Localidad Población Código N NF P H
Tenerife Guajara GUAT 3 0 0,00 0,000
Los Cachorros LCAT 1 0 0,00 0,000
La Palma Curva del Observatorio COBP 3 2 1,45 0,004
Espigón del Roque de los Muchachos ERMP 5 6 4,35 0,015
La Parcela Grande PAGP 5 5 3,62 0,015
El Espigón ESPP 1 0 0,00 0,000
Los Andenes LANP 30 15 10,87 0,039
El Espigón del Norte EPNP 3 0 0,00 0,000
Ciudad Real P.N. Cabañeros CABA 6 6 4,35 0,014
LLeida P.N. Augüestortes AUGÜ 36 83 60,14 0,093
Asturias P.N. Los Picos de Europa PEUR 51 97 70,29 0,097
Granada P.N. Sierra N evada SNEV 52 63 45,65 0,119
Todos 195 138 100 2,000
GENETIC DIFFERENTIATION AMONG Ilex perado ssp. lopezlilloi AND THEIR CONGENERS OF MACARONESIA González-González. E. A.1; M. A. González-Pérez1; E. Rivero1. M.J.B. Texeira-Pereira2; M. Moura2; L. Silva2 y P.A. Sosa1 1Biology Department, University of Las Palmas de Gran Canaria, Tafira Campus, 35017 Las Palmas de Gran Canaria, Spain. 2Biology Department, Universidade dos Açores, Rua Mãe de Deus 58, Apartado 1422. 9501-801, Ponta Delgada, Portugal. Introduction Description APAT-P The genus Ilex L. (Aquifoliacaea) has 4 taxa in Macaronesia: Ilex azorica Gand. 41 endemic of Azores; Ilex canariensis Poir. (Aceviño) endemic of Canary Islands and ENGO-L Madeira and Ilex perado Aiton is a Macaronesian endemism with two subspecies in the La Gomera 37 SUGO-P Canary Islands - Ilex perado ssp. platyphylla (Webb & Berthel.) Tutin and Ilex perado ssp. 50 lopezlilloi (G. Kunkel) A. Hansen & Sunding - the last, an endangered endemism of La 72 PARC-P Ilex perado Gomera Island (Fig. 1). CAFO-P 70 65 MOQU-P Tenerife 86 CRTA-P Ilex azorica TROQ-A 78 Ilex azorica 100 LAFO-A TAMA-C Gran Canaria VAHE-C La Gomera 57 TILO-C La Palma 44 Ilex canariensis 31 CALD-C Tenerife Ilex canariensis 73 MOQU-C 42 Ilex perado ssp. platyphylla LOJA-C Tenerife Nei´s Genetic Distance
0.6 0.5 0.4 0.3 0.2 0.1 0.0 Fig. 2 UPGMA based in Nei´s (1973) genetic distance to compare all taxa of Ilex Ilex perado ssp. lopezlilloi All populations are clustered according to their taxonomic range, clearly separating different groups (Fig. 2 and Fig. 3). Ilex perado populations are grouped obeying to the island of origin and not by the taxonomic classification.
Fig. 1 Origin of the samples Objectives Our main goal is to investigate the lever of genetic diversity of Ilex perado ssp. lopezlilloi and their congeners of Macaronesia. Materials and Methods Sampling Leaf tissue samples of 242 individuals [I. perado ssp. lopezlilloi (5), I. perado ssp. platyphylla (93), I. azorica (20) and I. canariensis (124)] were analyzed (Table 1). Microsatellites The genetic analyses used primers developed to Ilex leucoclada (Torimaru et al., 2004). DNA extraction following the Doyle and Doyle (1987) protocol and subsequent purification with Gene Elute PCR Clean-Up Kit. Loads to amplifications included 20 ng of DNA. 10 pmol of each primer and 24 μl of PCR Master Mix solution (Reddy-Mix. ABgene. Surrey. UK). Amplification protocol: 3' at 94 °C; 30 cycles 45" at 94 °C. 45" at 56 °C and 45" at 72 °C; and 5' 72 °C. Amplification products were detected using an ABI 3130 XL genetic analyzer and size of the fragments was determined using GenScan 2.02 and GenoTyper 1.1 programs (Applied Biosystems. Inc.). Data Analysis
All genetic diversity parameters (A, Ho, He and P), identity and genetic distance of Nei (Nei, 1973) were calculated using PopGene 3.2 (Yeh et al., 1997); Population 1.2.30beta (Langella, 2005) was used to construct a UPGMA dendrogram that displayed in Mega4 (Kumar et al., 2001); Structure 2.2 (Falush et al., 2007) was used to infer the populations structures through an implemented Bayesian analysis and GenAlEx 6.3 (Peakall and Smouse, 2006) for the principal coordinates analysis (PCoA). Fig. 3 Principal coordinates analysis (PCoA).. Table 1 Sampled populations and genetic diversity. TAXA LOCALITY CODE ISLAND Nº A Ho He P Conclusions Ilex perado ssp. lopezlilloi La Gomera ENGO-L La Gomera 5 2.12 0.50 0.44 100.00% ! Ancule, Bailadero y Reventón Both UPGMA and PCoA clearly separate populations and individuals of I. azorica and I. SUGO-P La Gomera 4 2.25 0.34 0.37 100.00% Oscuro canariensis from the rest of the populations; furthemore, they do not make a distinction Zona de Parcelas PARC-P La Gomera 5 2.50 0.38 0.39 87.50% between individuals of I. perado ssp. platyphylla and I. perado ssp. lopezlilloi. Ilex perado ssp. Apartacaminos APAT-P La Gomera 8 1.63 0.47 0.26 62.50% !The structure of Ilex perado populations is more related to the island of origin than with its platyphylla El Moquinal MOQU-P Tenerife 28 3.25 0.42 0.49 100.00% taxonomic category. Casa Forestal CAFO-P Tenerife 17 4.13 0.43 0.59 100.00% Cruz de Taganana CRTA-P Tenerife 31 3.00 0.44 0.47 100.00% Acknowledgements 93 5.38 0.43 0.60 100.00% This project has been funded by the Organismo Autónomo de Parques Nacionales (Nº Ref. Troqueira TROQ-A São Miguel 10 3.75 0.25 0.31 62.50% Ilex azorica Lago de Fogo LAFO-A São Miguel 10 4.13 0.28 0.34 75.00% 2/2005). An special recognition to Ángel Fernández and Cito Chinea (Parque Nacional 20 5.63 0.26 0.34 75.00% Garajonay), as well as to Manuel Marrero and Eduardo Carqué (Parque Nacional Teide) by their Vallehermoso VAHE-C La Gomera 7 2.38 0.49 0.38 75.00% assistance in samples collection. Lomo de la Jara LOJA-C Tenerife 11 3.43 0.57 0.56 75.00% El Moquinal MOQU-C Tenerife 31 3.57 0.69 0.53 75.00% References Ilex canariensis Las Calderetas CALD-C Tenerife 23 3.86 0.60 0.48 75.00% Doyle. J.J. & J.L. Doyle Phytochemical Bulletin of the Botanical Society of America. 19: 11-15. Los Tilos TILO-C La Palma 26 3.25 0.58 0.48 100.00% Falush. D.; Stephens. M. & J.K. Pritchard 2007 Molecular Ecology Note 7: 574-578. Available in: Tamadaba TAMA-C Gran Canaria 26 3.13 0.48 0.41 75.00% 124 5.88 0.60 0.55 100.00% http://pritch.bsd.uchicago.edu/structure.html. TOTAL 242 11.63 0.46 0.72 100.00% Kumar. S.; Tamura. K.; Jakobsen. I.B. & M. Nei 2001 Bioinformatics 17: 1244-1245.
Allele number (A). Observed heterozygosity (Ho). Expected heterozygosity (He) and Polymorphic loci percent (P). “Nº” is the number of individuals analyzed by location. Langella. O. 2005 Laboratoire Populations. Gènètique et Evolution. Centre Nacional de la Recherche Scientifique. CNRS UPR9034. Gif Sur Yvette. Available in: Results http://ftp.bioinformatics.org/pub/populations. Eight primers (ILE03-01 ILE03-38, ILE03-53, ILE03-86b, ILE04-02, ILE04-06, Peakall. R & P.E. Smouse 2006 Molecular Ecology Notes 6: 288-295. Available in: ILE04-10 and ILE05-81) were polymorphic. For the eight microsatellites analyzed a total http://www.anu.edu.au/BoZo/GenAlEx/genalex_download.php. of 94 different were observed. Nei. M. 1973 Proceeding of the National Academy of Science USA 155: 945-959. IIex perado ssp. platyphylla was the taxon that presented the highest level of genetic Torimaru. T.; Tani. N.; Tsumura. Y.; Hiraoka K. & N. Tomaru 2004 Molecular Ecology Notes 4: variation (Table 1). 531-533. UPGMA dendogram based in Nei´s distance shows that I. perado ssp. lopezlilloi is Yeh. F.C.; Yang R.C.; Boyle T.; Ye Z.H. & J.X. Mao 1997 Molecular Biology and Biotechnology more related to I. perado spp. platyphylla at La Gomera (Fig. 2) . Centre. University of Alberta.