Over last 130 years the American assh was often used for afforestation of Croatian lowland areas where the native narrow leafed ash (Fraxinus angustifolia Vahl) did not grow well. Due to unequivocal species identification of the American ash F. americana L. or F. pennsylvanica Marshall, pollen samples of concerned specimens were collected. Based on species pollen samples of F. americana and F. pennsylvanica, collected from trees growing at the Royal Botanical Gardens Hamilton in Canada, and on their distinctive morphological features, pollen samples of American ash trees from a plantation Črnovščak in Dugo Selo near Zagreb and from arboricultures in Zagreb were determined as Fraxinus pennsylvanica

D. Kremer

E. Stabentheiner

Ž. Borzan

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Acta Bot. Croat. 64 (1), 47–55, 2005 CODEN: ABCRA25ISSN 0365–0588Pollen traits of some American ashes investigatedby a scanning electron microscopeDARIO KREMER1, EDITH STABENTHEINER2, @ELIMIR BORZAN1*1 Faculty of Forestry, University of Zagreb, Sveto{imunska 25, HR-10000 Zagreb,Croatia.2 Institute of Plant Physiology, Karl-Franzens-University, Schubertstrasse, 51,A-8010 Graz, Austria.Over last 130 years the American assh was often used for afforestation of Croatian low-land areas where the native narrow leafed ash (Fraxinus angustifolia Vahl) did not growwell. Due to unequivocal species identification of the American ash F. americana L. or F.pennsylvanica Marshall, pollen samples of concerned specimens were collected. Basedon species pollen samples of F. americana and F. pennsylvanica, collected from treesgrowing at the Royal Botanical Gardens Hamilton in Canada, and on their distinctivemorphological features, pollen samples of American ash trees from a plantation ^rnov-{~ak in Dugo Selo near Zagreb and from arboricultures in Zagreb were determined asFraxinus pennsylvanica.Key words: pollen, morphology, scanning electron microscopy, Fraxinus americana L.,Fraxinus pennsylvanica MarshallIntroductionAmerican ash species Fraxinus americana L. and F. pennsylvanica Marshall were in-troduced to Europe in 1723 and in 1824 respectively (HEGI 1908). In Croatia these two spe-cies are present for more than 130 years. One of the first papers mentioning those speciesgrowing in Croatian parks was ETTINGER (1889, 1892). F. americana was recommended asspecies for afforestations in Croatia by ^ORDA[I] (1894) and KOZARAC (1898). Some de-cades after that first data about successful afforestation with F. americana and F. pen-nsylvanica in lowland area were reported by PERU[I] (1925), STREPA^KI (1931), [PANOVI](1931), CIVIDINI and MIRTH (1941) and FUKAREK (1956). Most of the authors have men-tioned F. americana as a species involved in afforestations, and only a few mentioned F.pennsylvanica as well ([PANOVI] 1954, ANI] 1954, DEKANI] 1974). More recent papers byRAU[ (1993), RAU[ et al. (1985), RAU[ and MATI] (1990), and MAJER (1994) were dealingwith the presence of F. americana in Croatian forests and arboricultures. KREMER (2001)and KREMER and BORZAN (2001) mentioned that F. pennsylvanica is primarily present inACTA BOT. CROAT. 64 (1), 2005 47* Corresponding author: e-mail: zelimir.borzan@zg.htnet.hrCroatian lowland forests in the Kupa river basin; the Sava river basin; and in part of theDanube river basin.In Croatian forestry practice North American ash species are known today under onename – American ash. The primary reason for performing investigations of morphologicaltraits of trees identified from the Royal Botanical Gardens, Hamilton in Canada was tocompare them with samples taken from Croatian forests and arboricultures. Owing tomicromorphological differences in the pollen of F. americana and F. pennsylvanica inves-tigation by use of scanning electron microscopy was applied.Materials and methodsPollen samples of Fraxinus pennsylvanica were collected in spring 2002 from the treeat the Botanical Garden of the Faculty of Sciences, University of Zagreb and in spring 2003from a tree at Royal Botanical Garden in Hamilton, Canada. Pollen of F. americana wasalso collected in spring 2003 from a tree at RBG, Hamilton, Canada.Samples of pollen of American ash were collected in spring 2002 from 12 trees in Man-agement unit ^rnov{~ak (subcompartment 4e, Forestry office Dugo Selo) and from 3 treesgrowing in arboricultures in Zagreb. Trees were randomly selected. The Management unit^rnov{~ak is situated about 20 km east from Zagreb. Borders of that unit are: railway lineSesvete – Bo`jakovina on the north, River Lonja on the east, villages Lipovec Lonjski,Trebovec and Rugvica on the southeast, the River Sava on the southwest and Ivanja Rekaon the west. The altitude ranges from 99 to 107 m, according to the Management plan forManagement unit ^rnov{~ak (ANONYMOUS 1997).At the beginning of bloom setting, flowering branches were cut and kept in the bottlewith water until shedding of pollen. Pollen samples were stored in glass vials at –20 °C.The SAM investigations were done at the Institute of Plant Physiology, Karl-Franzens--University, in Graz, Austria. Pollen samples were mounted on carbon-coated double-sidedtape, sputter coated with gold and investigated with a Phillips XL30 scanning electron mi-croscope (BORZAN and STABENTHEINER 2002, MÜLLER et al. 2003). A length of 35 pollengrains from each tree, along the longer pollen grain axis, was measured. Data were testedby Kolmogorov-Smirnov test and by ANOVA analysis. Differences among trees were es-tablished by means of Scheffé’s post-hoc test. The statistical analysis was performed usingStatistica 6.0 software.Pollen morphology was described using common terminology as in papers by ERDT-MAN (1952), MOORE and WEBB (1978), STRASBURGER et al. (1978), PUNT et al. (1994),RICCIARDELLI (1997), VON DER OHE and VON DER OHE (2000).ResultsStatistical results of pollen length measurements are presented in table 1 and Scheffé’spost-hoc test in table 2. Largest pollen grains were measured in samples of Fraxinuspennsylvanica from the RBG Hamilton (mean = 27.71mm). They were statistically longeronly from pollen grains belonging to 5 trees (trees no. 1, 6, 7,14, and 18), i.e. from treesmarked and identified as F. pennsylvaica (no. 1) in Botanical Garden Zagreb, and as Ameri-can ash from the forest plantation ^rnov{~ak (no. 6, 7, 14) and arboriculture (no. 18). The48 ACTA BOT. CROAT. 64 (1), 2005KREMER D., STABENTHEINER E., BORZAN @.smallest pollen grain (mean = 23.09 mm) was from tree no. 18 (= American ash from ar-boriculture in Zagreb), and was statistically smaller from pollen grains of all other trees ex-cept tree no. 1 (F. pennsylvanica, Botanical garden Zagreb) and no. 7 (forest plantation^rnov{~ak). Pollen grains from identified F. americana at the RBG Hamilton (tree no. 3)were statistically significantly different from the smallest pollen grains of the Americanash tree from the arboriculture in Zagreb (tree no. 18) only.Pollen grains of F. pennsylvanica (Fig. 1a) have irregular elliptic shape (more or less an-gular) in equatorial view with clearly visible apertures. In F. americana (Fig. 1d) pollengrains have irregular elliptic shape as well, somewhat less angular than the pollen of F.pennsylvanica. In both species the polar view is usually quadrangular with poorly visibleapertures. F. pennsylvanica and F. americana have commonly 4 (stephanocolpate) aper-tures. Apertures in both species are long and narrow (known as colpi), sunken with irregu-lar and diffuse margins (Fig. 1b, e). Mesocolpium is large in both species.In F. pennsylvanica and F. americana the outer layer (exina) of the sporodermis are re-ticulate (have reticulum-shaped sculptural elements). The inner areas of a reticulum mesh(lumina) are of similar size in both species. Ribs in the reticulum (muri), which limits thelumina, are firmly fused in both species and have the same width or are wider (seldom nar-ACTA BOT. CROAT. 64 (1), 2005 49POLLEN TRAITS OF AMERICAN ASHESTab. 1. Statistical analysis of pollen grain length. Thirthy five pollen grains were measured. Mini-mum and maximum values are in boldface. C. V. = coefficient of variability. 1 = Fraxinuspennsylvanica (Zagreb), 2 = F. pennsylvanica (Hamilton), 3 = F. americana, (Hamilton), 4 to18 = American ash = introduced ash species (not identified)Tree no: Mean(mm)Std. dev.(mm)C. V.(%)Minimum(mm)Median(mm)Maximum(mm)1 25.25 1.06 4.20 23.00 25.10 28.302 27.71 1.21 4.37 24.90 27.90 29.503 26.17 1.17 4.47 24.00 25.90 28.704 26.21 1.46 5.57 23.60 26.40 29.705 26.20 1.15 4.39 24.00 26.10 28.406 25.36 1.62 6.39 22.40 25.20 28.707 24.50 1.43 5.84 22.30 24.50 28.408 27.66 1.40 5.06 24.00 27.70 30.009 26.78 1.38 5.15 24.00 26.80 29.0010 26.05 1.21 4.64 24.00 25.90 28.6011 26.32 1.23 4.67 23.30 26.20 28.9012 26.50 1.27 4.79 22.30 26.40 28.8013 26.38 1.18 4.47 24.20 26.20 28.7014 25.31 0.98 3.87 23.00 25.10 28.0015 26.15 1.62 6.20 23.90 25.80 30.9016 27.06 1.25 4.62 24.70 26.90 29.7017 27.13 1.21 4.46 24.20 26.90 29.8018 23.09 1.41 6.11 20.00 22.90 25.9050 ACTA BOT. CROAT. 64 (1), 2005KREMER D., STABENTHEINER E., BORZAN @.Tab.2.ResultsofScheffé’spost-hoctestforlengthofpollengrains.*=statisticallysignificantdifference(p<0,05).1=Fraxinuspennsylvanica(Zagreb),2=F.pennsylvanica(Hamilton),3=F.americana(Hamilton),4to18=Americanash–introducedashspecies(notidentified).Treeno:1234567891011121314151617181 20.000*30.5860.46340.1470.8961.00050.5390.5091.0001.00061.0000.002*0.9990.9210.99871.0000.000*0.2970.040*0.2590.99780.000*1.0000.5390.9300.5860.003*0.000*90.025*0.9921.0001.0001.0000.6400.004*0.996100.4790.5691.0001.0001.0000.9960.2150.6441.000110.3650.6841.0001.0001.0000.9880.1420.7511.0001.000120.1590.8851.0001.0001.0000.9300.045*0.9211.0001.0001.000130.2800.7691.0001.0001.0000.9760.0970.8261.0001.0001.0001.000141.0000.000*0.9970.8880.9951.0000.9990.002*0.5690.9920.9800.8980.961150.6150.4331.0001.0001.0000.9990.3230.5091.0001.0001.0001.0001.0000.998160.002*1.0000.9951.0000.9970.2460.000*1.0001.0000.9980.9991.0001.0000.1950.993170.001*1.0000.9871.0000.9910.1740.000*1.0001.0000.9950.9981.0000.9990.1330.9841.000180.3530.000*0.000*0.000*0.000*0.005*0.6480.000*0.000*0.000*0.000*0.000*0.000*0.007*0.000*0.000*0.000*rower) than the lumina. On the muri of F. pennsylvanica there are supratectal transversalridges and sometimes granules, which are poorly visible because of the present ridges (Fig.1c). Muri of F. americana have small number of ridges so that the granules are more notice-able (Fig. 1f). The lumina in both species are different in size and irregularly shaped withobtuse angles and are not smaller toward apertures.The pollen morphology of trees under the name American ash was also investigated indetails. The muri of their pollen grains have supratectal transversal ridges and granules(Fig. 2). Because of that they were identified as pollen grains of F. pennsylvanica.ACTA BOT. CROAT. 64 (1), 2005 51POLLEN TRAITS OF AMERICAN ASHESFig. 1. SEM micrographs of pollen grains of Fraxinus pennsylvanica (a–c) and F. americana (d–f).Apertures in pollen grains of F. pennsylvanica (b) and F. americana (e). Reticulate pollengrain ornamentation in F. pennsylvanica (c) and F. americana (f). Ridges on muri of F.pennsylvanica pollen grains are abundantly present (c). Muri (ribs in reticulum which limitsthe lumina) are more smooth in F. americana with lesser ridges and granules (f).DiscussionPollen grains of Fraxinus pennsylvanica and F. americana, based on their averagelength, might be classified according to KREMP’S (1965) classification as middle large. Thelength of middle large pollen according to his classification is 25–50mm.Based on the pollen length it is not possible to distinguish the pollen of F. americanaand F. pennsylvanica. The variation of pollen length in F. pennsylvanica is broad so that therange of length includes ranges of the investigated pollen grains belonging to one identifiedF. americana tree and 13 (out of 15 investigated) American ash trees. Two smallest pollengrains are from two American ash trees, no. 7, and no. 18. This means that length of pollen52 ACTA BOT. CROAT. 64 (1), 2005KREMER D., STABENTHEINER E., BORZAN @.Fig. 2. SEM micrographs of pollen grains of two American ash trees: Tree no. 4 from forest planta-tion ^rnov{~ak (a–c), and tree no. 17 from arboricultures in Zagreb (d–f). Similar ornamen-tation on those and other American ash trees led to the conclusion that all those belong to F.pennsylvanica.is not a trait which could help in determining to which species the »American ashes« arebelonging.The number of colpi (apertures) on the pollen grains in F. americana and F. pennsyl-vanica is still not certain. JONES et al. (1995) have described the pollen of both species asstephanocolpate (with more than 3 apertures). On the contrary MARTIN and DREW (1970)described the pollen of F. pennsylvanica as tricolpate (with 3 apertures). Some of our pho-tos show that the pollen grains of F. pennsylvanica seen from a polar view have four aper-tures (Fig. 1a). Depending on the length of an aperture, if short, the aperture could be hid-den when viewed from the polar side. When seen from an equatorial side the aperture couldbe completely hidden under the pollen grain laying on a carbon-coated tape when beingprepared for microscopic research. On the other hand, if a pollen grain contains verysunken mesocolpi such a part can be misidentified as the aperture.The best possibility in differentiating pollen grains of F. americana from F. pennsyl-vanica is in their differences in ornamentation of surface. The surface of pollen grains inboth species is reticulate. Granules and ridges on the muri in F. pennsylvanica are more ex-pressed (Fig. 1c) than in F. americana pollen grains. Ridges on the muri are rare in F.americana (Fig. 1f) and surface of the muri seems more smooth, with rarely present ridgesand with fewer present, but with good visible granules. In all the samples of American ashridges on the muri on the pollen grain surfaces are readily visible (Fig. 2c, f) and abun-dantly present. Thus they have been identified as F. pennsylvanica.AcknowledgementsWe wish to thank arboriculturist Mr. Charles D. Holetich for sending us pollen samplesof Fraxinus pennsylvanica and F. americana from the RBG Hamilton, Canada, used in thisresearch.ReferencesANI], M., 1954: Dendrolo{ka i uzgojna va`nost nekoliko starih parkova u podru~juVara`dina. [um. list 78, 413–433.ANONYMOUS, 1997: Osnova gospodarenja za Gospodarsku jedinicu ^rnov{~ak od 1999. do2008. godine. J. p. »Hrvatske {ume« p. o. Zagreb, Uprava {uma Zagreb, [umarija DugoSelo, ZagrebBORZAN, @., STABENTHEINER, E., 2002: Biological and taxonomical investigations of someoak trees. Acta Bot. Croat. 61, 135–144.CIVIDINI, R., MIRTH, K., 1941: Apsolventska ekskurzija studenata {umarstva Poljoprivredno--{umarskog fakulteta u Zagrebu u mjesecu srpnju 1940. godine. [um. list 5, 187–203.]ORDA[I], F., 1894: Ne{to o onih vrstih ameri~koga drve}a, kojih gojitba bi i u nas koristnabila. 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CROAT. 64 (1), 2005 55POLLEN TRAITS OF AMERICAN ASHES

Pollen traits of some American ashes investigated by a scanning electron microscope

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