Abstract More than 200 research papers on the molecular phylogeny and phylogenetic biogeography of bryophytes have been published since the beginning of this millenium. These papers corroborated assumptions of a complex genetic structure of morphologically circumscribed bryophytes, and raised reservations against many morphologically justified species concepts, especially within the mosses. However, many molecular studies allowed for corrections and modifications of morphological classification schemes. Several studies reported that the phylogenetic structure of disjunctly distributed bryophyte species reflects their geographical ranges rather than morphological disparities. Molecular data led to new appraisals of distribution ranges and allowed for the reconstruction of refugia and migration routes. Intercontinental ranges of bryophytes are often caused by dispersal rather than geographical vicariance. Many distribution patterns of disjunct bryophytes are likely formed by processes such as short distance dispersal, rare long distance dispersal events, extinction, recolonization and diversification.
Bryophytes (liverworts, mosses and hornworts) comprise the three lineages of land plants with a life cycle in which the haploid gametophyte is the dominant photosynthetic active generation. In contrast to other land plants, the sporophyte is unbranched and not autonomously viable (Schofield, 1985). Bryophytes disperse frequently both by spores and by propagules that descend from the gametophyte, or by unspecialized gametophyte fragments with a high potential of regeneration (Correns, 1899). Bryophytes are the progeny of the first plants that successfully colonized terrestrial habitats (Qiu, 2008). Their evolution in space and time is still insufficiently known.
The increasing availability of DNA sequence data now enables the testing of morphology-based taxonomic and biogeographic concepts and the disclosure of the genotype structure of species. DNA sequence data also allow for an evaluation of different hypotheses concerning biogeographical patterns and processes.
1 Morphological species concepts in the light of molecular phylogenies: indications of non-monophyly and need for a revised taxonomy
An increasing body of published work points to many taxonomic incongruences of widespread morphologically circumscribed bryophyte species and phylogenies derived from molecular markers. Shaw & Allen (2000) resolved populations of morphologically circumscribed species of the aquatic moss genus Fontinalis Hedw. in widely diverging clades, as did Vanderpoorten et al. (2004) for species of Hygroamblystegium Loeske and Shaw et al. (2008) for representatives of the Sphagnum subsecundum complex. Stech & Wagner (2005) provided evidence for the polyphyly of several species of Campylopus Brid. Werner & Guerra (2004) resolved Tortula vahliana (Schultz) Mont. nested within Tortula muralis Hedw. Draper et al. (2007) showed the non-monophyly of the pleurocarpous moss species Isothecium alopecuroides (Dubois) Isov., Isothecium holtii Kindb. and Isothecium myosuroides Brid. Vanderpoorten & Goffinet (2006) identified several species of the moss Brachytheciastrum Ignatov & Huttunen as polyphyletic and documented parallel morphological evolution within this genus. Similarly, Cano et al. (2005) found incongruences between current classification schemes of the Tortula subulata complex, and a molecular topology. Incongruences between morphology-based classifications and molecular topologies have also been shown for several liverwort genera including Bryopteris (Nees) Lindenb. (Hartmann et al., 2006), Chiloscyphus Corda (Hentschel et al., 2006), Herbertus Gray (Feldberg et al., 2004), Lophozia (Dumort.) Dumort. (Vilnet et al., 2008), Plagiochila (Dumort.) Dumort. (Rycroft et al., 2004), and Porella L. (Hentschel et al., 2007b).
As a consequence of the numerous observations of species polyphyly, Vanderpoorten & Goffinet (2006) raised reservations regarding current species definitions. However, in many cases the molecular topologies allowed for a modified appraisal of morphological evidence including new circumscription of taxa or changes of rank. Vanderpoorten (2004) solved the problem of non-monophyletic Hygroamblystegium species by introducing a wide species concept for Hygroamblystegium varium (Hedw.) Mönk. Cano et al. (2005) proposed the binomen Tortula schimperi Cano et al. for a taxon that was usually treated as a variety of T. subulata Hedw. Rycroft et al. (2004) reinstated the leafy liverwort Plagiochila maderensis Steph. that was earlier placed in the synonymy of Plagiochila spinulosa (Dicks.) Dumort.
Redefinitions of species are often connected with changes of distribution range concepts. Pfeiffer et al. (2004) showed phylogeographic structure within the Australasian–South American simple thalloid liverwort Hymenophyton flabellatum (Labill.) Trev. Based on the outcome of their molecular phylogenetic analyses and accompanying morphological studies, they restricted the range of H. flabellatum to Australasia, and reinstated the New Zealand–Tasmanian Hymenophyton leptopodum (Hook.f. & Taylor) A.Evans as well as the southern South American Hymenophyton pedicellatum Steph. Based on molecular evidence, Feldberg et al. (2004, 2007) excluded Herbertus dicranus (Taylor) Trevis. from tropical America and proposed the application of the name Herbertus sendtneri (Nees) A.Evans, a taxon that was previously assigned to European and Asian populations only. Heinrichs et al. (1998, 2004, 2005a, b) lowered the European Plagiochila killarniensis Pearson to a synonym of the Neotropical Plagiochila bifaria (Sw.) Lindenb., included several African and Neotropical binomia in the European taxon Plagiochila punctata (Taylor) Taylor, and extended the range of the Neotropical Plagiochila boryana Steph. to tropical Africa. Hedenäs (2008a) excluded the African Antitrichia kilimandscharica Broth. and the western North American Antitrichia gigantea (Sull. & Lesq.) Kindb. from the synonymy of Antitrichia curtipendula (Hedw.) Brid.
It is assumed that ongoing studies into the molecular phylogeny of bryophytes will lead to numerous new appraisals of ranges.
2 Internal structure of bryophyte species: molecular variation versus morphological stasis
The complex thalloid liverwort Conocephalum F.H.Wigg. is possibly the most exhaustively studied example of a genetically heterogeneous bryophyte species. Isozyme studies in the Holarctic–temperate Asian Conocephalum conicum (L.) Dumort. s.l. revealed the presence of six partly sympatric taxa that were informally named using a letter system (Odrzykoski & Szweykowski, 1991). Subsequent detailed study of morphological and ecological traits led to the formal recognition of one of them as Conocephalum salebrosum Szweyk. et al. (Szweykowski et al., 2005). This species occurs sympatrically with C. conicum s.str. but tends to grow in dryer habitats. Boisselier-Dubayle et al. (1998) reported the presence of a morphologically indistinct Mediterranean sibling species besides the European–Asian–American–African complex thalloid liverwort Reboulia hemisphaerica (L.) Raddi s.str. Similarly, the subcosmopolitan simple thalloid liverwort Aneura pinguis (L.) Dumort. s.l. seems to include at least three reproductively isolated, sympatric cryptic species (Wachowiak et al., 2007). Evidence for cryptic speciation was also provided for the Holarctic simple thalloid liverworts Pellia epiphylla (L.) Corda and Pellia endiviifolia (Dicks.) Dumort. (Pacak & Szweykowska-Kulińska, 2000; Fiodorow et al., 2001).
Similar findings are available for several mosses. Bijlsma et al. (2000) provided evidence for the presence of two reproductively isolated cryptic species in the acrocarpous moss Polytrichum commune Hedw. [P. commune s.str., Polytrichum uliginosum (Wallr.) Schriebl, see also van der Velde & Bijlsma, 2004)]. McDaniel & Shaw (2003) recovered a deep split between the two morphologically weakly separated subspecies of the trans-Antarctic moss Hymenodontopsis mnioides (Hook.) N.E.Bell et al. Fernandez et al. (2006) analyzed amplified fragment length polymorphisms of Californian populations of the cosmopolitan species Grimmia laevigata (Bridel) Bridel. They identified two distinct geographically overlapping clades. Shaw (2000) published a nrITS phylogeny of Mielichhoferia elongata (Hoppe & Hornsch.) Nees & Hornsch. Based on the outcome of his analyses he proposed two cryptic species within the morphologically uniform taxon, one with a European–North American range, and the other strictly North American. Hedenäs & Eldenäs (2007) investigated nrITS and chloroplast DNA haplotype variation of the pleurocarpous moss Hamatocaulis vernicosus (Mitt.) Hedenäs. Based on their topologies Hedenäs & Eldenäs (2007) postulated the existence of two cryptic species, of which one is widespread in Europe, in addition to a few North American records. The other cryptic species was found south of the boreal zone in Europe, in northern-most Asiatic Russian Federation, and Peru.
3 Phylogeographic patterns, migration routes, and modes of reproduction as revealed from molecular data
Studies of haplotype variation do not only allow for the recognition of putative cryptic taxa but also for a reconstruction of the spatial structure of genetic diversity, potential bottleneck events, and modes of reproduction. Grundmann et al. (2007) studied DNA sequence and allozyme variation to resolve the spatial structure of Mediterranean accessions of the dioecious Holarctic moss Pleurochaete squarrosa (Brid.) Lindb. These authors observed a decline of intraspecific variation from west to east but no difference in gene diversity among populations from islands and mainland areas. Based on the latter observation, Grundmann et al. (2007) concluded that the large Mediterranean islands might function as “mainland” for bryophytes. Vanderpoorten et al. (2008) arrived at a similar conclusion when analyzing chloroplast markers of the moss Grimmia montana Bruch & Schimp. Madeiran and Mediterranean haplotypes of G. montana were identical or closely related to European or North American ones.
Grundmann et al. (2008) analyzed diversity patterns of European P. squarrosa using nuclear and chloroplast DNA sequences, and enzyme electrophoresis. These authors provided evidence for sexual reproduction, that is, recombination, of P. squarrosa in the Mediterranean Basin and the Kaiserstuhl Mountains in southwestern Germany, a region that is well known for its unusual climate with high monthly average temperatures and short, mild winter. In other regions of central and northwest Europe P. squarrosa disperses predominantly by vegetative propagules, a finding that is corroborated by a lack of evidence of recombination. Grundmann et al. (2008) also postulated a postglacial recolonization of central Europe from the Iberian Peninsula and the Balkans. Cronberg (2000) observed quite similar patterns for the epiphytic moss Leucodon sciuroides (Hedw.) Schwägr. Mediterranean populations reproduce sexually and are genetically diverse whereas more northern populations reproduce vegetatively and are genetically quite uniform. This pattern coincides with the expectation of a loss of genetic variation in populations at the northern limit of the glacial refugia. Glacial survival in southern Europe is obviously not a general pattern in bryophytes. Hedderson & Nowell (2006) recognized several unique Homalothecium sericeum (Hedw.) Schimp. haplotypes in the British Isles and adjacent mainland. Based on this observation Hedderson & Nowell (2006) assumed a survival of Homalothecium sericeum in this region during the last glacial period.
Szövéni et al. (2006) presented a chloroplast phylogeographic analysis of Sphagnum fimbriatum Wilson and Sphagnum squarrosum Crome. Their haplotype distribution patterns seem to support different dispersal scenarios for these species. S. fimbriatum seems to have survived the last glacial period along the Atlantic coast of Europe, and rapidly colonized Europe after the last glacial maximum. In contrast, S. squarrosum obviously had numerous scattered refugia throughout Europe.
Although most studies referring to the internal structure of widespread bryophyte species revealed molecular variation, a few examples contradicted this tendency. James et al. (2008) introduced the Diversity Arrays Technology, a hybridization-based genotyping method, to reproducibly detect largely low-copy genomic variation in ferns and mosses. Their study revealed a lack of phylogenetic pattern in the Australian moss Garovaglia elegans (Dozy & Molk.) Bosch & Sande Lac. Similarly, van der Velde & Bijlsma (2003) found nearly no genetic structure among European populations of several Polytrichum species (Polytrichum commune, Polytrichum uliginosum, Polytrichum formosum Hedw., and Polytrichum piliferum Hedw.). The lack of allozyme and microsatellite variation pointed to extensive spore dispersal and contradicted the hypothesis of a recolonization of Europe from southern refugia after the last glacial period.
Inter- or intraspecific variation of molecular markers might allow for the reconstruction of range expansion directions. Based on the recent distribution of taxa and their position in a phylogenetic framework, conclusions can be drawn as to the ranges of their ancestors. Heinrichs et al. (2005a) resolved an African accession of Plagiochila sect. Hylacoetes Carl within several tropical American accessions. Based on this topology, these authors proposed a Neotropical origin of the African Plagiochila sect. Hylacoetes populations. Hartmann et al. (2006) arrived at the same conclusion for the Madagascar–Réunion endemic Bryopteris gaudichaudii Gottsche. Feldberg et al. (2007) studied the phylogenetic biogeography of Herbertus and provided evidence for a colonization of Africa from Neotropical and Asian populations (Fig. 1). Huttunen et al. (2008) discovered evidence for a western North American origin of a Holarctic Homalothecium clade.
Taxonomic work combined with the reconstruction of migration routes provided new insights into the relationships between floristic regions. Contrary to the earlier belief, the Atlantic European and Macaronesian Plagiochila taxa are connected with Neotropical rather than Asiatic taxa (Heinrichs et al., 2006). Vanderpoorten & Long documented relationships of Macaronesian and Neotropical Leptoscyphus Mitt. Similarly, Stech et al. (2007) showed the close relationships of the Campylopus flora of tropical America and Madeira. This trend is contradicted by the example Porella with the Macaronesian endemic Porella inaequalis Perss. closely related to the Asian Porella grandiloba Lindb. rather than to Neotropical species (Hentschel et al., 2007b). Clearly more case studies are necessary to decide whether each species has its own history or whether there are recurring patterns.
4 Intercontinental range expansion versus conservation
Molecular phylogenies enable us to scrutinize evidence for dispersal or vicariance. One possibility concerning this matter is a critical comparison of phylogenies with breakup events of landmasses. Congruence of ancestral geographical distributions of clades and the sequence of breakup events rather supports vicariance. However, deviation of a phylogeny and a breakup sequence supports dispersal (Sanmartin & Ronquist, 2004).
The application of the molecular clock hypothesis allows for a transformation of a phylogram into a chronogram. Sequences usually do not exactly evolve at a constant rate but the tempo of mutations within a molecular marker seems to move in a more or less determined range. Accordingly, a comparison of sequence variation with that of dated phylogenies may shed some light on the likelihood of different scenarios (Les et al., 2003).
Divergence time estimates based on sequence variation and the fossil record will provide more reliable insights into the historical biogeography of lineages by enabling differentiation between coinciding events in time and pseudo-congruent patterns (Donoghue & Moore, 2003; Renner, 2005). The poor Paleozoic and Mesozoic fossil record of bryophytes (Oostendorp, 1987) is a serious challenge to any study on the timescale of bryophyte diversification. Cenozoic fossils are often preserved in the form of amber inclusions (Grolle & Meister, 2004; Frahm & Newton, 2005). Amber has not been formed continuously throughout the history of plants on land (Grimaldi, 1996). The infrequent generation of amber inclusions makes it difficult to use them as calibration points for dating approaches (Hartmann et al., 2006). Therefore we rely heavily on the variation of substitution rates.
Shaw et al. (2003) tested the likelihood of a Madrean–Tethyan origin of several western North American–Mediterranean disjunctions of mosses. These authors stated that “no plausible mutation rate” would link the disjunctions to early Miocene times, and favored recent dispersal as an explanation of the observed distribution ranges. Hentschel et al. (2006) verified the assumption of a recent introduction of the southern hemispheric Chiloscyphus semiteres (Lehm.) Lehm. & Lindenb. into Europe (Paton, 1965) by demonstrating nrITS sequence similarities of accessions from the Netherlands and Australia. Wall (2005) documented a clock-like behaviour of the nuclear glyceraldehyde 3-phosphate dehydrogenase gene of the moss Mitthyridium H.Rob., and identified clades that were related to oceanic archipelagos. Using island ages as calibration points, he provided evidence for a diversification of Mitthyridium within less than ten million years. Huttunen et al. (2008) used a mean nrITS substitution rate to deduce a late Miocene–Pliocene age of a split between North American and Eurasian Homalothecium. Hartmann et al. (2006) published a chronogram for the liverwort Bryopteris and provided hypothetical timescales based on assumptions of different scenarios, and nrITS mutation rates. These authors could clearly reject western Gondwanan vicariance for the Neotropical–African range of Bryopteris, and proposed a dispersal scenario. Heinrichs et al. (2006) reconstructed the molecular phylogeny of the cosmopolitan leafy liverwort Plagiochila and presented timescales based on putative fossil assignments. The results did not contradict a Gondwanan origin of Plagiochila, but the geographical distribution of clades (Fig. 2) and divergence time estimates rendered Gondwanan vicariance unlikely. Heinrichs et al. (2006) explained the observed distribution patterns as a result of short distance dispersal, rare long distance dispersal events, extinction, recolonization and diversification. It is not yet clear if the situation in Plagiochila represents a general pattern but several other studies seem to support this combination of mechanisms (Feldberg et al., 2007; Huttunen et al., 2008). Support also comes from comparisons of Southern Hemispheric ranges of bryophytes and main wind directions. Muñoz et al. (2004) found a stronger correlation of floristic similarities with wind connectivity than with geographic proximities, and therefore favored wind as a dispersal vector for many Southern Hemispheric biota. However, today's disjunct ranges of some Southern Hemispheric taxa such as Monoclea Hook. (Meissner et al., 1998) could be a result of short distance dispersal before the final disassembly of Gondwana, and subsequent range fragmentation as a result of climate changes (Schuster, 1979).
Dated chronograms, based on sequence variation plus the bryophyte fossil record, have been published for bryophytes in general (Newton et al., 2007, with a strong focus on the pleurocarpous moss lineage), the leafy liverworts (Heinrichs et al., 2007) and the leafy liverwort family Lejeuneaceae (Wilson et al., 2007). However, sampling within these studies was not sufficient to decide on species level disjunctions, and the results supported the idea of a reformation of bryophyte diversity throughout the Cretaceaous and Early Tertiary. Many recent genera seem to have originated not before the Late Cretaceous, rendering Gondwanan vicariance rather unlikely.
Although most current authors favor the adoption of infrequent long distance dispersal for disjunct ranges, this hypothesis needs to be tested by further studies that should focus both on a better understanding of the bryophyte fossil record and a more comprehensive taxon sampling.
5 State of the art and perspectives
A three-digit number of papers on the molecular phylogeny and phylogenetic biogeography of bryophytes have been published since Shaw's review on biogeographical patterns and cryptic speciation of bryophytes (Shaw, 2001). On the one hand, these papers corroborated assumptions of a complex genetic structure of bryophytes with a uniform morphology, and raised reservations against many morphologically justified species concepts, especially within the mosses. On the other hand, many molecular topologies allowed for corrections and modifications of morphological classification schemes.
The relationships of many deep clades of bryophytes have been clarified using molecular phylogenetic approaches plus morphology (Renzaglia et al., 2007). In contrast, boundaries and relationships of many species and genera of bryophytes are still unclear, as is the genetic structure of most species. Future studies should focus strongly on genus or species level relationships, and shed more light on reproduction modes of populations that are still insufficiently known. Population genetic studies using isozymes and microsatellites might result in reliable reconstructions of migration routes and refugia of bryophytes. These studies could also shed more light on the justification of assumptions of intercontinental ranges of bryophyte species.
Phylogenetic studies based on variable molecular markers and using divergence time estimates usually support dispersal scenarios rather than geographical vicariance as the preferred explanation of disjunct ranges of bryophytes. The number of studies is still insufficient for a general pattern to emerge, or to determine if there are also patterns that are indicative of geographical vicariance. Future studies should focus not only on sequence variation but also on the fossil record of bryophytes. The search of bryophyte inclusions in Cretaceous amber deposits may be a promising approach to our understanding of the origin of extant bryophyte diversity. Progress in the interpretation of the bryophyte fossil record is essential to achieve more reliable insights into the chronology of bryophyte diversification and distribution range formation.
Acknowledgements We thank Jun Wen (Washington DC), Yin-Long Qiu (Ann Arbor, Michigan) and Yan Liang (Beijing) for comments on the manuscript. Financial support from the German Research Foundation (grants HE 3584/1-4) is gratefully acknowledged.