Molecular Relationships in Encephalartos (Zamiaceae, Cycadales) Based on Nucleotide Sequences of Nuclear ITS 1&2, Rbcl, and Genomic ISSR Fingerprinting

Abstract

The cycad genus Encephalartos is restricted to Africa and is threatened with extinction in most of its range. Total DNA was extracted from 51, i.e., 78 %, of the described species of Encephalartos. The accessions were sampled from the furthest western occurrence of the genus in Nigeria, via Sudan and Uganda, to southern South Africa. The sequences of nuclear ribosomal internal transcribed spacer regions 1 and 2 (ITS 1&2), the chloroplast encoded rbcL gene, and ISSR genomic fingerprinting were employed to resolve the molecular history and the relationships within the genus. Sequence alignment, as well as ISSR fingerprinting, data show low genetic variation among all analysed accessions, indicating diversification within the Pliocene/Pleistocene. ITS 1&2 data agree well with morphological and geographical characters and resolved three major genetic clusters with overlapping distribution ranges in eastern South Africa. This area, that contains the largest diversity of genotypes of Encephalartos, may have served as a Pliocene/Pleistocene refugium.

Key words

Encephalartos - phylogeny - ribulose 1,5-bisphosphate carboxylase/oxygenase - ribosomal nuclear internal transcribed spacer region (ITS) - inter simple sequence repeat PCR (ISSR-PCR)

References

• 1 Doyle J. J., Doyle J. L.. Isolation of plant DNA from fresh tissue.  Focus. (1990);  12 13-15
• 2 Dyer R. A.. The cycads of Southern Africa.  Bothalia. (1965);  8 405-514
• 3 Goode D.. Cycads of Africa. D & E Publishers (2001)
• 4 Grobbelaar N.. Phylogeny of Encephalartos species.  Encephalartos (Journal of the Cycad Society of South Africa). (2001);  66 17-18
• 27 Newton L. E.. The citation of the name Encephalartos hildebrandtii. .  Journal of East African Natural History. (2002);  91 95-96
• 5 Norstog K. J., Nicholls T. J.. The Biology of the Cycads. Ithaca, USA; Cornell University Press (1997)
• 6 Osborne R., Salatino M. L. F., Salatino A.. Alkanes of foliar epicuticular waxes of the genus Encephalartos. .  Phytochemistry. (1989);  28 3027-3030
• 7 Pant D. D.. The fossil history and phylogeny of the Cycadales.  Geophytology. (1987);  17 125-162
• 8 Schneider D., Wink M., Sporer F., Lounibos P.. Cycads, their evolution, toxins, herbivores and insect pollinators.  Naturwissenschaften. (2002);  89 281-294
• 25 Sharma I. K., Jones D. L., Forster P. I., Young A. G.. The extent and structure of genetic variation in the Macrozamia pauli-guilielmi complex (Zamiaceae).  Biochemical Systematics and Ecology. (1998);  26 45-54
• 26 Sharma I. K., Jones D. L., Forster P. I., Young A. G.. Low isozymic differentiation among five species of the Macrozamia heteromera group (Zamiaceae).  Biochemical Systematics and Ecology. (1999);  27 67-77
• 9 Stevenson D. W.. A formal classification of the extant cycads.  Brittonia. (1992);  44 220-223
• 10 Swofford D. L.. “PAUP*: phylogenetic analysis using parsimony”. Version 4.0 beta 10. Sunderland, Massachusetts; Sinauer Associates (2003)
• 11 Treutlein J., Wink M.. Molecular phylogeny of cycads inferred from rbcL sequences.  Naturwissenschaften. (2002);  89 221-225
• 12 Van der Bank H., Vorster P., Van der Bank M.. Phylogenetic relationships, based on allozyme data, between six cycad taxa indigenous to South Africa.  South African Journal of Botany. (1998);  64 182-188
• 13 Van der Bank H., Wink M., Vorster P., Treutlein J., Brand L., Van der Bank M., Hurter J.. Allozyme and DNA sequence comparisons of nine species of Encephalartos (Zamiaceae).  Biochemical Systematics and Ecology. (2001);  29 241-266
• 14 Vorster P.. Hybridization in Encephalartos. .  Excelsa. (1986);  12 101-106
• 15 Vorster P.. Taxonomy of Encephalartos (Zamiaceae): taxonomically useful external characteristics. Stevenson, D. W. and Norstog, K. J., eds. Proceedings of the Second International Conference on Cycad Biology. Milton, Queensland; Palm & Cycad Societies of Australia (1993): 294-299
• 16 Vorster P.. Encephalartos aplanatus (Zamiaceae): a new species from Swaziland.  South African Journal of Botany. (1996 a);  62 57-60
• 17 Vorster P.. Encephalartos msinganus (Zamiaceae): a new species from KwaZulu-Natal.  South African Journal of Botany. (1996 b);  62 67-70
• 18 Vorster P.. Encephalartos senticosus (Zamiaceae): a new species from northern KwaZulu-Natal and Swaziland.  South African Journal of Botany. (1996 c);  62 76-79
• 19 Vorster P.. Classification concepts in Encephalartos (Zamiaceae). Walters, T. and Osborne, R., eds. Cycad Classification Concepts and Recommendations. Wallingford, UK; CABI Publishing (2004): 69-83
• 21 White F., Moll E. J.. The Indian Ocean coastal belt. Werger, M. J. A., ed. Biogeography and Ecology of Southern Africa. The Hague; Junk (1978): 561-598
• 22 Wink M., Sauer-Gürth H., Martinez F., Doval G., Blanco G., Hatzofe O.. The use of (GACA)₄-PCR to sex Old World vultures (Aves: Accipitridae).  Molecular Ecology. (1998);  7 779-782
• 23 Wink M., Guicking D., Fritz U.. Molecular evidence for hybrid origin of Mauremys iversoni Pritchard et McCord, 1991, and Mauremys pritchardi McCord, 1997 (Reptilia: Testudines: Bataguridae).  Zoologische Abhandlungen, Staatliches Museum für Tierkunde Dresden. (2001);  51 41-49
• 24 Zietkiewicz E., Rafalski A., Labuda D.. Genome fingerprinting by Simple Sequence Repeat (SSR)-anchored polymerase chain reaction amplification.  Genomics. (1994);  20 176-183

Editor: F. Salamini