The Hybrid Origin of Calamagrostis ×Gracilescens (Poaceae) in Poland Inferred from Morphology and Aflp Data
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Vol. 79, No. 1: 51-61, 2010 ACTA SOCIETATIS BOTANICORUM POLONIAE 51 THE HYBRID ORIGIN OF CALAMAGROSTIS ×GRACILESCENS (POACEAE) IN POLAND INFERRED FROM MORPHOLOGY AND AFLP DATA BEATA PASZKO1, MARCIN NOBIS2 1 W. Szafer Institute of Botany, Polish Academy of Sciences Lubicz 46, 31-512 Kraków, Poland e-mail: [email protected] 2 Department of Plant Taxonomy and Phytogeography, Jagiellonian University Kopernika 27, 31-501 Kraków, Poland (Received: March 23, 2009. Accepted: January 18, 2010) ABSTRACT The morphology of Calamagrostis canescens and C. stricta recorded in the Ma³opolska Upland (the vicinity of Zbijów Ma³y, ca. 10 km northeast of the town of Skar¿ysko-Kamienna, Central Poland) was examined due to in- termediate individuals found in sympatric populations of these species. Both putative parents as well as individu- als that appeared hybrid-like were found in an extensive, wet hay-meadow. Various vegetative and reproductive characteristics were studied to identify hybrids. Interestingly, Polish accessions of C. ×gracilescens exhibited so- me degree of morphological intermediacy but resembled C. stricta in spikelet morphology. Branching of the mid- culm, the number of nodes per culm, callus hair length and relative callus hair length are the best characters to di- stinguish the Polish C.×gracilescens. AFLP analysis proved to be suitable for detecting recent hybridization events between C. canescens and C. stricta. Analysis of the Bayesian clustering analysis showed that C. ×gracile- scens were subjected to gene flow from the C. canescens gene pool as well as from the C. stricta gene pool. KEY WORDS: Calamagrostis ×gracilescens, C. canescens, C. stricta, hybrid, habitat conditions, mor- phology, AFLP markers, gene flow. INTRODUCTION and was named C. gracilescens Blytt, a name which also included a number of apomictic derivatives (Nygren 1946, With approximately 270 species, Calamagrostis Adans. page 205). At present it is known as C. ×gracilescens sensu Clayton and Renvoize (1986) is one of the largest (Blytt) Blytt. It is a very rare hybrid in Central Europe and genera in the family Poaceae. As is the case with many the largest concentrations of its localities have been repor- grass genera, an important feature of Calamagrostis is the ted from southern Sweden and Finland (Palmgren 1933- prevalence of interspecific hybrids (Nygren 1962). There -1934; Nygren 1962; Hämet-Ahti et al. 1998; Karlsson and are at least 15 hybrids given by Conert (1989) in Central Christofferson 2007). Single localities of this hybrid taxon Europe and 20 listed by Tzvelev (1976) in the former are known from the Netherlands, Britain and Norway USSR within the genus Calamagrostis alone. The evidence (Holmberg 1922; Corporaal 1984; Crackles 1994, 1995). for hybrids is usually derived from studies on their mor- In Britain, C. ×gracilescens has the status of a rare species phology and reproductive biology (Nygren 1962; Scholz (Preston et al. 2002; Cheffings 2004) and is classified as 1964; Crackles 1994, 1995). Calamagrostis hybrids vary vulnerable by Cheffings et al. (2005). considerably in their frequency and distribution. Some are Populations of Calamagrostis canescens and C. stricta found at numerous localities across Europe, whereas others are known in the Ma³opolska Upland (Central Poland), are restricted to single locations. where they are distributed throughout the region. The two The hybrid between Calamagrostis canescens (Weber) species have usually been delimited from each other based Roth and C. stricta (Timm) Koeler [=C. neglecta (Ehrh.) on spikelet traits, especially the length of callus hairs sur- Gaertn.] was for a long time given the rank of a species rounding the florets, length of lemma awn and position of 52 HYBRID ORIGIN INFERRED FROM MORPHOLOGY AND AFLP Paszko B. et al. awn insertion on the lemma, as well as culm traits such as top of the culm. One well-developed spikelet per individu- the number of culm nodes, culm branching and the structu- al was used for measurement of spikelet characters. Measu- re of the upper leaf surface. Typical specimens can be clas- rements of spikelet characters were taken with a stereo mi- sified appropriately using these characters. C. canescens croscope (Nikon SMZ-10A) with ×20. and C. stricta occasionally occur sympatrically. At Zbijów Data were analyzed using Statistica ver. 8.0 for Win- Ma³y village (Ma³opolska Upland), plants having certain dows (StatSoft 2007). Differences in the means of three morphological features intermediate between C. canescens groups (two parental species and one hybrid population) and C. stricta were present together with the two species for morphological characters were examined using one- and were believed to be their hybrid: Calamagrostis ×gra- way analysis of variance (ANOVA). Principal Component cilescens (Blytt) Blytt (its occurrence in Poland was men- Analysis (PCA on the correlation matrix) was applied to tioned by Nobis (2007) and Paszko and Nobis (2007). examine morphological variation patterns in C. ×gracile- The aim of this paper is to evaluate characters used to di- scens and its putative parents. stinguish a hybrid between C. canescens and C. stricta. A population of the putative hybrid C. ×gracilescens is Amplified fragment length polymorphism (AFLP) examined to test the current view that this taxon arose from Fifty three leaf samples of C. ×gracilescens, C. cane- hybridization between C. canescens and C. stricta. The scens, and C. stricta from Poland, Czech Republik, Swe- structure of phenotypic variation in morphological charac- den, Finland, and Iceland were collected in silica gel prior ters is therefore analyzed to investigate the relationship be- to the AFLP analysis. Nineteen accessions of C. ×gracile- tween the hybrids and their putative parents. It should help scens were taken into AFLP analysis. Eight Polish acces- to exclude characters that were considered discriminatory sions came from the locality of Zbijów Ma³y (Central Po- by previous authors but display significant low-scale varia- land). Eleven Swedish accessions were descended from 3 tion. In this study, an AFLP marker system has been used localities: seven from Kiaholmen Island, three from Hunna with the objective of clarifying the taxonomic status of C. village (both in the vicinity of Lake Skatelövfjorden in ×gracilescens compared to its putative parental species. Småland Prov.), and one from the Singö Island (Upland Prov.). Eighteen accessions of C. canescens from Poland, Czech Republik, Sweden, and Finland, and 16 of C. stricta MATERIAL AND METHODS from Poland, Sweden, and Iceland were taken into account for comparison (see Appendix 1). Total genomic DNA was Specimens extracted from 20 mg of dried leaf tissue following DNea- Field observations were carried out in the years 2002- sy Plant Mini Kit protocol supplied by the manufacturer -2007. Detailed investigations were performed in early July (Qiagen). AFLP analysis was carried out according to Vos 2004 and repeated in the seasons of 2005, 2006, and 2007. et al. (1995) with minor modification. Preamplification and Phytosociological relevés were performed using either the selective amplification were performed as described by Braun-Blanquet method or floristic checklists compiled at Vos et al. (1995). Three primer pairs (E+AGT/M+CAG, the sites where C.×gracilescens plants and other accompa- E+ATG/M+CGC, and E+ATT/M+CTA) were chosen for nying plants formed homogenous patches of vegetation selective PCR based on a preliminary survey of 12 primer (Nobis 2007). The nomenclature follows relevant authors: pairs. The resulting PCR products were separated with the vascular plant species Mirek et al. (2002), mosses internal size standard GeneScan 500Rox (Applied Biosy- Ochyra et al. (2003); the names of syntaxa are given after stems) in an automated DNA sequencer ABI 3100-Avant Matuszkiewicz (2001). The herbarium material is deposited (Applied Biosystems) and analysed using GeneMapper soft- in the herbarium of the Institute of Botany, Polish Academy ware packages, version 4.0 (Applied Biosystems). AFLP of Sciences (KRAM) and in the herbarium of the Institute bands ranging from 50 to 500 base pairs were scored. of Botany, Jagiellonian University (KRA) in Kraków. Unweighted pair group method with arithmetic mean (UPGMA) was applied with restriction sites similarity co- Analysis of morphological characters efficiency method as described by Nei and Li (1979) using Morphological characters that were investigated are tho- Treecon for Windows, version 1.3b (Van de Peer & De se that commonly separate the two parental species. They Wachter 1994). Internal support for groupings was asses- included the following culm characters: culm length, pa- sed using the bootstrap procedure of Felsenstein (1985) nicle length, leaf length and width (at the second node with 2000 replications. Pairwise fixation index (FST) be- from the top), presence of long white hairs on upper leaf tween pairs of taxa was assed using Arlequin ver. 3.0 surfaces (0 not hairy, 1 sparse hairs, 2 hairy), number (Excoffier et al. 2005). Principal components analysis of culm nodes, culm branching (at the second node from (PCA on the variance-covariance matrix) was used as to the top), length of the basal branch of the panicle, peduncle explore the main patterns of genetic variation of C. ×graci- length, as well as the following spikelet characters: lower lescens in comparison with its putative parental species. and upper glume length and width (LGL, LGW, UGL, These analyses were conducted using Statistica ver. 8.0 for UGW), lemma and palea length (LL, PL), callus hair Windows (StatSoft 2007). Usually, the PCA of the cova- length (CHL), rachilla extension length (RL) (if absent, co- riance matrix is meaningful only if the variables are de: 0), rachilla extension length with hairs (RHL), awn expressed in the same units. Results of PCA were consi- length (AL) and awn insertion (AWNINS) (measured as stent with Principal Coordinates Analysis performed using the length from the base of the lemma to the point of awn Euklidean distances. The gene flow between the two puta- insertion).