A revision of the Baltic and Bitterfeld amber fossils
assigned to
Liverworts belong to the oldest lineages of plants on land and date back to
the early Paleozoic (Taylor et al., 2009). They are characterized by a life
cycle with a prominent leafy or thalloid gametophyte, an unbranched
sporophyte, and the frequent presence of oil bodies and elaters (Renzaglia
et al., 2007). Liverwort diversity today includes some 7000 species in
Using the geological age of
The amber inclusions (12 from Baltic and 6 from Bitterfeld amber) used in this study are housed at the Museum für Naturkunde at Berlin, the Georg August University of Göttingen (numbers preceded by GZG.BST), the SNSB-Bavarian State Collection for Palaeontology and Geology (numbers preceded by SNSB-BSPG), and the Carsten Gröhn amber collection. Specimens from the Museum für Naturkunde at Berlin were previously published under BHU-Palaeo collection numbers (e.g., Grolle and Meister, 2004). However, this acronym has recently been replaced by “MB.Pb”.
The surface of some of the amber pieces was polished manually with a series
of wet silicon carbide abrasive papers (grit size from FEPA P 600–4000
(particle size: 25.8 to 5
The Paleogene amber fossil
Divergence time estimates based on the DNA sequence variation obtained from
extant representatives of cephalozioid liverworts were conducted to assess
the level of congruence with our taxonomic placement of
jModelTest 2.1.7 (Guindon and Gascuel, 2003; Darriba et al., 2012) was
employed to choose a nucleotide substitution model for both nuclear and
plastid DNA datasets. With regard to the nuclear marker, the Bayesian
information criterion (BIC) supported the TIM3
Bayesian divergence time estimates were generated in BEAST 1.8.4 (Drummond
et al., 2012). The DNA dataset was split into a nuclear and a chloroplast
partition, with unlinked substitution and clock models, and linked trees. An
uncorrelated relaxed (lognormal) clock was employed for both partitions and
the substitution models were implemented according to the results of the
jModelTest analyses. A birth–death model for incomplete sampling was
employed. The root of the tree was calibrated at 202.01 Ma based on
estimates in Laenen et al. (2014) for the split between the Adelanthaceae
and Cephaloziaceae in an analysis not factoring
Plants small, prostrate or ascending, brown or reddish brown (sometimes
appearing whitish-green or yellowish as a result of shrinking subsequent to
embedding), creeping or forming dense mats; leafy shoots 1–14 mm long,
0.10–0.56 mm wide, sparingly ventral-intercalary branched
(gyrothecal), leafy, flagelliform or stoloniform; leafy shoots
often tapering into a long flagella or sectors with reduced, scaly leaves
alternating with sectors producing well-developed leaves. Rhizoids diffusely
distributed along ventral side of stem. Stems rigid, 0.05–0.11(–0.14) mm
in diameter, 3–6(–ca. 8) cells high, epidermal cells surrounding slightly
smaller or similar-sized inner cells (discernible in two broken edges of
stems), epidermal cells short rectangular to rectangular, 15–25
Bavarian State Collection for Palaeontology and Geology, Munich, Germany:SNSB-BSPG 1958 VIII 44 (Bachoven-Echt amber collection P44); SNSB-BSPG 1958 VIII 95 (Bachoven-Echt amber collection P95)Geoscientific collections, Georg August University Göttingen, Germany: GZG.BST.21957 (Hoffeins amber collection 5-43); GZG.BST.21959 (K7.319)Gröhn amber collection, Glinde, Germany:2015, 2038, 2082Museum für Naturkunde Berlin, Germany:MB.Pb.1979/654 (Künow amber collection 95);MB.Pb.1979/688 (Künow amber collection 145);MB.Pb.1979/689 (Künow amber collection 146);MB.Pb.1979/708 (Künow amber collection 165a)
Geoscientific collections, Georg August University Göttingen, Germany:GZG.BST.21958 (Hoffeins amber collection 930-3)Museum für Naturkunde Berlin, Germany:MB.Pb.1997/2 (Kutscher amber collection H006);MB.Pb.1997/16 (Kutscher amber collection M 8/6);MB.Pb.1997/24 (Grolle amber collection M 10/5);MB.Pb.1997/36 (Grolle amber collection M 12/8);MB.Pb.1997/36 (Grolle amber collection M 12/9)
The DNA-based divergence time estimates (Fig. 3) support a late Early
Cretaceous to early Eocene age of the
Phylogenetic chronogram of Cephaloziaceae based on DNA
sequence variation of extant species, with secondary calibration from Laenen
et al. (2014). Confidence age estimate intervals shown as horizontal bars.
Vertical bar indicates age interval of Baltic amber. Transfer of the fossil
Grolle and Meister (2004) transferred the Eocene amber fossil
Based on the presence of bilobed underleaves (Fig. 2c, g), we dismiss
assignment of
Based on the preceding considerations and comparison, we believe that
The extant
Deviations from DNA standard substitution rates are commonplace and have
been documented for seed plants (Bromham et al., 2013), ferns (Rothfels and
Schuettpelz, 2014; Zhong et al., 2014; Crusz et al., 2016), and liverworts
(Villarreal et al., 2016). As a result, age estimates for
Lineages of plants usually are somewhat older than their oldest indisputable
fossil representatives. Heinrichs et al. (2015a) and Schneider et al. (2016)
therefore proposed to involve age hypotheses from independently generated
molecular chronograms in the taxonomic treatment of fossils. These
integrative approaches, which focus on the integration of evidence from
different origins (Dayrat, 2005; Will et al., 2005), may be misleading if the
molecular clocks greatly vary; however, extreme rate variations (Rothfels
and Schuettpelz, 2014) have rarely been reconstructed for seed-free land
plants, and approaches involving secondary calibrations and standard
substitution rates have therefore been advocated (Villarreal and Renner,
2014). We present divergence time estimates of Cephaloziaceae based on a
secondary calibration obtained from the most comprehensive chronogram of
liverworts generated without using the fossil
The chronogram for Cephaloziaceae shown in Fig. 3 is the most comprehensive
assessment to date with regard to taxon sampling. The results are congruent
with the divergence time estimates provided in Feldberg et al. (2013, 2014)
and Laenen (2014), and support a Cretaceous to Paleogene age of most generic
crown groups, a recurrent pattern in the evolution of leafy liverworts
(Cooper et al., 2012). Similar hypotheses have been derived from amber
fossils of liverworts that usually match the morphology of extant genera
(Heinrichs et al., 2015b). Taxonomic conclusions drawn on the basis of the
gross morphology of incompletely preserved amber fossils are problematic,
and hence additional evidence is always intensively sought after and highly
welcome. Integrative approaches using a combination of morphological
evidence and evidence generated from the DNA variation of extant species
(Heinrichs et al., 2007, 2015a) have dismissed hypotheses on affinities of
certain Eocene amber fossils to the extant species
An integrative taxonomic approach using morphological and independent,
DNA-based evidence suggests that the fossil liverwort
All necessary data are available in the Supplement.
Taxa used in the divergence time estimates, including information about the origin of the studied material, voucher information, and GenBank accession numbers.
The authors declare that they have no conflict of interest.
We thank Christel and Hans Werner Hoffeins (Hamburg) for providing their liverwort fossils to the Geoscientific Museum Göttingen, and Alexander Gehler (Göttingen), Christian Neumann (Berlin) and Martin Nose (Munich) for making the amber collections of the Geoscientific Museum Göttingen, the Museum für Naturkunde at Berlin and the Bavarian State Collection for Palaeontology and Geology available for study. Edited by: C. Bickelmann Reviewed by: A. Hagborg and one anonymous referee