American Journal of Botany 98(8): 1252–1262. 2011.
FORMALIZING MORPHOLOGICALLY CRYPTIC BIOLOGICAL ENTITIES: NEW INSIGHTS FROM DNA TAXONOMY, HYBRIDIZATION, AND BIOGEOGRAPHY IN THE LEAFY LIVERWORT PORELLA PLATYPHYLLA (JUNGERMANNIOPSIDA, PORELLALES)1 Jochen Heinrichs2,7, Hans-Peter Kreier2, Kathrin Feldberg2, Alexander R. Schmidt3, Rui-Liang Zhu4, Blanka Shaw5, A. Jonathan Shaw5, and Volker Wissemann6 2Department
of Systematic Botany, Albrecht-von-Haller-Institute of Plant Sciences, Georg-August-University, Untere Karspüle 2, 37073 Göttingen, Germany; 3Courant Research Centre Geobiology, Georg-August-University, Goldschmidtstraße 3, 37077 Göttingen, Germany; 4Department of Biology, East China Normal University, 3663 Zhong Shan North Road, Shanghai 200062, China; 5Department of Biology, Duke University, Durham, North Carolina 27708 USA; and 6Arbeitsgruppe Spezielle Botanik, Justus-Liebig-Universität, Heinrich-Buff-Ring 38, 35392 Giessen, Germany • Premise of the study: Recognition and formalization of morphologically cryptic species is a major challenge to modern taxonomy. An extreme example in this regard is the Holarctic Porella platyphylla s.l. (P. platyphylla plus P. platyphylloidea). Earlier studies demonstrated the presence of three isozyme groups and two molecular lineages. The present investigation was carried out to elucidate the molecular diversity of P. platyphylla s.l. and the distribution of its main clades, and to evaluate evidence for the presence of one vs. several species. • Methods: We obtained chloroplast (atpB-rbcL, trnL-trnF) and nuclear ribosomal (ITS) DNA sequences from 101 Porella accessions (P. platyphylla s.l., P. × baueri, P. cordaeana, P. bolanderi, plus outgroup species) to estimate the phylogeny using parsimony and likelihood analyses. To facilitate the adoption of Linnean nomenclature for molecular lineages, we chose a DNA voucher as epitype. • Key results: Phylogenies derived from chloroplast vs. nuclear data were congruent except for P. platyphylla s.l., including a North American lineage that was placed sister to P. cordaeana in the chloroplast DNA phylogeny but sister to the Holarctic P. platyphylla s.str. in the nuclear DNA phylogeny. European and North American accessions of P. cordaeana and P. platyphylla form sister clades. • Conclusions: The genetic structure of P. platyphylla s.l. reflects morphologically cryptic or near cryptic speciation into Holarctic P. platyphylla s.str. and North American P. platyphylloidea. The latter species is possibly an ancient hybrid resulting from crossings of P. cordaeana and P. platyphylla s.str. and comprises several distinct molecular entities. Key words: cryptic speciation; epitype; hybridization; incongruence; Porella.
A general issue in natural sciences is the number of biologically relevant entities and how they should be recognized. Conventionally, a species description is a hypothesis about the discontinuous distribution of unique combinations of morpho1 Manuscript
received 11 March 2011; revision accepted 23 May 2011.
The authors thank J. Eckstein (Göttingen), J. Hentschel (Jena), A. Schäfer-Verwimp (Herdwangen Schönach), and J. Shevock (Berkeley) for plant material; H. Manitz (Jena, Archive of the Thuringian Botanical Society) for the digitalization of and permission to reproduce table 72 from Dillenius´ Historia Muscorum (1741) from the personal copy of Carl Haussknecht (Signature M8 Dillenius); as well as G. Wagenitz (Göttingen) and the late R. Grolle for helpful discussions. They are indebted to the curators of several herbaria (F, HIRO, JE, SAAR, UBC) for permission to take DNA samples from herbarium specimens. This research was supported by German Research foundation grants HE 3584/2 and 4 (to J.H.), NSF grant EF-0531730-002 (to A.J.S.), and NSFC grant (no. 30825004) and 211 Project (to R.L.Z.). This is publication number 67 from the Courant Research Centre Geobiology, which is funded by the German Initiative of Excellence. 7 Author for correspondence (e-mail:
[email protected]); phone: +49-551-39-22220; fax: +49-551-39-22329 doi:10.3732/ajb.1100115
logical characters (Wheeler, 2004). However, molecular studies point to the existence of numerous “cryptic” biological species that have accumulated genetic divergence without accompanying morphological disparities and thus cannot be identified using the traditional morphological species concept (Geiser et al., 1998; Shaw, 2001; Hebert et al., 2004; Witt et al., 2006; Bickford et al., 2007; Soltis et al., 2007). Because of the limited number of morphological traits and long evolutionary history (Heinrichs et al., 2007; Newton et al., 2007; Shaw et al., 2011), structurally simple plants such as bryophytes are likely to include morphologically indistinguishable entities that are nevertheless genetically distinct. Indeed, several examples of cryptic speciation in bryophytes have come to light. Among the mosses, complex evolutionary patterns and the presence of gametophytically haploid and diploid entities have been revealed in morphologically circumscribed species of Sphagnum L. using microsatellite and sequence data (Ricca and Shaw, 2010). The morphologically uniform acrocarpous moss Mielichhoferia elongata (Hoppe & Hornsch.) Nees & Hornsch. comprises two well-separated molecular lineages (Shaw, 2000), and the polyphyletic pleurocarpous moss Platyhypnidum riparioides (Hedw.) Dixon has a cryptic diversity that possibly reflects adaption to different aquatic habitats (Huttunen and Ignatov, 2010).
American Journal of Botany 98(8): 1252–1262, 2011; http://www.amjbot.org/ © 2011 Botanical Society of America
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Fig. 1. Maximum parsimony (MP) strict consensus trees (each compiled of >600,000 trees) of the nrITS and the chloroplast DNA data sets (all three markers complete for all accessions) with MP bootstrap percentage values (>50) above and maximum likelihood bootstrap percentage values (>50) below branches. Arrows indicate incongruent phylogenetic signals with bootstrap percentage values >70.
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Among the liverworts, isozyme and sequence studies point to six cryptic species in the complex thalloid liverwort Conocephalum conicum (L.) Dumort. (Odrzykoski and Szweykowski, 1991; Miwa et al., 2009), while further cryptic lineages have been found in the simple thalloids Metzgeria furcata (L.) Dumort. (Fuselier et al., 2009) and Aneura pinguis (L.) Dumort. (Wachowiak et al., 2007) and in the leafy liverworts Herbertus dicranus (Taylor) Trevis. (Feldberg et al., 2007), Marchesinia brachiata (Sw.) Schiffn. (Heinrichs et al., 2009b), and Frullania tamarisci (L.) Dumort. s.l. (Heinrichs et al., 2010; Ramaiya et al., 2010). With regard to cryptic speciation, Porella platyphylla (L.) Pfeiff. is possibly the most exhaustively studied leafy liverwort. The species was described by Linnaeus (1753, p. 1134) based on illustrations and descriptions of Morison (1699), Vaillant (1727), Micheli (1729), and Dillenius (1741) and was said to occur “in Europae & Americae septentrionalis sylvis” (in northern woods). Porella platyphylla is a large, dioicous liverwort that grows on bark and rock and exhibits considerable variation in lobule shape and dentition of the perianth mouth. De Schweinitz (1821) recognized the morphological plasticity of P. platyphylla and split off P. platyphylloidea (Schwein.) Lindb. on the basis of plant material from North Carolina. He distinguished P. platyphylloidea by lobules that are nearly equal in size to underleaves, in contrast to the narrower lobes of P. platyphylla. Howe (1897) was not able to discriminate the two taxa on the basis of gametophyte features, but later authors (Evans, 1916; Müller, 1957; Schuster, 1980; Hong, 1983) considered the perianth mouth and elaters to be distinctive characters for separating the two taxa, with P. platyphylla having a shortly dentate perianth mouth and bispiral elaters, and P. platyphylloidea having a perianth mouth with branched cilia and unispiral elaters. Boisselier-Dubayle and Bischler (1994) reported considerable differences among P. platyphylloidea populations from North America and P. platyphylla populations from Europe in terms of isozyme patterns. Those differences were confirmed by Therrien et al. (1998), who recognized three genotypes in North American populations, one of which also occurred in Europe. Accompanying morphological studies, however, demonstrated that the morphological characters used to identify P. platyphylla and P. platyphylloidea did not correlate with the molecular entities and that plants with a ciliate perianth mouth may have bispiral elaters. On the basis of these findings, Therrien et al. (1998) proposed to treat all genotypes as a single, intercontinentally distributed species, P. platyphylla. This treatment was rejected by Bischler et al. (2006), who regarded P. platyphylloidea as an American species and P. platyphylla as a Eurasian species with dubious occurrence in North America. They distinguished the taxa by the shape of the lobe apex and perianth mouth dentition. Hentschel et al. (2007) presented the first comprehensive Porella phylogeny based on nuclear and chloroplast DNA markers. In that study, eight accessions of the P. platyphylla/ P. platyphylloidea complex were resolved in two different lineages, with one lineage restricted to North America, and a second lineage including representatives from both Europe and North America. Another Holarctic species, P. cordaeana (Huebener) Moore, was placed sister to P. platyphylla s.l. This species differs from P. platyphylla by the crenulate-sinuous perianth mouth and acute, ventrally reflexed and twisted lobules. It is one parent of an allopolyploid hybrid, P. × baueri (Schiffn.) C. Jens., with P. platyphylla as the other parent (Boisselier-Dubayle et al., 1998).
Interspecific hybridization seems to occur less frequently in bryophytes than in angiosperms; however, growing evidence exists that many examples have been overlooked as a consequence of the rather conservative bryophyte morphology (Natcheva and Cronberg, 2004). Adding molecular evidence to morphological investigations points to examples of reticulate evolution in several bryophyte lineages, including thallose (Odrzykoski et al., 1996) and foliose liverworts (BoisselierDubayle et al., 1998) and mosses (Ricca and Shaw, 2010). These examples demonstrate complex evolutionary patterns in bryophytes and corroborate the need for further studies to gain greater understanding of speciation processes in bryophytes. In the current study, we produce phylogenies of the P. platyphylla/P. platyphylloidea complex using multiple accessions and sequences from the nuclear ribosomal internal transcribed spacer region (nrITS), along with the plastid DNA intergenic atpB-rbcL spacer and the trnL-trnF region. We address the following questions. (1) Are the topologies derived from the nuclear and chloroplast DNA markers congruent, and are the topologies in accordance with current morphology-based species concepts? (2) Does geographical structure in genetic variation exist within Porella species across their distribution ranges? (3) Are populations on different continents monophyletic? We provide evidence for at least three main clades within P. platyphylla s.l. as well as of incongruent phylogenetic signals derived from nuclear and chloroplast loci, and we choose an epitype to allow for an assignment of the binomials P. platyphylla and P. platyphylloidea to certain main clades. MATERIALS AND METHODS Sampling of plant material and outgroup selection—Regional ingroup sampling was based on the topology of Hentschel et al. (2007) and comprised three accessions of P. bolanderi (Aust.) Pears. (in Hentschel et al. [2007] erroneously as P. navicularis [Lehm. & Lindenb.] Pfeiff.), 60 accessions of the P. platyphylla/P. platyphylloidea complex, 29 accessions of P. cordaeana, and one accession of their allopolyploid hybrid P. × baueri (Szweykowska-Kulińska et al., 2002). Eight samples representing five other Porella species, including both close and distant relatives of the P. platyphylla/P. platyphylloidea complex, were chosen to represent outgroups. All samples are listed in the Appendix, which includes GenBank accession numbers and voucher details. DNA extraction, PCR amplification, and sequencing—Plant tissue from the distal portions of shoots was isolated from herbarium collections. Total genomic DNA was extracted and purified before amplification with the Invisorb Spin Plant Mini Kit (Invitek, Berlin, Germany). Protocols for PCR were carried out as described in previous publications, nrITS from Hentschel et al. (2006), trnL-F from Gradstein et al. (2006), and atpB-rbcL from Feldberg et al. (2010). Bidirectional sequences were generated with a MegaBACE 1000 automated sequencing machine and the DYEnamic ET Primer DNA Sequencing Reagent (Amersham Biosciences, Little Chalfont, UK). Sequencing primers were those used for PCR. Phylogenetic analyses—All sequences were aligned manually with BioEdit, version 5.0.9 (Hall, 1999). Ambiguous positions were excluded from the alignment; missing stretches were coded as missing. Maximum parsimony (MP) and maximum likelihood (ML) analyses were carried out using PAUP*, version 4.0b10 (Swofford, 2000). MP heuristic searches were conducted with the following options: heuristic search mode, 1000 random-addition–sequence replicates, tree bisection-reconnection (TBR) branch swapping, MULTrees option on, and collapse zero-length branches off. All characters were treated as equally weighted and unordered. Nonparametric bootstrapping values (Felsenstein, 1985) were generated as heuristic searches with 500 replicates, each with 10 random-addition replicates. The number of rearrangements was restricted to 10 million per replicate. A bootstrap percentage value (BPV) above 70 was regarded as good support (Hillis and Bull, 1993). Where more than one most
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Fig. 2. Maximum likelihood phylogeny (ln = −2997.59231) derived from an nrITS1-5.8S-ITS2 sequence alignment including 87 new sequences and 13 sequences from Hentschel et al. (2007). Maximum likelihood bootstrap percentage values (>50) in bold face, maximum parsimony bootstrap percentage values (>50) not bold.
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parsimonious tree were found, trees were summarized in a strict consensus tree. We first conducted analyses including all ingroup representatives for which at least nrITS 2 and one of the two chloroplast markers were available. We subsequently restricted our analyses to accessions for which complete sequences of all three markers were available. Nuclear and chloroplast genomic regions were first analyzed separately to check for incongruence. The strict consensus trees of the nonparametric bootstrap analyses were compared by eye to identify conflicting nodes supported by at least 70% (Mason-Gamer and Kellog, 1996). Conflicting nodes with BPV of >70 for each were taken as evidence for incongruent phylogenetic signals, and corresponding data sets were not combined. jModeltest 0.1 (Posada, 2008) in conjunction with PAUP* was used to select models of evolution for ML analyses. For the chloroplast DNA data set, the Akaike information criterion supported the model TIM2+G; for both nrITS data sets, the model GTR+G was supported. The analyses were performed as heuristic searches using five random-sequence addition replicates, MULTrees option on, collapse zero-length branches off, and TBR branch swapping. Clade support was assessed by nonparametric bootstrapping with 300 replicates, accomplished with GARLI, version 0.96 (Zwickl, 2008), using the model parameters estimated with jModeltest. Typification—Linnaeus (1753) described Porella platyphylla (as Jungermannia platyphylla) on the basis of descriptions and illustrations of Morison (1699), Vaillant (1727), Micheli (1729), and Dillenius (1741), rather than on his own studies of specimens. Accordingly, the P. platyphylla illustrations of the latter authors serve as syntypes, and a lectotype must be chosen from these. None of the illustrations assigned to P. platyphylla by Linnaeus (1753) allows for an identification of P. platyphylla in the broad sense (Therrien et al., 1998), and identification is even less possible for one of the main P. platyphylla clades identified in the current study. To allow for an assignment of the name Porella platyphylla to one of the molecular lineages identified in our study, we designate one of the figures cited by Linnaeus (1753) as lectotype and one of our DNA vouchers as epitype.
RESULTS We used 289 sequences from 101 samples, including 262 previously unpublished sequences. The sequences included 101 nuclear ribosomal ITS sequences (5.8 S ribosomal RNA gene and internal transcribed spacers 1+2), 100 chloroplast trnL-F sequences (trnL intron, trnL 3′-exon, trnL-trnF intergenic spacer), and 88 chloroplast atpB-rbcL sequences. We analyzed our nuclear and chloroplast DNA data sets, including both accessions for which sequences of all three markers were available and accessions with incomplete data (Appendix 1). This procedure allowed us to include several accessions from GenBank, especially nrITS2 and trnL-F sequences of P. × baueri and other accessions for which sequences from the atpB-rbcL spacer were lacking. Both nuclear and chloroplast DNA data identified P. × baueri as a representative of the P. cordaeana lineage (data not shown). Exclusion of the accessions with incomplete sequences led to an increased resolution with the chloroplast DNA data set. The MP strict consensus trees derived from the reduced nuclear and chloroplast DNA data sets are depicted in Fig. 1. Chloroplast DNA data set (Fig. 1, right tree)— Accessions of the P. platyphylla/P. platyphylloidea complex form two independent lineages. A mainly Eurasian clade of the complex is placed in a sister relationship to the rest of the ingroup; in the following, this clade is named the P. platyphylla (s.str.) clade. A North American clade of the complex is placed sister to a monophyletic P. cordaeana lineage in a robust sister relationship (MP: BPV 89; ML: BPV 94). This clade is referred to as the P. platyphylloidea clade. Nuclear DNA data set (Fig. 1, left tree)— Porella platyphylla is resolved as sister to P. platyphylloidea with BPVs
above 70 (MP: BPV 76; ML: BPV 71); hence, the nrITS phylogeny is not congruent to the chloroplast DNA phylogeny. We therefore did not combine the nuclear and chloroplast DNA data sets. The P. bolanderi and P. cordaeana clades form a polytomy with the P. platyphylla/P. platyphylloidea clade. Extended nuclear DNA data set— Maximum likelihood analyses of the nrITS data set resolved two trees with ln = −2997.59231, of which one is depicted in Fig. 2. Porella cordaeana is placed sister to the P. platyphylla/P. platyphylloidea clade and consists of a North American and a European lineage. Porella platyphylla is made up of a North American and a Eurasian lineage in a well-supported sister relationship. It is resolved as sister to P. platyphylloidea with BPVs of 67 (ML) and 78 (MP). Porella platyphylloidea splits into two clades (A, B; Fig. 2). Clade A occurs over large parts of North America. The P. platyphylloidea clade B is made up of several accessions from northeastern North America plus two accessions from North Carolina and West Virginia in a polytomous topology, and subclade B1 has accessions from southeastern North America. Typification— We designate Dillenius’ (1741) Fig. 32B of Plate 72 as lectotype of Jungermannia platyphylla L. (Linnaeus, 1753, p. 1134). This figure (Fig. 3) shows the ventral side of a Porella shoot with several sporophytes. To allow for an assignment of the name Porella platyphylla (L.) Pfeiff. to one of the P. platyphylla/P. platyphylloidea clades identified in our study, we choose the DNA voucher Heinrichs and Feldberg 4600 (Germany, Lower Saxony, Plesse near Göttingen, GOET) as epitype of Jungermannia platyphylla L. DISCUSSION Congruence— Mason-Gamer and Kellog (1996) introduced the 70% bootstrap threshold to identify incongruent phylogenetic signal from different markers. Our analyses with several combined or uncombined data sets demonstrated an increase of resolution and bootstrap percentage values after exclusion of incomplete sequences. The combination of the two congruent chloroplast data sets likewise increased the robustness of the related topologies. We therefore propose that the 70% BPV criterion be adopted for complete data sets. We also propose that all available congruent markers from one genomic compartment be combined before they are compared with the phylogenetic signal from other genomic compartments rather than comparing BPVs for all individual markers. Inferences made under the two optimality criteria MP and ML for data partitions were congruent except for the position of P. platyphylloidea, being sister to P. platyphylla in the nrITS phylogeny but placed sister to P. cordaeana in the chloroplast DNA phylogeny (Fig. 1). Topological incongruence between nuclear and chloroplast DNA phylogenies may be caused by lateral gene transfer through hybridization (Wendel and Doyle, 1998; Soltis and Soltis, 2009). Hybridization is not uncommon in bryophytes (Natcheva and Cronberg, 2004), but within leafy liverworts it has so far only been shown for the European Porella × baueri, which is interpreted as an allopolyploid derivative of P. cordaeana and P. platyphylla (Boisselier-Dubayle et al., 1998). According to available evidence, both P. cordaeana and P. platyphylla have haploid gametophytes with a base chromosome number of N = 8; however, all published chromosome
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counts were conducted on European material (Fritsch, 1991; Boisselier-Dubayle et al., 1998). It is therefore unclear whether North American accessions of the P. platyphylla/P. platyphylloidea complex have the same number or are in fact polyploids. Nuclear as well as chloroplast and mitochondrial sequences place the allopolyploid P. × baueri within P. cordaeana (SzweykowskaKulinska et al., 2002; Jankowiak and Szweykowska-Kulinska, 2004); accordingly, Jankowiak and Szweykowska-Kulinska (2004) propose a uniparental transmission of the latter organelles. Our data do not allow for a reconstruction of the hybridization process that may have caused the topological incongruence in P. platyphylloidea, but morphological evidence relates P. platyphylloidea to P. platyphylla rather than to P. cordaeana. A hybrid origin could also explain the unstable perianth and elater morphology of P. platyphylloidea (Therrien et al., 1998). It can be hypothesized that the ancestor of P. cordaeana is the chloroplast donor of P. platyphylloidea, whereas the nucleus represents the paternal lineage. This hypothesis is also supported by the morphology of P. platyphylloidea, which does not show any influence of P. cordaeana. The considerable sequence differences between P. platyphylloidea and related lineages indicate an ancient hybridization event, whereas the allopolyploid P. × baueri shares its nrITS2 and trnL-F sequences with P. cordaeana. This hybrid seems to have a recent origin and appears to have originated more than once (BoisselierDubayle and Bischler, 1994). Álvarez and Wendel (2003) provide evidence that the nrITS region is among the most homoplasious sources for molecular phylogenetic data. This observation is confirmed in our study, with a consistency index of 0.76 for the nrITS data set vs. 0.94 for the combined chloroplast data sets (Fig. 1). Follow-up studies therefore should consider additional nuclear markers, which we hope will lead to a better understanding of the processes that caused the observed incongruence. Species identification problem— Species identification is still largely based on morphology, though growing evidence exists that many biologically relevant entities will be missed in studies that rely solely on morphological traits (Bickford et al., 2007; Soltis et al., 2007). An increasing number of studies point to a complex genetic and phylogenetic structure of morphologically uniform bryophytes (Feldberg et al., 2007; Vanderpoorten et al., 2008; Wickett and Goffinet, 2008; Fuselier et al., 2009; Kreier et al., 2010; Ramaiya et al., 2010; Laenen et al., 2011), and it is now obvious that the morphological species concept is neither suited for a reliable approximation of bryophyte diversity nor for reconstruction of species origins and range formation (Hedenäs and Eldenäs, 2008; Heinrichs et al., 2009b). Molecular data do not solve all questions; however, they improve our understanding of the patterns and processes that have led to the current bryophyte diversity (Derda and Wyatt, 2000; Jankowiak-Siuda et al., 2008; Szövényi et al., 2009; Karlin et al., 2010). DNA sequences have become increasingly important for the identification of plant species (CBOL Plant Working Group, 2009), but it is evident that not all land plant species can be identified by a unified molecular barcode (Roy et al., 2010), especially because recently emerged species may be too young to have accumulated mutations in barcode regions. A simple transfer of current species concepts into a molecular code allows for a rapid identification without morphological skills. However, it does not lead to a deeper understanding of morphologically cryptic entities. Hence, the pivotal question associated with molecular taxonomy is what kind of species or entities
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should be identified and how should they be named (Bachmann, 1995; Rieseberg et al., 2006)? Some authors adopt a wide species concept in bryophyte complexes with unstable morphology (Hodgetts, 2003; Heinrichs et al., 2004), especially when morphological character states are diffusely distributed on a molecular topology (Therrien et al., 1998; Hartmann et al., 2006; Fuselier et al., 2009). It becomes more and more evident that this approach depicts diversification processes incompletely (Heinrichs et al., 2009b). In the near future, we therefore will need to find taxonomic solutions for biological entities that have not accumulated morphological disparities. Integrating these entities into a formal nomenclature system will alienate botanists without access to molecular tools; ignorance of these entities can lead to stagnation in making scientific findings. Phycologists often distinguish between morphological species and sexually isolated but morphologically cryptic strains (Coleman, 2005; Amato et al., 2007). Similar entities within the thalloid liverworts Conocephalum and Pellia have been informally named using a letter system (Odrzykoski and Szweykowski, 1991; Pacak and Szweykowska-Kulinska, 2003). Such an informal taxonomy is possibly a practical temporary solution. Allopatric morphologically cryptic entities can be integrated into a formal taxonomy without much difficulty. Biological species sensu Mayr (1942) are generalizable entities that fulfill the requirements of a “natural kind” (Bunge, 1979; Mahner, 1993; Ax, 1995); however, to date we do not possess an approach that allows for an efficient identification of reproductive communities in bryophytes. Therefore we can merely approximate the species recognition problem and make some proposals toward a better understanding of Porella species diversity. Our sampling of 60 accessions of the P. platyphylla/P. platyphylloidea complex does not allow for a comprehensive estimation of clade diversity and distribution, especially with regard to the limited presence of Asian accessions. However, according to our data, P. platyphylla s.l. consists of a North American main clade and a main clade that is widespread in Europe but also present in North America and in Asia. Therrien et al. (1998) were not able to separate these clades by character states of the sporophyte and the perianth mouth. We also were not able to clearly discriminate our gametophytic material using morphology. However, we were able to confirm the tendency of larger leaf lobules in the purely North American clade (Bischler et al., 2006). Taking into consideration the different distributions and evolutionary histories of our two main clades and the availability of several binominals (P. platyphylla, P. platyphylloidea), we adopt an epitypification approach to relate the Linnean name P. platyphylla (s. str.) to the mainly European clade. The North American main clade corresponds to P. platyphylloidea, which was based on herbarium material gathered in North Carolina (de Schweinitz, 1821). Therrien et al. (1998) distinguished three isozyme groups within their broadly defined P. platyphylla. These are likely consistent with our P. platyphylla s.str. and the clades A and B of P. platyphylloidea (Fig. 2). However, a formalization of the latter two entities appears premature, especially in light of our limited sampling and the presence of a third entity that deserves attention (subclade B1 of clade B, Fig. 2). Extension of the population sampling and utilization of more variable marker systems such as microsatellites are necessary to gain deeper insights into speciation in P. platyphylloidea. North American and European accessions of both P. platyphylla and P. cordaeana are placed sister to each other in our
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Lectotype of Jungermannia platyphylla L. (reproduced from Dillenius, 1741, Plate 72, Fig. 32 B).
nuclear data set. Our data set does not allow the determination of whether these geographically separated populations still hold the potential to interbreed successfully or represent genetically incompatible entities that have kept the morphology of their common ancestor. Morphologically less conservative angiosperms include examples of closely related species with North American vs. Eurasian distributions (Ekenäs et al., 2009), and it is possible that such entities also occur within bryophytes. Biogeography—The clear phylogeographical structure of many bryophyte lineages contradicts a general panmixis hypothesis (Heinrichs et al., 2009b). Available molecular evidence instead supports the emergence of disjunct ranges of bryophyte species by short-distance dispersal, rare long-distance dispersal events, extinction, recolonization, and diversification (Heinrichs et al., 2009a). This series of events could explain the distribution ranges of the investigated Porella species, which were likely subjected to range contradictions and expansions in consequence of the Pleistocenic glacial cycles. The substructure of P. platyphylloidea clade B (Fig. 2) may be the result of a range fragmentation of the eastern North American metapopulation into northern and southern groups, with subsequent sporadic dispersal events of the southern group into northern regions. A similar pattern has been recognized within the eastern North American liverwort Frullania asagrayana Mont. (Ramaiya et al., 2010).
The sister relationship of North American and European clades of P. platyphylla and P. cordaeana seems to contradict recent intercontinental gene flow. However, rare long-distance dispersal may not necessarily lead to recombination and the recoalescence of diverging lineages. Genetic drift may instead eliminate haplotypes from populations soon after their arrival (Hock et al., 2009). Thus, it would be worthwhile to conduct crossing experiments with North American and Eurasian populations. Porella platyphylla seems to be rather rare in North America, and so far it has been confirmed from only a single locality in New Mexico (Figs. 1, 2). However, subsequent studies that focus on an extension of the sampling will demonstrate whether P. platyphylla is really an anomaly of the North American Porella flora or whether it occurs sporadically throughout larger parts of the continent (Schuster, 1980). Perspectives— Our study demonstrates the limited suitability of the morphological species concept for the recognition of biologically relevant entities of liverworts. The conservative morphology of many liverwort lineages leads to underestimations of species diversity, and we therefore should test the existing classifications using variable molecular markers with extensive population sampling. We also should study the distribution and ecology of the newly detected molecular entities to adequately
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recognize their importance as biological indicators (Hedenäs and Eldenäs, 2007; Heinrichs et al., 2010). Our study emphasizes the need for a revised species classification concept that adequately considers morphologically cryptic biological entities. Failure to include molecular entities into Linnean nomenclature (Benton, 2000; Wheeler, 2004) will result in estrangement of morphologists and evolutionary biologists in the near future. LITERATURE CITED Álvarez, I., and J. F. Wendel. 2003. Ribosomal ITS sequences and plant phylogenetic inference. Molecular Phylogenetics and Evolution 29: 417–434. Amato, A., W. H. C. F. Kooistra, J. H. L. Ghiron, D. G. Mann, T. Pröschold, and M. Montresor. 2007. Reproductive isolation among sympatric cryptic species in marine diatoms. Protist 158: 193–207. Ax, P. 1995. Das System der Metazoa I. Fischer, Stuttgart, Germany. Bachmann, K. 1995. Progress and pitfalls in systematics: Cladistics, DNA and morphology. Acta Botanica Neerlandica 44: 403–419. Benton, M. J. 2000. Stems, nodes, crown clades, and rank-free lists: Is Linnaeus dead? Biological Reviews of the Cambridge Philosophical Society 75: 633–648. Bickford, D., D. J. Lohman, N. S. Sodhi, P. K. L. Ng, R. Meyer, K. Winkler, K. K. Ingram, and I. Das. 2007. Cryptic species as a window on diversity and conservation. Trends in Ecology & Evolution 22: 148–155. Bischler, H., M. C. Boisselier-Dubayle, S. Fontinha, and J. Lambordière. 2006. Species boundaries in European and Macaronesian Porella L. (Jungermanniales, Porellaceae). Cryptogamie, Bryologie 27: 35–57. Boisselier-Dubayle, M. C., and H. Bischler. 1994. A combination of molecular and morphological characters for delimitation of taxa in European Porella. Journal of Bryology 18: 1–11. Boisselier-Dubayle, M. C., J. Lambordière, and H. Bischler. 1998. The leafy liverwort Porella baueri (Porellaceae) is an allopolyploid. Plant Systematics and Evolution 210: 175–197. Bunge, M. 1979. Treatise on basic philosophy, vol. 4. Ontology II: A world of systems. Reidel, Dordrecht, Netherlands. CBOL Plant Working Group. 2009. A DNA barcode for land plants. Proceedings of the National Academy of Sciences, USA 106: 12794–12797. Coleman, A. W. 2005. Paramecium aurelia revisited. Journal of Eukaryotic Microbiology 52: 68–77. de Schweinitz, L. D. 1821. Specimen florae Americae septentrionalis cryptogamicae. J. Gales, Raleigh, North Carolina, USA. Derda, G. S., and R. Wyatt. 2000. Isozyme evidence regarding the origins of three allopolyploid species of Polytrichastrum (Polytrichaceae, Bryophyta). Plant Systematics and Evolution 220: 37–53. Dillenius, J. J. 1741. Historia muscorum. Oxford, UK. Ekenäs, C., J. Rosén, S. Wagner, I. Merfort, A. Backlund, and K. Andreasen. 2009. Secondary chemistry and ribosomal DNA congruencies in Arnica (Asteraceae). Cladistics 25: 78–92. Evans, A. W. 1916. Notes on New England Hepaticae, XIII. Rhodora 18: 74–85, 103–120. Feldberg, K., J. Hentschel, R. Wilson, D. S. Rycroft, D. Glenny, and J. Heinrichs. 2007. Phylogenetic biogeography of the leafy liverwort Herbertus (Jungermanniales, Herbertaceae) based on nuclear and chloroplast DNA sequence data: Correlation between genetic variation and geographical distribution. Journal of Biogeography 34: 688–698. Feldberg, K., J. VáŇa, D. G. Long, J. Shaw, J. Hentschel, and J. Heinrichs. 2010. A phylogeny of Adelanthaceae (Jungermanniales, Marchantiophyta) based on nuclear and chloroplast DNA markers, with comments on classification, cryptic speciation and biogeography. Molecular Phylogenetics and Evolution 55: 293–304. Felsenstein, J. 1985. Confidence limits on phylogenies: An approach using the bootstrap. Evolution; International Journal of Organic Evolution 39: 783–791.
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Appendix 1. Voucher information and GenBank accession numbers for species of Porella. A dash indicates the region was not sequenced for the sample. Sequences in bold were obtained from GenBank. Abbreviations of herbaria are as follows: DUKE, Duke University; E, Royal Botanic Garden Edinburgh; F, Field Museum; GOET, University of Göttingen; HSNU, East China Normal University; JE, University of Jena; UBC, University of British Columbia. Taxon Porella arboris-vitae (With.) Grolle
Locality Georgia
Spain, Tenerife Porella × baueri (Schiffn.) C. E. ? O. Jensen Porella bolanderi (Aust.) Pears. USA, California, Santa Cruz Co. USA, California, San Mateo Co. USA, California, San Mateo Co. Spain, Canary Isls., La Palma Porella canariensis (F. Weber) Underw. Austria, Tyrol, Silvretta Porella cordaeana (Huebener) Moore Bulgaria, Rila Mts., Borowez Canada, British Columbia, Graham Isl. Czech Republic, Moravia France, Dép. Haut-Rhin, Kruth Germany, Rhineland-Palatinate, Birkenfeld Co. Germany Germany, Thuringia, Schmalkalden Great Britain, Scotland Italy, the Abruzzi, Prov. Pescara Italy, Sicily, Prov. Palermo Italy, Piedmon, Prov. Cúeno Spain, Tenerife USA, Alaska, Anchorage USA, Alaska, Duck Isl. USA, Alaska, Duck Isl. USA, California, Marin Co. USA, California, Mariposa Co. USA, California, Mariposa Co. USA, California, Mariposa Co. USA, California, Shasta Co USA, California, Shasta Co. USA, California, Trinity Co. USA, Montana, Granite Co. USA, Montana, Missoula Co. USA, Montana, Sweet Grass Co. USA, Nevada, White Pine Co. USA, Washington, Columbia Co. USA, Washington, Snohomish Co. Porella obtusata (Taylor) Trevis. Porella pinnata L. Porella platyphylla (L.) Pfeiff.
France, Provence, Grasse Greece, Samos, Kosmadei USA, North Carolina, Wake Co. Austria, Salzkammergut Bulgaria China, Xinjiang, Bogedashan France, Haute Saône, Faucogney Georgia Georgia Germany, Bavaria, Garmisch-Partenkirchen Germany, Bavaria, Mellrichstadt Germany, Lower Saxony, Göttingen Germany, North Rhine-Westphalia, Mechernich Germany, Rhineland-Palatinate, Ahrweiler Co. Germany, Rhineland-Palatinate, Birkenfeld Co. Germany, Saarland, St. Wendel Co. Germany, Thuringia, Werra Valley
Voucher
nrITS
trnL-trnF
atpB-rbcL
Zündorf 24757 (JE)
JF734465
JF734472
JF734457
Eckstein 7110 (JE) ?
JF734466 AF499014
JF734473 AF462148
JF734458 —
Shevock 31794 (GOET) Whittemore 4393 (GOET) Whittemore 4405 (GOET) Schäfer-Verwimp & Verwimp 24763 (GOET) Heinrichs 1437 (GOET)
JF734585 EF545480 EF545481 EF545539
JF734665 EF545388 EF545389 EF545447
JF734505 — — JF734464
JF734559
JF734639
JF734479
Huneck BG-H-85-4 (JE) Schofield et al., 100739 (F)
JF734560 JF734561
JF734640 JF734641
JF734480 JF734481
Buryová 4390 (DUKE) Frahm Bry. Vog. Exs. 97 (GOET) Heinrichs et al., 3767 (GOET)
JF734562 JF734563 JF734564
JF734642 JF734643 JF734644
JF734482 JF734483 JF734485
Hentschel 1730 (GOET) Hentschel 2460 (GOET) Long 34092 (E) Lübenau 346 (JE) Raimondo 117L (JE) Schäfer-Verwimp 27338 (GOET) Eckstein 2322 (JE) Schofield 117519 (UBC) Schofield & Talbot 98853 (F) Schofield & Talbot 98861 (F) Ornduff 10254 (DUKE) Shevock 29087 (GOET) Shevock 29117 (GOET) Shevock 29979 (GOET) Norris 72907 (GOET) Shevock 32509 (GOET) Shevock 33023 (GOET) Schofield et al., 120975 (DUKE) Schofield 120841 (DUKE) Vitt 35492 (F) Shevock 27633 (GOET) Schofield et al., 116985 (DUKE) Schofield & Harpel 120651 (DUKE) Asakawa H-0001F (GOET) Düll 24 (JE) Majestyk & Wilbur 10469 (DUKE) Huneck s.n. (JE) Hentschel 763 (GOET) Gao & Fu 399 (HSNU) Frahm Bry. Vog. Exs. 120 (GOET) Zündorf 23906 (JE) Zündorf 24561 (JE) Hentschel 264 (JE)
EF545474 JF734564 EF545473 JF734566 JF734568 JF734567 JF734569 EF545475 JF734570 JF734571 JF734572 JF734573 JF734574 JF734575 JF734578 JF734576 JF734577 JF734579 JF734580 JF734581 JF734582 JF734583 JF734584
EF545382 JF734644 EF545381 JF734646 JF734648 JF734647 JF734649 EF545383 JF734650 JF734651 JF734652 JF734653 JF734654 JF734655 JF734658 JF734656 JF734657 JF734659 JF734660 JF734661 JF734662 JF734663 JF734664
— JF734484 — JF734486 JF734488 JF734487 JF734489 — JF734490 JF734491 JF734492 JF734493 JF734494 JF734495 JF734498 JF734496 JF734497 JF734499 JF734500 JF734501 JF734502 JF734503 JF734504
JF734467 JF734468 JF734469
JF734474 JF734475 JF734476
JF734459 JF734460 JF734461
JF734586 EF545477 JF734587 JF734588 JF734589 JF734590 JF734599
JF734666 EF545385 JF734667 JF734668 JF734669 JF734670 JF734679
JF734506 — JF734507 JF734508 JF734509 JF734510 JF734519
Hentschel 3575 (JE) Heinrichs & Feldberg 4600 (GOET)
JF734597 JF734594
JF734677 JF734674
JF734517 JF734514
Heinrichs 854 (GOET)
JF734593
JF734673
JF734513
Heinrichs 840 (GOET)
JF734592
JF734672
JF734512
Heinrichs & Caspari 2573 (GOET)
JF734595
JF734675
JF734515
Heinrichs & Caspari 2705 (GOET) Hentschel 1567 (GOET)
JF734596 EF545476
JF734676 EF545384
JF734516 —
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Appendix 1. Continued. Taxon
Locality
nrITS
trnL-trnF
atpB-rbcL
Hentschel & Heinrichs 3138 (GOET) Hentschel & Busch 3131 (JE)
JF734598
JF734678
JF734518
JF734600
JF734680
JF734520
EF545478 JF734601 JF734602 JF734591
EF545386 JF734681 JF734682 JF734671
— JF734521 JF734522 JF734511
Poland, Western Carpathians, Gorce Mts. Poland, Western Carpathians, Przybedza Sweden, Södermanland, Södertälje Switzerland, Neuenburg, Neuchâtel USA, New Mexico, Catron Co. USA, New Mexico, Catron Co. Canada, New Brunswick, Albert Co.
Eckstein 4632 (GOET) Düll s.n. (JE) Marstaller s.n. (JE) Klama & Zarnowiec Hep. Polon. Exs. 105 (GOET) Stebel Hep. Polon. Exs. 317 (GOET) Stebel Hep. Polon. Exs. 264 (JE) Huneck S92-5 (JE) Huneck s.n. (JE) Worthington 32690 (GOET) Worthington 32690a (GOET) Schofield 124481 (DUKE)
JF734604 JF734603 JF734605 JF734606 EF545479 JF734607 JF734609
JF734684 JF734683 JF734685 JF734526 EF545387 JF734687 JF734689
JF734524 JF734523 JF734525 JF734526 — JF734527 JF734529
Canada, New Brunswick, Fundy N. P. Canada, Nova Scotia, Kings Co. Canada, Nova Scotia, Kings Co. Canada, Nova Scotia, Inverness Co. Canada, Nova Scotia, Kings Co. Canada, Nova Scotia, Inverness Co. Canada, Nova Scotia, Kings Co. Canada, Ontario, Algoma Distr. Canada, Ontario, Frontenac Co. Canada, Ontario, Manitoulin Dist. Canada, Quebec, Rimouski USA, Alabama, Covington Co. USA, Arkansas, Conway Co. USA, Arkansas, Montgomery Co. USA, Kentucky, Caroll Co. USA, Kentucky, Knott Co. USA, Maine, Hancock Co. USA, Michigan, Keeweenaw Co. USA, Michigan, Emmet Co. USA, Mississippi, Amite Co. USA, Missouri USA, Missouri, St. Louis Co. USA, Missouri, Reynolds Co. USA, New Hampshire, Bennington USA, New York, Green Co. USA, North Carolina, Orange Co. USA, North Carolina, Haywood Co. USA, South Dakota, Meade Co. USA, Vermont, Essex Co. USA, Virginia, Lunenburg Co. USA, Virginia, Scott Co. USA, West Virginia, Hardy Co. USA, Wisconsin, Door Co. USA, Oregon, Douglas Co. USA, Oregon, Hood Co.
Belland et al., 17828 (DUKE) Schofield 106589 (UBC) Schofield 88211 (F) Schofield et al., 88549 (F) Schofield 89524 (F) Schofield 91871 (F) Schofield 95317 (F) Williams & Cain s.n. (GOET) Ley 379 (GOET) Ireland 23230 (F) Faubert & Grenier 238 (GOET) Shaw 6143 (DUKE) Majestyk 5125 (DUKE) Majestyk 7186 (DUKE) Risk 1460 (DUKE) Risk 11427 (DUKE) Briscoe 810 (GOET) Janssens 7336 (DUKE) Taub 21 (DUKE) Alford 47 (DUKE) Holmberg s.n. (GOET) Holmberg & Darigo 22 (GOET) Redfearn 28852 (GOET) Raabe 2707 (GOET) Smith 51864 (GOET) Shaw 4915 (DUKE) Singh 227 (DUKE) Churchill 9221 (GOET) Shaw 6997 (DUKE) Breil 6028 (DUKE) Nelson 17498 (DUKE) Bachmann 416 (DUKE) Brown s.n. (GOET) Schofield 100589 (DUKE) Schofield 119304 (DUKE)
JF734608 EF545484 JF734610 JF734611 JF734612 JF734613 JF734614 EF545483 JF734615 JF734616 JF734617 JF734618 JF734619 JF734620 JF734621 JF734622 JF734623 JF734624 JF734625 JF734626 JF734627 EF545482 JF734628 JF734631 JF734632 JF734629 JF734630 JF734633 JF734638 JF734634 JF734635 JF734636 JF734637 JF734470 JF734471
JF734688 EF545391 JF734690 JF734691 JF734692 JF734693 JF734694 — JF734695 JF734696 JF734697 JF734698 JF734699 JF734700 JF734701 JF734702 JF734703 JF734704 JF734705 JF734706 JF734707 EF545390 JF734708 JF734711 JF734712 JF734709 JF734710 JF734713 JF734718 JF734714 JF734715 JF734716 JF734717 JF734477 JF734478
JF734528 — JF734530 JF734531 JF734532 JF734533 JF734534 — JF734535 JF734536 JF734537 JF734538 JF734539 JF734540 JF734541 JF734542 JF734543 JF734544 JF734545 JF734546 JF734547 — JF734548 JF734551 JF734552 JF734549 JF734550 JF734553 JF734558 JF734554 JF734555 JF734556 JF734557 JF734462 JF734463
Germany, Hesse, Bad Sooden-Allendorf Germany, Mecklenburg-West Pomerania, Rügen Italy, Sicily, Sciacca Italy, South Tyrol, Prov. Bozen Italy, Umbria, Lago Trasimento Poland, Krakóv Upland
Porella platyphylloidea (Schwein.) Lindb
Porella roellii Steph.
Voucher
