Hladmkia 12-13: 145-157 (2001) 159 Iris cengialti Ambr. (Iridaceae) in Slovenia - karvological analyses Iris cengialti Ambr. (Iridaceae) v Sloveniji - kariološka analiza Božena Mrrič, Zinka Pavletic Botanički zavod PMF-a, Sveučilišta u Zagrebu. Marulicev trg 20/11, HR - 10 000 Zagreb E-mail: bozena@croatica.botanic.hr bvlecek: članek obravnava kanološke značilnosti alpske vrste Iris cengialti Ambr. iz serije Pallida, (A Sni Z' , V° S!0Venije komparativni mater,al pa iz Italje Ceng,o Alto -1, cengialti s. s ). Rezultat, so pokazali in.crpopolacijsku variab.lnost znotraj vrste in up- SS navajanja nSk!h P°PUlaCij k°r POdVrStC L CenSialnAmh<- ^ ^chinLis (Paulin) Abstract: The article discusses the kaiyological features of the Alpine species /. cengialti Ambr from die senes PaUdae (A. Kern.) Trinajstic. The samples were from S,oven,a (Komarča. Kobarid) and „m parative matenal was from Italy (Ceng.o Alto - I cengialti s. s.). The results showed interpopulZi varablhtywithm spee.es and the justifiability of trealing the Slovenian populations as subsperies7 c"" gialti Ambr. subsp. vochinensis (Paulin) Trinajstic. 1. Introduction A group of connected taxa of the Genus Iris L„ from the Alpine-Dinaric region of Europe characterised by dryskinned spathes was described by TRINAJSTIC (1976) as a separate series Pallidae (A. Kern.) Trinajstih. Following this opinion, the series comprehends the species Iris cengialti Ambr. (AMBROSI 1854), Iris illyrica Tomra (TOMMASINI 1875), his pseudopallida Trinajstic (TRINAJSTIC 1976) and the horticultural Iris pallida Lam. (LAMARCK 1789). The Iris with dryskinned spathe and violet flowers in the Alpine region of Italy was named after its "locus classicus" (Cengio Alto) Iris cengialti Ambr. (AMBROSI 1854). Botanists considered it a species (AMBROSI 1854, KERNER 1871, PAMPANIN1 1909, DYKES 1913, LAWRENCE 1953, TRINAJSTIČ 1976, KOHLEIN 1981, PIG-NATTI, 1982; SCHULZE, 1988, SCORT-EGAGNA et al. 1996, MITIČ 1998) or a subspecies from the species I. pallida (WEBB & CHATER 1980, MATHEW 1981 WRABER 1998, 1999). In the Slovenian Alps, plants that are different but nevertheless related to the Italian irises, have been described as /. cengialti f. vochinensis Paulin (PAULIN 1917). MAYER (1952) described these plants as a variety (/. cengialti Ambr. var. vochinensis (Paulin) Mayer), TRINAJSTltf (1976) as a subspecies (/. cengialti Ambr. subsp. vochinensis (Paulin) Trinajstic, SUSNIK (1984) as I. illyrica, TRP1N & VRES (1995) take over the Paulin name, and WRABER (1998, 1999) considered them I. pallida Lam. subsp. cengialti (Ambr.) Forster. as did WEBB & CHATER (1980), With regard to the controversy about the taxonomic status of the series Pallidae and subcategories (WEBB & CHATER 1980, MATHEW 1981, WRABER 1998, 1999), taxonomic investigations of its Alpine-Di-naric populations were carried out (MITIC 1998, MITIC et al. 1999). The presence of four separate groups was established, and to those groups a separate taxonomic status 160 B. Mri* & Z. Pavi.etic: 1ns cengialti Ambr. (Iridaceae) m Slovenia - karyological ailalYses can be ascribed, which tends to confirm the opinion of TRINAJSTIC (1976). One of those groups corresponds to the description of the /. cengialti species (Alpine populations from Italy and Slovenia). Because of the taxonomical importance of cytological data for the genus Iris and the Pallidae series (SIMONET 1934, MITRA 1956, MITRA & RANDOLPH 1959, RANDOLPH & MITRA 1959, LAUSI 1964, RICCI 1970/71, TRINAJSTIC & LOVASEN-EBERHARDT 1977, SAUER & LEEP 1979, TRINAJSTIC et al. 1980, MITIC 1991), in this paper we present results of karyologica! analyses of the Alpine populations of I. cengialti, with the intention of further explanation of their relationships and completing the re- classicus" for /. cengialti) were analysed (tab. 1). For each population, four plants were analysed. A study of somatic chromosome morphology was made using root-tip squash technique and aceto-carmine stain, after pre-treatment with p-dichlorobenzene. The most satisfactory procedure was found to be a 3hour pre-treatment in the p-dichlorobenzene at 4°C. Slides were made permanent by first removing the cover glass, with the freezing technique (C02) and the mounting of the material in Canada balsam. Morphometrical characteristics of chromosomes have been worked out based on the analyses of ten metaphase plates (according to RAFFINSKI & PASSAKAS 1976. SAUER & LEEP 1979). For each Tab. 1: The populations sampled: name of locality; co-ordinates: Latitude (N) and Longitude (E). Co-ordinates N E Locality Cengio Alto 45° 45' 30" IIe 40' 0" Komarča 46e 17' 47" 13' 47' 40" Kobarid 46° 16' 30" 13° 33' 5" suits of morphological analyses (MITIC et al. 1999). There are few data about I. cengialti chromosome morphology and number. We have only a few references about I, cengialti chromosome studies (SIMONET 1934, MITRA 1956, SUSNIK 1962, SCORTE-GAGNA et al. 1996). For Slovenia, only SUSNIK (1962) has given any information about the number of chromosomes. 2. Material and methods Two populations from Slovenia (Ko-marca and Starijski vrh, near Kobarid) and one from Italy (Cengio Alto - the "locus population and for each chromosome pair the following parameters were estimated: the arithmetical mean of relative chromosome length (RL), arithmetical mean of relative length of longer chromosome arm (LA), arithmetical mean of relative length of shorter arm (SA), arm ratio (AR) and position of centromere (PC: M-metacentric, SM-submetacentric, ST-subtelocentric chromosome). Photomicrographs were taken for the most successful metaphase plates with a microscope OPTON III, and camera lucida drawings of karyotypes were made with ZEISS apparatus. For idiograms the homologous chromosomes have been arranged in decreasing order of size. Hladnikia 13: 159-166 (2001) 161 3. Results For all the populations analysed, the mitotic number of chromosomes 2n=24 and the interpopulation variability in chromosome morphology have been established. Two groups of kaiyotypes (fig. 1) and idio-grams (fig. 2) have been established, but all karyotypes have relatively large chromosomes (tab. 2). All karyotypes have one metacentric pair of chromosomes and the others are submetacentric or subtelocentric. Interpopulation variability in chromosome morphology is obvious in the number of chromosome pairs with satellites. That is, the Slovenian populations have shown uniformity and have two chromosome pairs with satellites, white the Italian population has three chromosome pairs with satellites. CENGIO ALTO KOMARČA KOBARID ■ i i i i i M i i 11 Ii M i I I i i I H IM i i (i I i i i, i 2 3 4 5 6 7 8 9 10 11 12 10 fmi Fig. 2: idiograms of somatic chromosomes of the si specics r. cengialti populations. 162 B. Mme & Z. PaVLETIc: Iris cengiaM Ambr. (lridaceae) in Slovenia - karyoiogica) analyses advb: t„Rr"f cngiolti populations: relative length of chromosomes (RL) rel- Tmere?PC M °T ^ ^ °f shor,er ™ (SA>' . of ce - 'ZZ I1 plT SM-submetacentric, ST-subtetacentric chromosome), Chrom. no. - the ordinal number of Chrom, no. 1 2 3 4 5 6 7 8 9 io li 12 Population C. Alto KL 9,95 6,30 7,30 6,60 6,45 6,00 5,85 5,50 5,05 4,50 4,05 3,85 LA 5,13 4,40 5,48 5,05 4,59 4,22 4.75 4,30 3,94 3,55 3.23 3,15 SA 4,82 1,90 1,82 1,55 1,86 1,78 1,10 1,20 1.11 0,95 0,82 0,70 AR 1,06 2,32 3,01 3,23 2,47 2,37 4,32 3,58 3,55 3,74 3.94 4,50 PC M SM* ST* ST* SM SM ST ST ST ST ST ST Komarča RL 9.83 7,08 6,60 6,23 6,05 6.55 6,13 5,75 5,08 4,55 4,08 3,80 LA 4,98 4,98 4,95 4,35 4,23 4,77 5,03 4,43 3,87 3,68 3,28 3,16 SA 4.85 2,10 1,65 1,88 1,82 1,78 1,10 1,32 1,21 0,87 0.80 0,64 AR 1,03 2,37 3.00 2,31 2,32 2,68 4.57 3,36 3,20 4.23 4,10 4.94 PC M SM* ST* SM SM SM ST ST ST ST ST ST Kobarid RL 9,94 7,12 6.56 6,16 6,38 6,40 6,06 5.64 5.16 4,62 4,08 3.86 LA 5,04 4,80 5.01 4,33 4,47 4,60 4,93 4,48 4,02 3.72 3,18 3,06 SA 4,90 2,32 1.55 1,83 1.91 1,80 1.13 1.16 1,14 0,90 0.90 0,80 AR 1,03 2,07 3,23 2,37 2,34 2,56 4,36 3,86 3,53 4,13 3,53 3.82 PC M SM* ST* SM SM SM ST ST si- si- ST ST I * - Chromosome pairs with satellite Both Slovenian populations have the first chromosome pair metacentric; the second, fourth and sixth pairs submetacentric; the third, and seventh to twelfth pairs are subtelocentric. The second and third pairs have satellites. In the Italian population the first chromosome pair is also metacentric. The second, fifth and sixth pairs are submetacentric; the third, fourth and seventh to twelfth pairs are subtelocentric. The second, third and fourth pairs have satellites. 4. Discussion For all three populations analysed, mitotic number of chromosomes 2n=24 has been established, which corresponds to pre- vious studies of somatic chromosomes of the species I. cengialli (SIMONET 1934, M1TRA 1956, SUSNIK 1962, SCORTE-GAGNA et al. 1996) and of the Paliidae series (SIMONET 1934, MITRA 1956, I-OVE & LOVE 1974, M1T1C 1991, MITJC 1998). AH karyotypes have been characterised as having relatively large, morphologically variable chromosomes, with one metacentric pair of chromosomes (the first pair), the others being submetacentric or subtelocentric. Also, morphometrical analysis of chromosomes has shown up variability at the population level, where two groups can be distinguished. That is, in one group there are the Slovenian populations, which have revealed uniformity and have two chromosome pairs with satellites, and one submeta- Hladnikia 13: 159-166 (2001) 163 centric pair more than Italian population. The other group is made up of the Italian population with three chromosome pairs with satellites. Comparison of all the idiograms from this investigation with those shown in former papers (MITRA 1956, LAUSI 1964, MITIC 1991, SCORTEGAGNA et al 1996), shows chromosome morphology similarities among some of the populations. For example, the idiogram of the Italian population Cengio Alto resemble that of the Italian population Passo di San Uboldo (SCORTEGAGNA et al. 1996). Idiograms of Slovenian populations resemble those of 1. illyrica from the north Adriatic littoral (MITIC 1998). The Italian population of I. cengialti has an idiogram resembling those of I. illyrica from the northeast Adriatic littoral (MITIC 1998), with a very similar fourth satellite chromosome pair. The existence of chromosome morphological variability has already been established for different species of the genus Iris (MITRA 1956, SAUER & LEEP 1979), the reason for which might be affinity of large chromosomes to structural mutations SAUER & LEEP 1979). For the investigated populations of the /. cengialti species, five or six pairs of symmetrical (metacentric and submetacentric) chromosomes have been established. Because of the consideration that the number of (a)symmetric chromosomes can reflect more or less of a range of speciation (SI-MONET 1934, MITRA 1956), and with regard to the more symmetric chromosomes in /. cengialti populations and the whole Pallidae series (MITIC 1998), we can presume that the speciation process within the series is not complete. The results of our analyses complement the results of morphological analyses of the Alpine-Dinaric populations of the Pallidae series (MITKi et al. 1999) and tend to bear out the opinion about the taxonomical range of the Slovenian dry skinned Irises as constituting a separate subspecies named I. cengialti Ambr. subsp. vochinensis (Paulin) Trinajstic, 5. Summary Within taxonomical investigations of the Alpine-Dinaric populations of the series Pallidae (A. Kern.) Trinajstic, Genus Iris L. (MITIC 1998, MITIC et al. 1999), the presence of four separate groups has been established. A separate taxonomic status can be ascribed to these groups, which tends to confirm the opinion (TRINAJSTIC 1976) that there are four species in this complex: I. pallida Lam. (LAMARCK 1789), 7 cengialti Ambr. (AMBROSI 1854), I. illyrica Tomm. (TOMMASINI 1875) and /. pseudopallida Trinajstic (TRINAJSTIC 1976). In this paper we present Ihe results of karyological investigations of the Alpine species /. cengialti, with the accent on the Slovenian populations named I. cengialti Ambr. subsp. vochinensis (Paulin) Trinajstic. Two populations from Slovenia - Ko-marca and Kobaricl (Starijski Vrh) were analysed. The results were compared with those of investigations into typical I. cengialti from the Italian population from Cengio Alto (the "locus classicus" for /. cengialti). A study of somatic chromosome morphology was made using root-tip squash technique and aceto-carmine stain, after pre-trcatment with p-dichlorobenzene. For each population and for each chromosome pair, chromosome length (with length of longer and shorter arm), arm index and type of chromosomes were estimated. The results are shown in a table (tab. 2), in photomicrographs of karyograms (fig. 1) and by idiograms (fig. 2). For all the populations analysed, the mitotic number of chromosomes 2n=24 _B. Mine & z. Pavleti* IriscengialU Ambr. (Iridaceae) in Slovenia - karyological analyses with relatively large chromosomes has been established. All karyotypes have one metacentric pair of chromosomes, while the others are submetacentric or subtelocentric. Interpopulation variability in chromosome morphology and a different number of chromosome pairs with satellites have been established. The Slovenian populations revealed uniformity and have two chromosome pairs with satellites and one submetacentric pair more than the Italian population. The Italian population has an idiogram with three chromosome pairs with satellites. Those results tend to confirm the opinion that the taxonomical range of Slovenian dryskinned irises constitutes a separate subspecies, named I cengialti Ambr. subsp. vochinensis (Paulin) Trinajstič. Karyogram shape similarity in the populations analysed and in other populations of the Pallidae series point at their relationship and indicate that the procession of spe-ciation within the series is still incomplete. 6. Sažetak U okviru taksonomskih istraživanja alp-sko-dinarskih populacija serije Pallidae (A. Kem.) Trinajstič, roda his L. (MITIČ 1998, MITIČ et al. 1999) utvrdena je prisutnost Četiriju odvojenih skupina, kojima se može pripisati rang vrste, što potvrduje mišljenje (TRINAJSTIČ 1976) o postojanju četinju vrsta tog kompleksa: /. pallida Lam. (LAMARCK 1789), / cengialti Ambr. (AM-BROSI 1854), /. iUyrica Tomm. (TOMMASINI 1875) i I. pseudopallida Trinajstič (TRINAJSTIČ 1976). Ovim radom prezentiramo komparativna kariološka istraživanja alpske vrste i cengialti. Istražene su dvije populacije iz Slovenije - Komarča i Kobarid (Starijski vrh), opisane kao I. cengialti subsp. vochi- nensis (Paulin) Trinajstič. Rezultati su kom-parirani s rezultatima istraživanja tipične vrste I. cengialti iz talijanske populacije Cengio Alto ("locus classicus" za I. cengialti). Analizirani su mitotski kromosomi iz meristema korjenovih vršaka, dobiveni standardnem "squash" metodom, bojenjem ace-tokarminom, nakon pretretmana p-diklor-benzenom. Za svaku populacijti i kromosomski par izračunati su relativna duljina kromosoma (s duljinom duljeg i kračeg kraka), indeks krakova i tip kromosoma. Rezultati su prikazani tabelarno (tab. 2), te fotografijama kariograma (fig. 1) i idiogrami-ma (fig. 2). Za sve tri istražene populacije vrste /. cengialti utvrden je mitotski broj kromosoma 2n=24, a sve kariotipove karakterizira-ju relativno veliki kromosomi varijabilne morfologije. S vi kariotipovi imajupojedan metacentričan par kromosoma (prvi par), a ostali kromosomi su ili submetacentric™ ili subtelocentrični. Takoder je utvrdena inter-populacijska varijabilnost morfologije kromosoma i broja satelitskih kromosoma Slovenske populacije pokazuju uniform-nost idiograma u kojima su utvrdena dva para satelitskih kromosoma i jedan submetacentrics par više, dok su u talijanskoj populaciji utvrdena tri para satelitskih kromosoma. Ovakvi rezultati, uz rezultate morfološke analize (MITIČ et al. 1999), doprinos su mišljenju da se slovenskim populacijama perunika suhokožičastih spa-ta može pripisati taksonomski status podvrste - I. cengialti Ambr. subsp. vochinensis (Paulin) Trinajstič. Sličnost u izgledu kariograma istraženih populacija s kariogramima ostalih populacija serije Pallidae ukazuje na njihovu povezanost i nezavršen proces specijacije un-utar serije. Hladnikia 13: 159-166 (2001) 165 7. References Ambrosi, F., 1854: Flora del Tirolo Méridionale 1 Padova Dykes, W. H. 1913: The Genus Iris. London Kerner, A., 1871 : Über Iris cengialti Ambrosi. Österr. Bot z 21-225-231 Köhlein, F., 1981: Iris. 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WR Ljubljana 9991 L' ^ ^ ^ **** S'°Venije' TdlniŠka založba Sk,venije-