Leaf Morphology for Ramet Identification of Tilia cordata Mill . (Small Leaved Lime ).

1. Leaf Morphology for Ramet Identification of Tilia cordataMill. (Small Leaved Lime). Jon Tomkinson, Samuel A. Logan, Kirsten Wolff and Paul Ashton • Introduction • Cloning is an important mode of reproduction in many species • The extent of clonal reproduction may vary between populations of the same species. In particular range edge populations often contain lower heterozygosity and lower allele richness suggesting clonality more likely. • Tiliacordata is a long-lived tree species restricted to ancient woodlands with capacity for sexual and clonal reproduction. • The extent of sexual reproduction in T. cordatais determined by climatic factors with fertility declining with increasing latitude. Almost all seeds found at T. cordata’s northern limit, in the UK Lake District, are sterile (Pigott & Huntley 1981). • Henceclonal reproduction may be a factor in the persistence of northern populations which will be reflected in lower genetic diversity than those populations further south. • Pigott (1991) claims that clones of T.cordata are usually recognisable by distinctive leaves. If this is the case, clones of T.cordata could be identified in the field, helping determine population compositions without the need for molecular analysis. • Project Aims • Utilise morphometric and DNA approaches to investigate if cloned individuals of T. cordata can be identified by similar leaf morphology. • Undertake a preliminary investigation to find if northern range edge populations have more clones compared to those in the range centre. • Morphometric Methods • Material was collected from two sites: • RoudseaWood NNR, Cumbria (SD335823) at T. cordata’s northern limit. • CollywestonGreat Wood NNR, Lincs. (TF013007) in the middle of T. cordata’sUK distribution. • At each site all trees within a 20mx20m area were sampled. Hence approx 40 trees per site were sampled. Ten leaves per trees and ten morphological measurements taken per leaf (see Fig 1). PCA was used to assess how similar leaf morphology was among trees . Results PCA output of morphological scores from individual trees at Roudsea Wood (Figure 2) and Collyweston Great Wood (Figure 3) are shown below. Each point represents a single tree. Genetically identical individuals are represented with the same colour. Diamonds with no colour are genets with only one ramet and no clonal reproduction. Triangle points of the same colour are identical at all but one loci. The closer individuals are to one another the more similar they are morphologically, but this is not always the case genetically. Genetic analysis revealed twelve clonal groups in the Roudsea population and nine in the Collyweston population (α2= 0.52 NS). The modal size of clones is the same at both sites (M=2). The maximum distance that two clones of the same genet where located was 11 metres. Results from Roudsea Wood and CollywestonGreat Wood are very similar. Clones have many morphometric similarities to each other, but there are several trees which are not clones that also as similar. Observations with this study show that the variation within clones is due to various small morphological differences rather than any particular characters. Fig. 1: . • Molecular Methods • A multiplex PCR was carried out to amplify 14 variable microsatellite loci. • Microsatellites were genotyped using an ABI 3031XL genetic analyser, and scored using Genemapper v3.5 software. • Individuals with identical multi locus genotypes (MLG) were recognised using GenClone 2.0 (Arnaud-Haoud & Belkir, 2007) and GenAlEx 6.5 (Peakall & Smouse, 2012). • Individuals sharing MLG were considered to be clones. Fig. 2: Principal Component Analysis of morphometric analysis of Roudsea Wood. Fig. 3: Principal Component Analysis of morphometric analysis of Collyweston Great Wood. Discussion Clones of T. cordataare not unified by distinctive leaf morphology. Leaf characters of the same clone are often less similar than leaf characters of different clones. Hence Pigott’s (1991) assertion that clones can be identified by distinct leaf characters is not upheld by this study.Variation within a clone may be due to phenotypic plasticity or somatic mutation.There is some evidence from the microsatellite data that somatic mutation has occurred. Both Collyweston GW and Roudsea possessed clonal limes. At both sites they were less than 11m apart (results not shown). There is no significant difference between clone numbers between the two sites or mean clonal size. Hence T. cordataclone abundance and size is not influenced directly related to fertility levels between the two sites. Further work is needed to determine if this pattern is typical. However it is possible that all populations of T. cordata have persisted at least partially due to their ability to clone. References. Arnaud-Haond S. and K. Belkhir. (2007). GENCLONE:acomputer program to analyse genotypic data, test for clonality and describe spatial clonal organization. Molecular Ecology Notes 7. p15-17 . Peakall, R and Smouse, P, E. (2012)GenAlEx 6.5: genetic analysis in Excel. Population genetic software for teaching and research-an update. Bioinformatics 28 (19), p: 2537-2539. Pigott, C. (1991). Biological Flora of the British Isles. Journal of Ecology. 79 p.1147-1207. Pigott, C. and Huntley, J. (1981). Factors Controlling the Distribution of Tilia cordata at the Northern Limits of its Geographical Range. 2 History in North-West England. New Phytologist. 84 p.145-164. Further Information This project was a joint study conducted by Jon Tomkinson (jon.tomkinson@go.edgehill.ac.uk) who is a third year undergraduate student at Edge Hill University and Samuel Logan (s.logan@newcastle.ac.uk) who is a PhD student at Newcastle University. This study was part of Jon’s dissertation and the data will be used towards Samuel's PhD. We would like to thank Dr Mary Dean and our respective departments for their support and assistance.