollen germination in the corresponding parental species. The same tendency holds for the pollen tube length reaching 38.68 \u0026micro;m in the putative hybrids, 31.39 \u0026micro;m in \u003cem\u003eJ. communis\u003c/em\u003e var. \u003cem\u003ecommunis\u003c/em\u003e and 28.08 \u0026micro;m in \u003cem\u003eJ. sibirica\u003c/em\u003e. At the taxa level, the above given data were statistically highly significant in both pollen viability characteristics. Highest viability of \u003cem\u003eJ. communis\u003c/em\u003e var. \u003cem\u003eintermedia\u003c/em\u003e pollen does not corroborate the widespread notion on reduced fertility of the plant interspecific hybrids. Additional tests are suggested to verify the hybrid nature of the putative hybrid population using DNA based population study and artificial crossing experiments involving the putative parental species \u003cem\u003eJ. communis\u003c/em\u003e var. \u003cem\u003ecommunis\u003c/em\u003e and \u003cem\u003eJ. sibirica\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Polien viability in three Juniperus taxa","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-12-21 15:55:42","doi":"10.21203/rs.3.rs-3748501/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-01-14T10:46:33+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-12-19T10:34:37+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Biologia","date":"2023-12-15T11:31:44+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-12-14T01:44:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biologia","date":"2023-12-12T04:13:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biologia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"biol","sideBox":"Learn more about [Biologia](http://link.springer.com/journal/11756)","snPcode":"11756","submissionUrl":"https://www.editorialmanager.com/biol/default2.aspx","title":"Biologia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"04a88528-2091-4ebf-81d1-119787c7dcb9","owner":[],"postedDate":"December 21st, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-02-10T16:09:23+00:00","versionOfRecord":{"articleIdentity":"rs-3748501","link":"https://doi.org/10.1007/s11756-024-01795-2","journal":{"identity":"biologia","isVorOnly":false,"title":"Biologia"},"publishedOn":"2025-02-05 15:58:08","publishedOnDateReadable":"February 5th, 2025"},"versionCreatedAt":"2023-12-21 15:55:42","video":"","vorDoi":"10.1007/s11756-024-01795-2","vorDoiUrl":"https://doi.org/10.1007/s11756-024-01795-2","workflowStages":[]},"version":"v1","identity":"rs-3748501","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3748501","identity":"rs-3748501","version":["v1"]},"buildId":"i_I_jhV-QviBjPLXJga9V","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}