DOI: 10.5061/dryad.qnk98sfcb
title: Extremely low genetic diversity in the European clade of the model bryophyte Anthoceros agrestis

description: The hornwort Anthoceros agrestis is emerging as a model system for the
 study of symbiotic interactions and carbon fixation processes. It is an
 annual species with a remarkably small and compact genome. Single
 accessions of the plant have been shown to be related to the cosmopolitan
 perennial hornwort Anthoceros punctatus. We provide the first detailed
 insight into the evolutionary history of the two species. Due to the
 rather conserved nature of organellar loci, we sequenced multiple
 accessions in the Anthoceros agrestis-A.  punctatus complex using
 three nuclear regions: the ribosomal spacer ITS2, and exon and intron
 regions from the single-copy coding genes rbcS and phytochrome. We used
 phylogenetic and dating analyses to uncover the relationships between
 these two taxa. Our analyses resolve a lineage of genetically near-uniform
 European A. agrestis accessions and two non-European A. agrestis lineages.
 In addition, the cosmopolitan species Anthoceros punctatus forms two
 lineages, one of mostly European accessions, and another from India. All
 studied European A. agrestis accessions have a single origin, radiated
 relatively  recently (less than 1 million years ago), and are
 currently strictly associated with agroecosystem habitats
description: <b><i>Molecular
 methods </i></b>We sequenced 51 accessions representing
 <i>Anthoceros punctatus</i> and <i>A. agrestis
 </i>from Europe, the Americas, Australia and Asia (Table S1, Figure
 1).<i> </i>Given some uncertainty as to what the closest
 extant relative of <i>A. agrestis</i> and <i>A.
 punctatus </i>is, we included several related species of
 <i>Anthoceros</i> as outgroup taxa, based on previous studies
 (Duff et al. 2007; Villarreal et al. 2017). We included<i>
 </i>five accessions of <i>A.
 </i><i>neesii</i>, three accessions of
 <i>A.</i><i> cristatus,</i>
 and<i> </i>one accession each of <i>A. venosus, A.
 lamellatus </i>and <i>A. fragilis</i>. All those species
 share similar spore morphology and have been associated with the
 <i>A. punctatus</i> complex (Schuster 1992; Villarreal et al.
 2015). Voucher information and GenBank accession numbers are available in
 Supplementary Table 1. The DNA extraction and PCR amplification followed
 standard protocols (available upon request). The data set includes
 nucleotide sequences from three nuclear loci: part of the RuBisCO small subunit
 (<i>rbc</i>S), which has been shown to be single copy in
 European accessions of <i>A. agrestis, </i>part of the
 phytochrome gene, which again is single copy in European <i>A.
 agrestis</i> (Szövényi unpublished
 data), and the internal transcribed spacer 2 region (ITS2). Primers to
 amplify <i>rbc</i>S and phytochrome were newly generated as
 part of this study (Table S2). We used Geneious 9.0.5 (Biomatters Limited)
 to generate an initial alignment of the nucleotide sequences, with
 subsequent manual augmentation. Due to some failed amplifications, the
 final data matrix (hereafter called dataset I) comprises forty-seven
 accessions that have sequence data for at least two loci, including thirty
 accessions of <i>A. agrestis</i>, twelve accessions of
 <i>A. punctatus</i>, two accessions of <i>A.
 neesii</i>, and one each of <i>A.</i>
 <i>cristatus, A. lamellatus, A. venosus </i>and <i>A.
 fragilis.</i> A subset of the matrix used for the dating analyses
 (dataset II) contains 20 accessions and includes representatives of
 <i>A. agrestis</i>, <i>A. punctatus,</i> and the
 outgroup species <i>A. neesii, A. cristatus </i>and
 <i>A. fragilis</i>. <b>            Phylogenetic
 analyses </b>¾ We analysed each locus separately, and also as a
 single partitioned dataset with models suggested by PartitionFinder
 (Lanfear et al. 2012), under the maximum likelihood criterion (ML), using
 RAxML black box (Stamatakis 2014) with 500 bootstrap replicates (MLB).
 Basic statistics on each dataset were retrieved using PAUP* (Swofford
 2002). Bayesian analyses on the combined dataset were conducted in MrBayes
 3.2 (Ronquist et al. 2012), using the default two runs and four chains,
 with default priors on most parameters. To assess burn-in and convergence
 we compared the bipartitions across the two runs. Convergence was usually
 achieved in MrBayes after 2 ´ 10<sup>7</sup> generations, with
 trees sampled every 10,000<sup>th</sup> generation for a total
 length of 10 x10<sup>6</sup> generations; we discarded 25% of
 each run and then pooled runs. All analyses were run under the CIPRES
 platform (Warnow 2010).            
 <b><i>Molecular Clock Dating </i></b>We used a
 root prior from a previous study (Villarreal et al. 2015) to date a
 phylogeny produced using dataset II, with <i>A. cristatus</i>,
 <i>A. fragilis </i>and <i>A.</i>
 <i>neessii</i> as outgroup taxa. There is no reliable fossil
 described from <i>Anthoceros</i>, although spores similar to
 those of <i>A. punctatus</i> and referred to as
 <i>Rudolphisporis</i> <i>rudolfii</i> have been
 reported from Late Miocene (Machnín, Bohemia),
 Pliocene and Pleiostocene deposits from Europe (Krutzsch 1963). We have
 decided to use a root prior because of the uncertainty around this fossil
 spore and the lack of precise dating of the stratum. We gave the root a
 normal prior, with a mean of 20 Ma and sd of 3, to account for the highest
 posterior density (HPD) from a previous study (Villarreal et al. 2015). 
 In addition, we used the crown age of the <i>A. punctatus –
 agrestis</i> group from this previous study, applying it to the
 ingroup with a mean of 6 Ma and sd of 1. The last calibration used was the
 age of Ascension Island, as a proxy age for the neo-endemic <i>A.
 cristatus</i>. Ascension Island is the tip of an undersea volcano
 that is thought to have emerged from the ocean one million years ago
 (Ashmole and Ashmole 2000). We used a normal prior with a mean of 1 and sd
 of 0.25. To explore the effective priors, we ran analyses with either one
 or two calibrations at a time, and one on an empty alignment, to compare
 the frequency distribution of age estimates for each calibrated node with
 the prior. Bayesian divergence time estimation used a Yule process tree
 prior with unlinked data partitions, using same substitution models
 suggested by PartitionFinder. The analyses were done using an uncorrelated
 log-normal (UCLN) relaxed clock model. The MCMC chains were run for 900
 million generations, with parameters sampled every
 10,000<sup>th</sup> generation using BEAST 1.8.3 (Drummond et
 al. 2012). Tracer 1.5 was used to assess effective sample sizes (ESS) for
 all estimated parameters and to decide the appropriate percentages of
 burn-in. We verified that all ESS values were &gt;200. Trees were
 combined in TreeAnnotator 1.8 (part of the BEAST package), and maximum
 clade credibility trees with mean node heights were visualized using
 FigTree 1.4.0. We report the HPD intervals (the interval containing 95% of
 the sampled values).