Molecular evidence for convergent evolution and allopolyploid speciation within the Physcomitrium-Physcomitrella species complex

Abstract

Background: The moss Physcomitrella patens (Hedw.) Bruch & Schimp. is an important experimental model system for evolutionary-developmental studies. In order to shed light on the evolutionary history of Physcomitrella and related species within the Funariaceae, we analyzed the natural genetic diversity of the Physcomitrium-Physcomitrella species complex.

Results: Molecular analysis of the nuclear single copy gene BRK1 reveals that three Physcomitrium species feature larger genome sizes than Physcomitrella patens and encode two expressed BRK1 homeologs (polyploidization-derived paralogs), indicating that they may be allopolyploid hybrids. Phylogenetic analyses of BRK1 as well as microsatellite simple sequence repeat (SSR) data confirm a polyphyletic origin for three Physcomitrella lineages. Differences in the conservation of mitochondrial editing sites further support hybridization and cryptic speciation within the Physcomitrium-Physcomitrella species complex.

Conclusions: We propose a revised classification of the previously described four subspecies of Physcomitrella patens into three distinct species, namely Physcomitrella patens, Physcomitrella readeri and Physcomitrella magdalenae. We argue that secondary reduction of sporophyte complexity in these species is due to the establishment of an ecological niche, namely spores resting in mud and possible spore dispersal by migratory birds. Besides the Physcomitrium-Physcomitrella species complex, the Funariaceae are host to their type species, Funaria hygrometrica, featuring a sporophyte morphology which is more complex. Their considerable developmental variation among closely related lineages and remarkable trait evolution render the Funariaceae an interesting group for evolutionary and genetic research.

Figure 1

Electropherograms of the BRK1 intron region from Physcomitrium eurystomum. (A) Direct sequencing product of BRK1 amplified from P. eurystomum (Schleiz, Europe) genomic DNA. Sequence polymorphisms are indicated with black arrows. (B) Clonal sequencing products of the BRK1 showing two distinct homeologs of BRK1 in P. eurystomum. Figure generated using Gentle (http://gentle.magnusmanske.de/).

Figure 2

Phylogenetic tree of BRK1. Phylogenetic tree of the nuclear gene BRK1 (Pp1s35_157V6.1) from selected Funariaceae. Three Funaria species (F. hygrometrica, F. flavicans, and F. americana) were used as the outgroup (not shown, see Additional file 6: Figure S3, for details). The cluster representing the Physcomitrium-Physcomitrella species complex shown here is supported by all methods. Due to differing or unresolved branching order, depending on inference method used, the backbone is shown as multifurcating. The three distinct clades of Physcomitrella representing P. readeri (Japan, Australia), P. magdalenae (Africa) and P. patens (Europe and North America) are shown in bold. The distinct paralogous loci of BRK1 for each hybrid species are highlighted with colored brackets. Red brackets represent distinct loci of five P. pyriforme accessions from Europe. Blue brackets show multiple loci of two capsules of a P. collenchymatum accession from North America. Green brackets mark distinct loci of BRK1 within three accessions of P. eurystomum from Europe. The symbols at the nodes represent support values > 95 for Maximum Likelihood (ML), Bayesian Inference (BI) and Neighbor Joining (NJ), respectively. They are derived from 1,000 NJ bootstrap samples, respectively show posterior probabilities of Bayesian inference or maximum likelihood quartet puzzling support.

Figure 3

Electropherograms of a polymorphic region within the BRK1 transcript from six selected Funariaceae. Sequencing of an amplified region within the BRK1 transcript from (A)P. patens (Gransden, Europe), P. eurystomum (Schleiz (B), Neukirch (D) and Neustadt (F), Europe) and P. collenchymatum (Shaw Nature Reserve, Franklin County, MO, USA, K1 (C) and K2A (E), USA). Black arrows indicate sequence polymorphisms.

Figure 4

High resolution melting analysis of BRK1. (A) Normalized and temperature shifted high resolution melting curve and (B) difference plot of BRK1 from P. patens (mauve), the two P. collenchymatum accessions K1 (green) and K2A (red) and the three P. eurystomum accessions Neustadt (gray) and Neukirch/Schleiz (both blue); see Additional file 3: Figure S1 for alignment with marked polymorphisms.

Figure 5

SSR-based neighbor-joining tree. Unrooted neighbor-joining tree constructed using Nei’s DA-distance on datasets derived from 64 SSRs of 21 Physcomitrella accessions. Accessions are named with their designation and country. Gray ellipses indicate three clades of Physcomitrella accessions, supporting a revised classification into the three distinct species P. patens (Europe and North America), P. readeri (Australia and Japan) and P. magdalenae (Africa). The numbers at the nodes are derived from 1,000 bootstrap samples.

Figure 6

SSR-based NeighborNet. Unrooted, equal angled Splitstree NeighborNet based on presence/absence of 170 different sized SSR bands derived from 49 loci. Numbers at the edges represent bootstrap support, shown only for values > 50. Accessions are named with their designation and country.

Figure 7

Overview of molecular features supporting the proposed speciation within the Physcomitrium-Physcomitrella species complex. The re-classified species (subspecies following the former classification of Tan 1979 in brackets) are arranged as supported by data presented in this study, modified after [12]. Dashed lines indicate hybridization events resulting in the three species of Physcomitrium. The dotted line illustrates the clustering of A. serratum together with P. patens. The requirement of editing at particular positions in nad3 and nad4 is shown. The RNA editing site nomenclature consists of affected gene, position in the reading frame and the resulting amino acid codon change induced by RNA editing [32]. C (Cytidine) at the DNA level indicates an editing site, whereas no editing is required in the case of a thymidine (T) at the appropriate position. The number of BRK1 loci (homeologs) is depicted for all analyzed species, while *indicates SSR-based support for the three Physcomitrella species.