Geographic isolation has long been viewed as a critical step in the process of speciation and the generation of biodiversity (Mayr 1963; Barraclough and Vogler 2000; Coyne and Orr 2004). Within many moss species, however, mounting evidence from floristics (van Zanten 1978; Miller and McDaniel 2004; Hutsemekers et al. 2008), molecular population genetics (Shaw et al. 2003; Van der Velde and Bijlsma 2003; McDaniel and Shaw 2005; Vanderpoorten and Long 2006; Heinrichs et al. 2007; Huttunen et al. 2008; Szovenyi et al. 2008), and phylogenetics (Shaw et al. 2005a) indicates that geographic barriers may not cause a long-term impediment to migration. This observation suggests that genetic, demographic, or ecological factors, in addition to geographic isolation, cause the cessation of gene flow between nascent species. The nature of these biological factors, however, is largely unknown. The moss Physcomitrella patens and its relatives have long been model systems for studies of hybrid interfertility (von Wettstein 1924; Bryan 1957). P. patens is now emerging as a model plant for comparative genomics, and studies including P. patens have provided insights into the evolution of land plant gene function and genome structure (Nishiyama et al. 2003; Lang et al. 2005; Rensing et al. 2005; Stenoien 2005; Richardt et al. 2007; Rensing et al. 2008; reviewed in Quatrano et al. 2007). As a consequence, P. patens also holds promise as a model for understanding the process of speciation (von Stackelberg et al. 2006; Kamisugi et al. 2008).
Phylogenetic studies have shown that the monotypic genus Physcomitrella is closely related to the genus Physcomitrium, based on analyses of DNA sequences from the chloroplast (Goffinet and Cox 2000; Goffinet et al. 2007; Werner et al. 2007). The two genera are most easily distinguished by the morphology of the diploid sporophyte; the Physcomitrella sporophyte is nestled among the gametophyte leaves, rather than elevated on a seta like in Physcomitrium and most other mosses, and lacks the specialized abscission layer around the opening of the sporangium. Both genera are cosmopolitan in distribution, but Physcomitrium is more diverse than Physcomitrella, with a total of seven species found in Europe and temperate North America (Crum and Anderson 1981; Hill et al. 2006). Populations containing species of both genera are frequent in some habitats, and hybrids are widely reported (Britton 1895; Andrews 1918; Loeske 1929; Andrews 1942; Pettet 1964; Tan 1978). Therefore, the evolutionary relationships within the Physcomitrella–Physcomitrium complex may not be captured by a single gene or organelle, because ongoing hybridization may cause different parts of the genome to have different histories.
In spite of the evidence for hybridization, the species of Physcomitrella and Physcomitrium are phenotypically distinct, even in sympatric sites (e.g., Crum and Anderson 1981), suggesting that the evolutionary effects of hybridization may be limited. In a landmark series of experiments, von Wettstein (1924, 1928, 1932) and colleagues (Schmidt 1931; Bauer and Brosig 1959) generated artificial crosses among members of the Funariaceae, including P. patens and three European species of Physcomitrium (the cosmopolitan P. pyriforme, and the endemics P. sphaericum and P. eurystomum). In general, the recombinant haploid progeny (i.e., spores that develop into gametophytes) of most intergeneric hybrids were inviable or developmentally abnormal, making it unlikely that natural recombinants would contribute to subsequent generations (reviewed in Bryan 1957). However, occasionally a few recombinants were viable and vigorous, suggesting that the fortuitous segregation of species-difference loci (either karyotypic differences or epistatically interacting factors) may allow for the production of fertile progeny. Von Wettstein (1932) reported that the frequency of such vigorous recombinants was greatest in crosses in which P. patens was the maternal parent, although this frequency also depended upon the paternal species. These data have engendered a considerable amount of speculation regarding the role of introgressive hybridization in the evolution of the Funariaceae, but neither additional crossing studies nor molecular population genetics analyses of hybridization have been undertaken in the Physcomitrella–Physcomitrium complex.
In principle, the signature of introgressive hybridization is readily observable by comparing the genealogical patterns at multiple independent loci (Sang and Zhong 2000; McBreen and Lockhart 2006). Ongoing gene flow should cause allele sharing between distinct species and elevated levels of genetic variation within the hybridizing species, because allelic variants coalesce before the origin of either species. Historical gene flow may be indicated by conflicting genealogical relationships among loci. However, genealogical conflict due to ancient introgression can be difficult to distinguish from the retention of ancestral polymorphism (incomplete lineage sorting). This problem is particularly acute where multiple speciation events have occurred over a short period of time relative to the amount of segregating variation in the ancestral population (Maddison 1997). The coalescence times in large populations are long, increasing the probability that the branching order in a particular gene genealogy will not reflect the true order of speciation events. Moreover, because a given genealogy may arise under a broad range of demographic processes, inferences based on genealogical shape alone are generally insufficient to rule out particular demographic histories (Kuhner 2009). Recently, however, researchers have developed approaches that estimate historical demographic parameters over a range of probable genealogies, providing a means to explicitly distinguish gene flow from incomplete lineage sorting (Hey and Nielsen 2004, 2007).
To test for introgressive hybridization, we examined patterns of genetic variation in primarily European isolates of Physcomitrium and Physcomitrella, using molecular evolutionary analyses of the DNA sequence variation from one chloroplast spacer (atpB-rbcL), the nuclear ribosomal internal transcribed spacers (ITS), and intron-containing portions of four nuclear protein-coding loci (adenosine kinase, adk; adenosine 5′-phosphosulfate reductase, apr; phosphoadenosine–phosphosulphate reductase, papr; and heme oxygenase, ho). We included isolates from geographically widespread populations of four species of Physcomitrium, and four subspecies of P. patens, focusing in particular on the species where fertility estimates from interspecific hybrids were available in natural (Pettet 1964) and experimental crosses (von Wettstein 1924, 1928). Our results indicate that P. patens has arisen at least three times from within the genus Physcomitrium, and although the species shows no history of introgression, other species within Physcomitrium show a clear signature of hybridization. We discuss these findings in the context of the patterns of interfertility in the Physcomitrella–Physcomitrium complex, and patterns of speciation and diversification in general.