Phylogenetic data
Does the phylogenetic history of Syntrichia suggest diversification (possibly convergent) in physiological traits, OR conservation of physiology and subsequent colonization of suitable environments by specialized lineages?
We plan to take advantage of the two high quality reference genomes to develop a set of sequence capture probes (http://www.mycroarray.com/index.html), using customized target enrichment MYbait TM technology for next-generation sequencing, to a large number (empirically determined from the genomes) of orthologous conserved nuclear genes. The use of sequence capture technology allows us to not only broadly sample the genomes of the living collections of Syntrichia we will collect within the western US but also to sample broadly world-wide using herbarium specimens. The fragmented DNA that can be extracted from herbarium specimens lends itself easily to nextgeneration sequencing and has been exploited in several organisms including plants (Jones and Good 2015).
DNA isolation from fresh materials will be accomplished using our standard protocols (described above) and from herbarium specimens using protocols described by Drábková (2014). The sequence capture enrichment will be achieved using the protocols described by Mandel et al. (2014) which generate bar-coded libraries that are suitable for pooling and sequencing using standard Illumina sequencing protocols. Once sequences are obtained, sequence reads will be sorted by individual barcode and aligned to the Syntrichia genomes for validation after removal of restriction site and barcode sequences. See section D5 for analysis.
Phylogenetic and Systematic Analyses
The functional components of this study will be set into a phylogenetic context, which will serve to integrate them (Question 4, Table 1). Phylogenies are fractal; there are lineages within lineages (Mishler 2005) and all are of interest for functional questions focused at different levels (i.e., different dimensions of biodiversity). To examine these different dimensions while under time and budget constraints, we will focus the intensity of our sampling at two levels:
(1) narrowly within S. caninervis and S. ruralis in order to understand microevolutionary patterns and processes in detail;
(2) and broadly across the genus Syntrichia including its worldwide range, but with an emphasis on North America in order to complement our population genetic and ecological studies.
This stratified sampling approach bridges the different dimensions of the proposed research, allowing an integrated view of how genomic and physiological variation relates to population genetic structure, how differences in ecological performance and biogeographic history result in assembly of lineages into functioning communities at the ecosystem level, how macroevolutionary patterns and processes result in (and from) lineage divergence, and how best to reflect these in a natural taxonomic classification.
Phylogeny reconstruction and classification
(1) Aligned nucleotide sequence data will be generated as described in section D2.
(2) A comparable morphological matrix will be built, beginning with the data set of Mishler (1985).
(3) Phylogenies will be estimated using both MP (maximum parsimony) and ML (maximum likelihood) methods, assessing congruence among trees built with separate genes, as well as with combined analyses of morphological and molecular data. Because concatenation of numerous loci can sometimes be positively misleading, we will also use methods to estimate the species tree that account for discordance between individual gene trees in a maximum likelihood coalescent framework (MP-EST; Liu et al. 2010).
(4) The resulting trees will be used to evaluate existing classifications (Mishler 1994, 2007; Gallego 2005). The overall goal for this portion of the study is to decide what the terminal clades really are in this group, to work out their relationships, and develop a phylogenetic classification.
Comparative methods
The resulting phylogenies will be used as the basis for applications of comparative methods, e.g., to understand evolution of individual traits (morphological and physiological) as well as correlations among traits through evolution, the role of biogeography in lineage differentiation, and the evolution of ecology including community assembly of the biocrust and tree bark communities where Syntrichia dominates. Specific methods to be employed include:
(1) Blomberg’s K tests for phylogenetic signal (Blomberg et al. 2003),
(2) independent contrasts for examining correlation of continuous characters (Felsenstein 1985),
(3) concentrated changes tests for examining correlation of discrete characters (Maddison 1990),
(4) Lagrange (http://blackrim.org/programs/lagrange/) and Biogeobears (http://phylo.wikidot.com/biogeobears) tests to evaluate likelihood models for geographic range evolution on phylogenetic trees,
(5) comparative analyses of character state evolution using functions within BayesTraits (http://www.evolution.rdg.ac.uk/BayesTraits.html), and
(6) randomization tests to evaluate community assembly parameters and patterns of diversity and endemism on the landscape (Webb et al. 2002; Mishler et al. 2014).