Molecular Phylogenetics and Evolution 36 (2005) 509–522 www.elsevier.com/locate/ympev 1055-7903/$ - see front matter  2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2005.04.018 AmpliWcation of noncoding chloroplast DNA for phylogenetic studies in lycophytes and monilophytes with a comparative example of relative phylogenetic utility from Ophioglossaceae Randall L. Small a,¤ , Edgar B. Lickey a , Joey Shaw a , Warren D. Hauk b a Department of Ecology and Evolutionary Biology, The University of Tennessee, Knoxville, TN 37996, USA b Department of Biology, Denison University, Granville, OH 43023, USA Received 14 June 2004; revised 6 April 2005 Available online 1 June 2005 Abstract Noncoding DNA sequences from numerous regions of the chloroplast genome have provided a signiWcant source of characters for phylogenetic studies in seed plants. In lycophytes and monilophytes (leptosporangiate ferns, eusporangiate ferns, Psilotaceae, and Equisetaceae), on the other hand, relatively few noncoding chloroplast DNA regions have been explored. We screened 30 lycophyte and monilophyte species to determine the potential utility of PCR ampliWcation primers for 18 noncoding chloroplast DNA regions that have previously been used in seed plant studies. Of these primer sets eight appear to be nearly universally capable of amplifying lycophyte and monilophyte DNAs, and an additional six are useful in at least some groups. To further explore the application of noncoding chloroplast DNA, we analyzed the relative phylogenetic utility of Wve cpDNA regions for resolving relationships in Bot- rychium s.l. (Ophioglossaceae). Previous studies have evaluated both the gene rbcL and the trnL UAA –trnF GAA intergenic spacer in this group. To these published data we added sequences of the trnS GCU –trnG UUC intergenic spacer + the trnG UUC intron region, the trnS GGA –rpS4 intergenic spacer + rpS4 gene, and the rpL16 intron. Both the trnS GCU –trnG UUC and rpL16 regions are highly variable in angiosperms and the trnS GGA –rpS4 region has been widely used in monilophyte phylogenetic studies. Phylogenetic resolution was equivalent across regions, but the strength of support for the phylogenies varied among regions. Of the Wve sampled regions the trnS GCU –trnG UUC spacer + trnG UUC intron region provided the strongest support for the inferred phylogeny.  2005 Elsevier Inc. All rights reserved. Keywords: Botrychium; Chloroplast DNA; Ferns; Lycophytes; Ophioglossaceae; Pteridophytes; Monilophytes 1. Introduction Chloroplast DNA (cpDNA) sequences are the pri- mary source of characters for phylogenetic studies in plants. Many early studies focused on protein-coding gene sequences such as rbcL and were designed to eluci- date phylogenetic relationships among higher-level taxa (e.g., Chase et al., 1993). Subsequently, the potential util- ity of noncoding regions of the chloroplast genome was recognized for lower-level (intergeneric, interspeciWc, and intraspeciWc) studies (e.g., Taberlet et al., 1991). Noncoding regions such as introns and intergenic spac- ers often display more variation on a per site basis than coding regions, presumably due to fewer functional constraints. In angiosperm systematics the application of noncod- ing cpDNA sequences to low-level phylogenetic studies is now routine (e.g., Shaw et al., 2005; and references therein). A large number of diVerent noncoding regions of the chloroplast genome have been investigated in angiosperms, some of which are highly variable while others show relatively little variation (Shaw et al., 2005). These investigations have been facilitated by the large * Corresponding author. Fax: +1 865 974 2258. E-mail address: rsmall@utk.edu (R.L. Small).