Cladistics Cladistics 27 (2011) 559–580 10.1111/j.1096-0031.2011.00352.x A phylogenetic analysis of morphological and molecular characters of Lithospermum L. (Boraginaceae) and related taxa: evolutionary relationships and character evolution James I. Cohen* Department of Biology and Chemistry, Texas A&M International University, LBVSC 379E, 5201 University Blvd, Laredo, TX 78041, USA Accepted 12 January 2011 Abstract Lithospermum (Boraginaceae) includes ca. 60 species and exhibits a wide range of floral, palynological, and vegetative diversity. Phylogenetic analyses based on 10 chloroplast DNA regions and 22 morphological characters were conducted in order to (i) examine evolutionary relationships within Lithospermum and among related genera of Boraginaceae, and (ii) investigate patterns of morphological evolution. Several morphological features, such as long-funnelform corollas, faucal appendages, reciprocal herkogamy, and evident secondary leaf venation, have evolved multiple times within the genus. In contrast, other morphological features, including the presence of glands and the position and number of pollen pores, are less plastic and tend to characterize larger clades. Some features, including the presence of glands, are interpreted as symplesiomorphic for Lithospermum, while others, such as evident secondary leaf venation, appear to have originated repeatedly. The range of structural diversity that occurs among the species of Lithospermum suggests the potential utility of this genus as a model for integrative studies of evolution, development, and molecular biology. Ó The Willi Hennig Society 2011. Lithospermum L., a genus in the family Boraginaceae, the other New World members of Lithospermeae, previ- comprises ca. 60 species, with a centre of diversity in ously placed in Lasiarrhenum I.M. Johnst., Macromeria Mexico and the south-western United States. Recently, D. Don, Nomosa I.M. Johnst., Onosmodium Michx., Cohen and Davis (2009) and Weigend et al. (2009) Perittostema I.M. Johnst., and Psilolaemus I.M. Johnst. reconstructed phylogenies of Lithospermum and related (Cohen and Davis, 2009). With this broader generic genera of Lithospermeae, the tribe to which Lithosper- circumscription, Lithospermum exhibits a wide range of mum belongs. Cohen and Davis (2009) utilized 10 floral, palynological, and vegetative diversity. chloroplast DNA (cpDNA) regions, and Weigend et al. In light of the broad range of structural diversity (2009) concatenated two cpDNA regions along with the among its species, Lithospermum may be a useful taxon nuclear ribosomal internal transcribed spacer (ITS). Both in which to investigate character evolution. Some of these analyses resolved Lithospermum as non-mono- features, such as flowers with long-funnelform corollas phyletic, with members of related genera nested among its (> 4 cm in length) and exserted anthers and stigmas species. In light of these relationships, Cohen and Davis (termed Macromeria-type flowers), appear to have (2009) expanded the circumscription of Lithospermum, originated multiple times (Cohen and Davis, 2009). and under this broader interpretation the genus appears However, the phylogenetic distributions of other char- to be monophyletic. Lithospermum currently includes the acters, including type of herkogamy, pollen shape, and species that traditionally have been recognized as mem- pattern of leaf venation, remain unexamined. bers of the genus (Johnston, 1952, 1954a,b) as well as all The goals of the present study are twofold: (i) to reconstruct a phylogeny of Lithospermum (herein *Corresponding author: the ingroup) and other members of Lithospermeae E-mail address: [email protected] and Boraginaceae (herein the outgroup) through a Ó The Willi Hennig Society 2011 560 J.I. Cohen / Cladistics 27 (2011) 559–580 combination of cpDNA sequence data and morpholog- www.ebi.ac.uk/Tools/muscle/index.html) using the de- ical character data, and (ii) to investigate patterns of fault settings, with subsequent adjustments made by eye. morphological character evolution. The results of the The matrices that include the 10 cpDNA regions are present study will allow for the identification of char- missing ca. 20% of the sequence data. This number is due, acters that diagnose larger clades and that may prove in part, to failed amplifications for some cpDNA regions predictive for future taxonomic classifications. for a limited number of species, but the majority of missing A great deal of discussion has taken place during the data are confined to a set of 10 species for which data were past 20 years regarding the inclusion of morphological obtained from GenBank. With the exclusion of these characters in phylogenetic analyses (e.g. Eernisse and species, only 5.7% of the data are missing. The topology of Kluge, 1993; Luckow and Brunneau, 1997; Scotland the consensus tree, with respect to the remaining species, et al., 2003; Wiens, 2004). I believe that the most does not differ when these 10 species are excluded. comprehensive tests of phylogenetic relationships and Sequences generated for the present study are deposited patterns of character evolution result from the integra- in GenBank (GenBank numbers in Table S1), and the tion of multiple types of critically examined data (e.g. matrix is available at TreeBASE (http://purl.org/phylo/ Eernisse and Kluge, 1993; Luckow and Brunneau, treebase/phylows/study/TB2:S1130). 1997), and the present study employs this holistic approach to phylogenetic analysis and the investigation Morphological coding of character evolution. The morphological matrix includes 22 characters (Table 1). Half of the characters are binary, while the Materials and methods other half are multistate. Morphological character data were gathered from Taxon sampling living plant material, herbarium specimens from BH, CAS, F, GH, MEXU, MICH, NY, TEX ⁄LL, US, and The taxon sampling employed in the present study WISC, and digital images of species. Published descrip- includes 67 species (Table S1). Thirty-seven belong to the tions also were consulted (Johnston, 1952, 1953a,b, ingroup, and this sampling represents both the morpho- 1954a,b; Valentine and Chater, 1972; Al-Shehbaz, 1991; logical and geographical range of variation within Litho- Zhu et al., 1995), as were other peer-reviewed publica- spermum sensu Cohen and Davis (2009). The outgroup tions (Dı´ez et al., 1986; Jian-Chang et al., 1995; Boyd, comprises 30 species from related genera of Boragina- 2003; Selvi and Bigazzi, 2003; Aytas Akc¸ in and Ulu, 2007; ceae: three from Boragineae, seven from Cynoglosseae, Thomas et al., 2008; Ferrero et al., 2009). I attempted to one from Echiochileae, and 19 from Lithospermeae. observe at least 20 specimens for each species; however, given the limited quantity of representative material for DNA sequence data some taxa, fewer specimens were sometimes examined. This occurred more frequently with outgroup than with Sequence data from one protein-encoding cpDNA ingroup species. If a species included multiple states for a region, matK; and from nine non-encoding cpDNA character, the species was scored with all applicable states regions: ndhF–rpl32, psbA–trnH, psbJ–petA, the rpl16 for that character. In a few cases where it was not possible intron, trnK–rps16, trnL–rpl32, trnQ–rps16, ycf6– to collect data (often related to pollen) for a particular psbM, and trnL–trnF, were included in the present species, information for a specific character was based on study. The majority of the species were collected from observations of congeneric species. For the morpholog- wild populations. For these taxa, herbarium specimens ical matrix, 1.5% of the cells are scored as missing. were collected and deposited at the Bailey Mortorium Herbarium (BH) at Cornell University, and leaf tissue Phylogenetic analysis was dried and preserved in silica gel for subsequent DNA extraction. Taxa not collected from natural Three matrices were constructed: the cpDNA matrix populations were obtained from gardens (Cornell Plan- comprises cpDNA sequence data plus scores for tations, Missouri Botanical Garden, and National structural features of DNA regions (gaps, inversions, Botanic Garden of Belgium) as leaf samples preserved and unusual nucleotide motifs1); the morphological in silica gel, or as DNA isolations from either the DNA bank of the Royal Botanic Gardens, Kew, UK or the 1An unusual nucleotide motif is defined as a short (between seven and South African National Biodiversity Institute (SANBI). 10 nucleotides in length) non-inverted, non-length-variable (or shorter DNA isolation and PCR and sequencing protocols are by one nucleotide) sequence of nucleotides that occurs in two or more taxa and differs substantially in composition (more than 50% of the the same as those described in Cohen and Davis (2009). aligned nucleotides) from the sequences of most other species. Each Sequence data were aligned using the MUSCLE server unusual nucleotide motif was treated as a single character, as each is at the European Bioinformatics Institute (EBI) (http:// treated provisionally as having arisen from a single event. Table 1 Morphological characters, their states, and additional information Character Character states Comments 1 Position of leaves (0) cauline and basal (1) only cauline (2) cauline and A pseudobasal rosette is defined as a rosette that is sometimes present and pseudobasal may be ephemeral. This type of rosette includes leaves that, although they may have short internodes between them, are not necessarily from the base of