Comparing three complete mitochondrial genomes of the moss genus Orthotrichum Hedw.

Abstract Here, we present a comparative analysis of the mitochondrial genome of three representatives of Orthotrichum Hedw (Bryophyta): two populations of O. diaphanum and one of the related species, namely O. macrocephalum. Their mitochondrial genomes share the same gene content and gene order, and are furthermore structurally identical to those of other arthrodontous mosses. The mitogenome of the allopatric samples of O. diaphanum differ in 0.1% of their sequence, with protein coding genes holding five mutations, including two non-synonymous changes. The divergence between the mitogenomes of the two species, O. diaphanum and O. macrocephalum, is 0.4%. Within a broader sampling of the Orthotrichaceae, patterns of genome divergence are consistent with phylogenetic relationships.

The genus Orthotrichum is one of the most species-rich moss genera, with $163 species (Medina et al. 2013). Orthotrichum diaphanum Brid. and O. macrocephalum F. Lara, Garilleti and Mazimpaka are two related epiphytic species of section Diaphana Vitt. (Lara et al. 1994) with distinct but overlapping geographic distributions: O. diaphanum occurs throughout the Western Palearctic-Western Nearctic, whereas O. macrocephalum is restricted to the Mediterranean areas in the Northern Hemisphere.
The number of moss mitochondrial (mt) genomes announced has dramatically increased in recent years (Liu et al. 2011(Liu et al. , 2014Sawicki et al. 2014Sawicki et al. , 2015Alonso et al. 2015), but only one study (Lewis et al. 2016) has targeted the mt genome of several conspecific populations. We sought to assess the types and distribution of substitutions between the genome from two populations of O. diaphanum and between this species and the related O. macrocephalum.
To confirm the phylogenetic identity of the samples, we inferred their relationships with other 13 moss species publicly available, including members of the Orthotrichaceae (see Figure 1 for GenBank accession numbers). Protein-coding genes sequences were aligned using the progressive Mauve algorithm (Darling et al. 2004) in Geneious, in order to perform phylogenetic analyses under maximum likelihood and Bayesian inference. The phylogenetic inferences (Figure 1) are congruent with the phylogenetic structure among moss genera (Liu et al. 2014;Young-Jun et al. 2015). Orthotrichum is known to be polyphyletic, which is confirmed here with species of Orthotrichum with superficial stomata more closely related to Ulota D. Mohr than to species with immersed stomata (Goffinet et al. 2004).
Within O. diaphanum, the variable sites are relatively scarce and widely dispersed along the mt genome. Sixtythree substitutions occur within non-coding regions, and five (three transitions and two transversions) within proteincoding regions. Among the latter, two substitutions result in non-synonymous changes (i.e. in the rps1 gene: A<->C, 3rd codon position of the 211th codon, Asparagine to Lysine; ccmFN gene: A<->G, 1st codon position of the 175th codon, Asparagine to Aspartic acid). The only concentration of mutations occurs in the cox1 group II intron cox1i1064g2, which holds two mononucleotide substitutions, one 6 bp indel and either five or three TATAT microsatellite repeats in O. diaphanum #1 and #2, respectively. The alignment of both O. diaphanum and O. macrocephalum mitogenomes, and that of all Orthotrichaceae, reveals noticeable interspecific variation, most of it in non-coding regions, such as cox1 and cox2 group II introns, and also within coding regions such as ccmFN gene. Those regions could potentially be evaluated as new markers for phylogenetic analyses within this moss family.

Funding information
Financial support was received to B. Vigalondo from the Spanish ''Ministerio de Econom ıa y Competitividad'' (EEBB-I-15-09739) for a three month stay at UConn. Financial support for the sequencing and laboratory analyses was provided by the Spanish ''Ministerio de Ciencia e Innovaci on'' (Project CGL2011-28857) and by US National Science Foundation (DEB-1146295 and 1240045 to BG).