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Systematic Botany (2008), 33(2): pp. 229–236 © Copyright 2008 by the American Society of Plant Taxonomists Phylogenetic Relationships of the Neotropical Genus Dioon (Cycadales, Zamiaceae) Based on Nuclear and Chloroplast DNA Sequence Data Dolores González,1 Andrew P. Vovides, and Cristina Bárcenas Instituto de Ecología, A.C., Apartado Postal 63, Xalapa Veracruz, 91070 México 1Author for correspondence ([email protected]) Communicating Editor: Lena Struwe Abstract—Previous hypotheses of relationships within Dioon Lindl. indicated the presence of two large clades within the genus. However, relationships among species still remained unresolved. In this study, molecular phylogenetic analyses were performed with individual and combined data sets from the ITS region of the ribosomal DNA and the trnL–F region of the chloroplast DNA. To explore whether indels were a source of phylogenetically informative characters, indels were analyzed by excluding them from the analyses (Coding A); including them as ambiguous data (Coding B); as multistate (Coding C) and as binary characters (Coding D). We found that the rate of mutation in the ITS region is appropriate to solve most relationships at the species level. This is in contrast with the trnL–F rgion, which showed little variation. Our results show that most clades obtained during analyses correspond with previously recognized species within Dioon. This phylogeny groups the genus into two main clades that show clear biogeographic relationships between the species occurring along the Pacific Sierra Madre Occidental and the Atlantic Sierra Madre Oriental. Keywords—Cycads, molecular phylogeny, Dioon biogeography, ITS, trnL–F. The Neotropical cycad genus Dioon Lindl. (Zamiaceae) in- ous distribution was interrupted by the Laramide orogeny cludes 12 species from Mexico and one from Honduras (Hill giving rise to species such as D. edule developing to the east et al. 2004). Dioon is distributed mostly within mountain sys- and D. tomaselli to the west (Moretti et al. 1993). tems along the Sierra Madre Oriental and Sierra Madre Oc- DNA sequence-based studies have provided insights into cidental. On the eastern seaboard, the range is from north- phylogenetic relationships in a variety of cycads (e.g. De eastern Nuevo León to southern Veracruz and northern Luca et al. 1995; González and Vovides 2002; Treutlein and Oaxaca. There is a vicariant Dioon in Honduras (D. mejiae Wink 2002; Chaw et al. 2005). This study was conducted to Standl. & L.O. Williams) on the Caribbean coast. Along the explore the phylogenetic relationships within Dioon using Pacific seaboard, the range of Dioon extends from northwest- nucleotide sequence data from the internal transcribed spacer ern side of the Sierra Madre Occidental from central Sonora (ITS) of the nuclear ribosomal DNA (nrDNA) and the trnL–F State to southern Chiapas (Fig. 1). The genus is commonly of cpDNA. Alignment of sequences of different length is a divided into two distinct morphological groups. One group concern in phylogenetic analyses because gaps generated af- contains species characterized generally by large fronds, ter alignment can be interpreted as artifacts or as phyloge- large trunks, and massive cones, and the other group has netically informative characters. There are many studies species with generally shorter trunks, considerably shorter where gaps are ignored as ambiguous characters or are sim- fronds, and smaller cones (Norstog and Nicholls 1997). These ply deleted from the data sets arguing that they are uninfor- groups were also recovered in phylogenetic studies per- mative (e.g. Olsen 1988; Olsen and Woese 1993). However, in formed with morphology and chloroplast DNA (cpDNA) other studies gaps are considered as putative insertion or deletion events (indels) and are incorporated in the analyses RFLPs (Moretti et al. 1993) and with sequence data (Bogler as a class of phylogenetic characters using different coding and Francisco-Ortega 2004). However, species relationships schemes. Some authors include multibase gaps as binary have not been clearly established because several morpho- characters or as fifth state (e.g. Milinkovitch et al. 1994; logical characters overlap among species. In the consensus Kropp et al. 1997; Coetzee et al. 2003). Others explore levels tree based on morphology, all species of Dioon were divided of variation among indels using a coding scheme to represent into two major clades. The Spinulosum clade was fully re- such variation (e.g. Hibbett et al. 1995; González 1996; Lut- solved whereas the Edule clade collapsed in a polytomy from zoni et al. 2000; Simmons and Ochoterena 2000; Simmons et which only D. edule and D. tomasellii remained as separate al. 2001; Grubisha et al. 2002; Aagesen et al. 2005; González et clades (Moretti et al. 1993). In addition, there was lack of al. 2006; Müller 2006). Unfortunately, there is no consensus support obtained for some clades recovered by the phyloge- agreement as to how indels should be coded for phylogenetic netic analysis with cpDNA RFLPs. Discrepancies found be- analyses in spite of the big impact they have on the outcome tween morphological analysis and proposed molecular phy- of the analysis (e.g. Simons and Mayden 1997; González et al. logeny were interpreted by Moretti et al. (1993) as a fast series 2006). In this study we also aim to test the potential utility of of vicariance effects caused by new orogenies thus not allow- gaps, as characters, by assessing the effects of different cod- ing sufficient time for the accumulation of synapomorphies. ing schemes on clade support. In addition, we will explore Their molecular phylogeny was congruent with a model for the congruence between trees obtained in this study with recent species radiation and vicariance biogeography (Mor- previous phylogenetic hypothesis. etti et al. 1993), thereby setting a working hypothesis for their two main clades dividing the Dioon taxa into two groups; the Spinulosum clade composed of D. mejiae, D. spinulosum, and MATERIALS AND METHODS D. rzedowskii of the Caribbean (northern Honduras) and Gulf Taxon Sampling and Molecular Techniques—Thirty-three exemplars representing all 13 recognized species of Dioon (twelve from Mexico and of Mexico seaboard respectively and the Edule clade includ- one from Honduras) and one exemplar of Cycas rumphii were used in this ing the rest of the species. Cladogenesis being the result of study. All of the exemplars are held in the Living National Cycad Col- vicariance where a putative common ancestor with continu- lection of the Clavijero Botanic Garden at Xalapa (Appendix 1). The se- 229 230 SYSTEMATIC BOTANY [Volume 33 FIG. 1. Map of Mexico showing geographical distribution of Dioon species. Shaded areas: 1, Sierra Madre Occidental; 2, Sierra Madre Ori- ental; 3, Mexican Transverse volcanic range; 4, Sierra Madre del Sur. lection of Cycas rumphii Miq. as an outgroup was based on previous phylogenetic analyses within Cycadales where Cycas was always placed as the basal group to the remaining genera. In contrast, the sister group of Dioon has not been clearly defined. Zamia, Cycas, and Stangeria have all been proposed as putative sister groups of Dioon (Caputo et al. 1991; Moretti et al. 1993; De Luca et al. 1995; González and Vovides 2002; Rai et al. 2003; Bogler and Francisco-Ortega 2004). We did not perform any analyses to find the sister group of Dioon because the alignment gener- ated with sequences from the ITS region was ambiguous among all genera. DNA extraction, amplification and sequencing were performed as de- scribed in González and Vovides (2002). Four new primers were designed to amplify and/or sequence the ITS spacers and the trnL–F spacer of cpDNA for all the exemplars of Dioon. For the ITS spacer, a larger product including the 3Ј part of the 18S rDNA was amplified for most species using primers NS7 and ITS4 (White et al. 1990). Sequencing of the whole product was performed using the primers NS7, ITS4 and two internal Dioon-specific primers called Dioon-ITS/350 (5Ј-ATTTCGCTAC- GTTCTTCATCG-3Ј) and Dioon-ITS/180 (5Ј-GAATGCCGTGCGTATG- CAA-3Ј). For the trnL–F spacer, primer f (Taberlet et al. 1991) and a Dioon-specific primer called Dioon-c 5Ј-CGGAATTGGTAGACGCTACG- 3Ј were used for amplification. Sequencing was performed with primers f and the Dioon-specific primers Dioon-c and Dioon-trnL–F/455 5Ј- TAGAGTCCAGTTCTACATGTC-3Ј. Difficulties were encountered in ob- taining sequence data for the trnL–F regions in Dioon spinulosum A, D. tomasellii B, and D. angustifolium C. Consequently, the combined data matrix (ITS + trnL–F) including outgroup, consisted only of thirty-one exemplars (Appendix 1). Alignment of DNA Sequence Data—Sequences were aligned using the Clustal V program (Higgins et al. 1992) within the Megalign software package (Lasergene DNASTAR Inc). The alignment of the combined data set consisted of 31 OTUs. In total 2196 nucleotide positions from the ITS and trnL–F regions were used. A survey of primary homology assessment was performed on eight alignments with combinations of penalties of 10, 20, and 50 for introducing gaps and penalties of 5, 10, 20, and 50 for gap extension. Six gaps of one nucleotide and 16 of two or more contiguous nucleotides were required to align the studied taxa. Eight analyses were performed in total, utilizing single and combined data sets. Four analyses FIG. 2. Coding schemes used for indels of two or more contiguous corresponded to the ITS sequence data alone and four to the combined nucleotides. A) excluded; B) included as ambiguous characters; C) in- data set (ITS + trnL–F). The trnL–F data matrix was not analyzed indi- cluded as multistate and D) coded as binary characters (asterisks indicate vidually because there were only 13 informative characters. All matrices informative characters). were deposited in TreeBASE (study number S2004). To ascertain if gaps are useful phylogenetic characters in our data set, phylogenetic analyses were performed utilizing several different coding Lutzoni et al.

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