Phylogenetic Analysis: How Old are the Parts of Your Body?

According to the National Academy of Sciences, biology students in the USA are not being adequately prepared for successful futures. Of paramount concern is a lack of sufficient training in quantitative and computational skills, which are needed to compete effectively for an array of educational and occupational opportunities. In this paper, we introduce a classroom exercise that invites students to solve a simple biological problem and illustrates the need for a computer-assisted strategy to arrive at a solution. The exercise invites students to consider the question “How old are the parts of your body?” Some features of the human body are more ancient than others. For example, our bodies have both hair and backbones, but backbones arose much earlier in evolutionary history. Our exercise relies upon MEGA 4.0, a free, visually appealing, and intuitive computer program that allows students to gather DNA or protein sequences from electronic databases, then use them to infer phylogenetic trees. Student-inferred phylogenies are used to explore the relative order in which diverse aspects of the human form evolved. In the process, students are trained to use powerful features of MEGA and encouraged through group discussion to consider additional applications of the technology they have learned. Our lesson plan includes a brief video, a web site with essential terminology and links for further exploration, a hands-on experience using MEGA, and a follow-up discussion.     

DISCORDANCE BETWEEN NUCLEAR AND CHLOROPLAST PHYLOGENIES IN THE HEUCHERA GROUP (SAXIFRAGACEAE)

Various factors, including taxon density, sampling error, convergence, and heterogeneity of evolutionary rates, can potentially lead to incongruence between phylogenetic trees based on different genomes. Particularly at the generic level and below, chloroplast capture resulting from hybridization may distort organismal relationships in phylogenetic analyses based on the chloroplast genome, or genes included therein. However, the extent of such discord between chloroplast DNA (cpDNA) trees and those trees based on nuclear genes has rarely been assessed. We therefore used sequences of the internal transcribed spacer regions (ITS-1 and ITS-2) of nuclear ribosomal DNA (rDNA) to reconstruct phylogenetic relationships among members of the Heuchera group of genera (Saxifragaceae). The Heuchera group presents an important model for the analysis of chloroplast capture and its impact on phylogenetic reconstruction because hybridization is well documented within genera (e.g., Heuchera), and intergeneric hybrids involving six of the nine genera have been reported. An earlier study provided a well-resolved phylogenetic hypothesis for the Heuchera group based on cpDNA restriction-site variation. However, trees based on ITS sequences are discordant with the cpDNA-based tree. Evidence from both morphology and nuclear-encoded allozymes is consistent with the ITS trees, rather than the cpDNA tree, and several points of phylogenetic discord can clearly be attributed to chloroplast capture. Comparison of the organellar and ITS trees also raises the strong likelihood that ancient events of chloroplast capture occurred between lineages during the early diversification of the Heuchera group. Thus, despite the many advantages and widespread use of cpDNA data in phylogeny reconstruction, comparison of relationships based on cpDNA and ITS sequences for the Heuchera group underscores the need for caution in the use of organellar variation for retrieving phylogeny at lower taxonomic levels, particularly in groups noted for hybridization.     

Molecular systematics of Saxifragaceae sensu stricto

To circumscribe Saxifragaceae sensu stricto better, as well as to elucidate generic relationships within this group, we sequenced the chloroplast gene rbcL and its 3' flanking region (yielding 1,471 bp) from 19 genera considered to represent core members of Saxifragaceae. In addition, we conducted a restriction site analysis of chloroplast DNA (cpDNA) for 21 core genera using 23 restriction endonucleases. Phylogenetic analyses using both data sets corroborate the results obtained from surveying the distribution of the loss of the intron in the chloroplast gene rp/2 in delimiting a well-defined Saxifragaceae sensu stricto. Within the Saxifragaceae s.s. clade, a number of poorly resolved, basal phylogenetic branches supports the hypothesis that Saxifragaceae s.s. radiated rapidly very early in its evolutionary history. Molecular data also indicate the presence of several strongly supported groups of genera, such as the Boykinia group (Boykinia, Suksdorfia, Bolandra, Sullivantia, Jepsonia, and Telesonix), the Heuchera group (Heuchera, Bensoniella, Conimitella, Eìmera, Lithophragma, Mitella, Tellima, Tiarelia, and Tolmiea) the Leptarrhena/Tanakaea group, and the Darmera group (Darmera, Astilboides, Mukdenia, Bergenia, and Rodgersia). Significantly, molecular data suggest that the very large, taxonomically complex genus Saxifraga may not be monophyletic. DNA data have also helped to resolve the generic relationships of problematic taxa, indicating, for example, that Telesonix and the enigmatic Jepsonia are sister taxa. In addition to its phylogenetic implications, this study provides insight into basic trends in morphological, chemical, and cytological evolution within Saxifragaceae s.s. The molecular-based phylogenies suggest multiple origins and/or losses of several classes of flavonoid compounds, as well as several independent instances of reduction in stamen and petal number, hypanthium-ovary fusion, and aneuploidy. This study also illustrates the ability of rbcL sequence data to resolve generic-level relationships in some taxonomic groups.     

Mechanisms of derived unitegmy among Impatiens species

Morphological transitions associated with ovule diversification provide unique opportunities for studies of developmental evolution. Here, we investigate the underlying mechanisms of one such transition, reduction in integument number, which has occurred several times among diverse angiosperms. In particular, reduction in integument number occurred early in the history of the asterids, a large clade comprising approximately one-third of all flowering plants. Unlike the vast majority of other eudicots, nearly all asterids have a single integument, with the only exceptions in the Ericales, a sister group to the other asterids. Impatiens, a genus of the Ericales, includes species with one integument, two integuments, or an apparently intermediate bifid integument. A comparison of the development of representative Impatiens species and analysis of the expression patterns of putative orthologs of the Arabidopsis thaliana ovule development gene INNER NO OUTER (INO) has enabled us to propose a mechanism responsible for morphological transitions between integument types in this group. We attribute transitions between each of the three integument morphologies to congenital fusion via a combination of variation in the location of subdermal growth beneath primordia and the merging of primordia. Evidence of multiple transitions in integument morphology among Impatiens species suggests that control of underlying developmental programs is relatively plastic and that changes in a small number of genes may have been responsible for the transitions. Our expression data also indicate that the role of INO in the outgrowth and abaxial-adaxial polarity of the outer integument has been conserved between two divergent angiosperms, the rosid Arabidopsis and the asterid Impatiens.     

The phylogenetic potential of entire 26S rDNA sequences in plants

18S ribosomal RNA genes are the most widely used nuclear sequences for phylogeny reconstruction at higher taxonomic levels in plants. However, due to a conservative rate of evolution, 18S rDNA alone sometimes provides too few phylogenetically informative characters to resolve relationships adequately. Previous studies using partial sequences have suggested the potential of 26S or large-subunit (LSU) rDNA for phylogeny retrieval at taxonomic levels comparable to those investigated with 18S rDNA. Here we explore the patterns of molecular evolution of entire 26S rDNA sequences and their impact on phylogeny retrieval. We present a protocol for PCR amplification and sequencing of entire (approximately 3.4 kb) 26S rDNA sequences as single amplicons, as well as primers that can be used for amplification and sequencing. These primers proved useful in angiosperms and Gnetales and likely have broader applicability. With these protocols and primers, entire 26S rDNA sequences were generated for a diverse array of 15 seed plants, including basal eudicots, monocots, and higher eudicots, plus two representatives of Gnetales. Comparisons of sequence dissimilarity indicate that expansion segments (or divergence domains) evolve 6.4 to 10.2 times as fast as conserved core regions of 26S rDNA sequences in plants. Additional comparisons indicate that 26S rDNA evolves 1.6 to 2.2 times as fast as and provides 3.3 times as many phylogenetically informative characters as 18S rDNA; compared to the chloroplast gene rbcL, 26S rDNA evolves at 0.44 to 1.0 times its rate and provides 2.0 times as many phylogenetically informative characters. Expansion segment sequences analyzed here evolve 1.2 to 3.0 times faster than rbcL, providing 1.5 times the number of informative characters. Plant expansion segments have a pattern of evolution distinct from that found in animals, exhibiting less cryptic sequence simplicity, a lower frequency of insertion and deletion, and greater phylogenetic potential.      

matK and rbcL gene sequence data indicate that Saxifraga (Saxifragaceae) is polyphyletic

The large genus Saxifraga, which consists of ≈400 morphologically and cytologically diverse species, has long been considered taxonomically complex. Phylogenetic analysis of over 2500 bp of chloroplast sequence data derived from matK and rbcL was employed to examine relationships among sections of Saxifraga, the segregate genera Zahlbrucknera, Saxifragopsis, and Cascadia, and the relationships of these taxa to other Saxifragaceae sensu stricto. Phylogenetic trees resulting from separate analyses of the matK and rbcL sequences were highly congruent; phylogenetic analysis of a combined matK–rbcL data matrix was therefore also conducted. Our analyses indicate that Saxifraga is polyphyletic, comprising two well-differentiated clades. One clade, Saxifraga sensu stricto, is the sister to the remainder of the family and consists of Saxifraga sections Irregulares, Heterisia, Trachyphyllum, Cymbalaria, Mesogyne, Xanthizoon, Porphyrion, Ciliatae, Cotylea, Ligulatae, Saxifraga, and Gymnopera. With the exception of Gymnopera, the species-rich sections of this clade are monophyletic. Also part of this clade is the problematic Zahlbrucknera paradoxa, which is allied with members of section Saxifraga. A second major clade of Saxifraga species, Micranthes sensu lato, comprises the large section Micranthes, as well as the segregate genus Cascadia, and S. tolmiei of section Merkianae. This clade is allied with the Heuchera, Darmera, and Chrysosplenium-Peltoboykinia groups of genera. The segregate genus Saxifragopsis is only distantly related to species of Saxifraga, and is instead the sister to Astilbe. The monotypic Oresitrophe is confirmed as a member of the Darmera group of genera. These results suggest that the floral features used to define Saxifraga may simply be symplesiomorphic in these well-separated Saxifraga lineages. Furthermore, the enormous cytological diversity encompassed by Saxifraga likely represents two independent instances of extensive aneuploidy and polyploidy in Saxifragaceae.     

Reduced leaf complexity in tomato wiry mutants suggests a role for PHAN and KNOX genes in generating compound leaves

Recent work on species with simple leaves suggests that the juxtaposition of abaxial (lower) and adaxial (upper) cell fates (dorsiventrality) in leaf primordia is necessary for lamina outgrowth. However, how leaf dorsiventral symmetry affects leaflet formation in species with compound leaves is largely unknown. In four non-allelic dorsiventrality-defective mutants in tomato, wiry, wiry3, wiry4 and wiry6, partial or complete loss of ab-adaxiality was observed in leaves as well as in lateral organs in the flower, and the number of leaflets in leaves was reduced significantly. Morphological analyses and expression patterns of molecular markers for ab-adaxiality [LePHANTASTICA (LePHAN) and LeYABBY B (LeYAB B)] indicated that ab-adaxial cell fates were altered in mutant leaves. Reduction in expression of both LeT6 (a tomato KNOX gene) and LePHAN during post-primordial leaf development was correlated with a reduction in leaflet formation in the wiry mutants. LePHAN expression in LeT6 overexpression mutants suggests that LeT6 is a negative regulator of LePHAN. KNOX expression is known to be correlated with leaflet formation and we show that LeT6 requires LePHAN activity to form leaflets. These phenotypes and gene expression patterns suggest that the abaxial and adaxial domains of leaf primordia are important for leaflet primordia formation, and thus also important for compound leaf development. Furthermore, the regulatory relationship between LePHAN and KNOX genes is different from that proposed for simple-leafed species. We propose that this change in the regulatory relationship between KNOX genes and LePHAN plays a role in compound leaf development and is an important feature that distinguishes simple leaves from compound leaves.     

Arabidopsis SHORT INTEGUMENTS 2 is a mitochondrial DAR GTPase

The Arabidopsis short integuments 2-1 (sin2-1) mutant produces ovules with short integuments due to early cessation of cell division in these structures. SIN2 was isolated and encodes a putative GTPase sharing features found in the novel DAR GTPase family. DAR proteins share a signature DAR motif and a unique arrangement of the four conserved GTPase G motifs. We found that DAR GTPases are present in all examined prokaryotes and eukaryotes and that they have diversified into four paralogous lineages in higher eukaryotes. Eukaryotic members of the SIN2 clade of DAR GTPases have been found to localize to mitochondria and are related to eubacterial proteins that facilitate essential steps in biogenesis of the large ribosomal subunit. We propose a similar role for SIN2 in mitochondria. A sin2 insertional allele has ovule effects similar to sin2-1, but more pronounced pleiotropic effects on vegetative and floral development. The diverse developmental effects of the mitochondrial SIN2 GTPase support a mitochondrial role in the regulation of multiple developmental pathways.     

Recent progress in reconstructing angiosperm phylogeny

In the past year, the study of angiosperm phylogeny has moved from tentative inferences based on relatively small data matrices into an era of sophisticated, multigene analyses and significantly greater confidence. Recent studies provide both strong statistical support and mutual corroboration for crucial aspects of angiosperm phylogeny. These include identifying the earliest extant lineages of angiosperms, confirming Amborella as the sister of all other angiosperms, confirming some previously proposed lineages and redefining other groups consistent with their phylogeny. This phylogenetic framework enables the exploration of both genotypic and phenotypic diversification among angiosperms.