Rewinding the molecular clock in the genus Carabus (Coleoptera: Carabidae) in light of fossil evidence and the Gondwana split: A reanalysis

Molecular clocks have become powerful tools given increasing sequencing and fossil resources. However, calibration analyses outcomes depend on the choice of priors. Here, we revisited the seminal dating study published by Andújar and coworkers of the genus Carabus proposing that prior choices need re-evaluation. We hypothesized that reflecting fossil evidence and the Gondwanan split properly significantly rewinds the molecular clock. We re-used the dataset including five mitochondrial and four nuclear DNA fragments with a total length of 7888 nt. Fossil evidence for Oligocene occurrence of Calosoma was considered. Root age was set based on the fossil evidence of Harpalinae ground beetles in the Upper Cretaceous. Paleogene divergence of the outgroup taxa Ceroglossini and Pamborini is introduced as a new prior based on current paleontological and geological literature. The ultrametric time-calibrated tree of the extended nd5 dataset resulted in a median TMRCA Carabus of 53.92 Ma (HPD 95% 45.01–63.18 Ma), roughly 30 Ma older than in the Andújar study. The splits among C. rugosus and C. morbillosus (A), C. riffensis from the European Mesocarabus (B), and Eurycarabus and Nesaeocarabus (C) were dated to 17.58 (12.87–22.85), 24.14 (18.02–30.58), and 21.6 (16.44–27.43) Ma. They were decidedly older than those previously reported (7.48, 10.93, and 9.51 Ma). These changes were driven almost entirely by constraining the Carabidae time-tree root with a Harpalinae amber fossil at ~99 Ma. Utilizing the nd5 dating results of three well-supported Carabus clades as secondary calibration points for the complete MIT-NUC dataset led to a TMRCA of Carabus of 44.72 (37.54–52.22) Ma, compared with 25.16 Ma (18.41–33.04 Ma) in the previous study. Considering fossil evidence for Oligocene Calosoma and Late Cretaceous Harpalini together with the Gondwanan split as a new prior, our new approach supports the origin of genus Carabus in the Eocene. Our results are preliminary because of the heavy reliance on the nd5 gene, and thus will have to be tested with a sufficient set of nuclear markers. Additionally, uncertainties due to dating root age of the tree based on a single fossil and outgroup taxon affect the results. Improvement of the fossil database, particularly in the supertribe Carabitae, is needed to reduce these uncertainties in dating Carabus phylogeny.

Analysis of Genetic Variability Amongst Polyploid Genotypes of Pteris vittata L. From Various Geographic Locales of India

Pteris vittata L. is very common and a widely distributed species belongs to the family Pteridaceae. Various cytotypes from diploid to octaploid is available in this fern species. The present work has been carried out for genetic diversity in this fern both within and between the cytotypes. The molecular analysis at inter- as well as intra-species has been carried out with 57 accessions of P. vittata as well as of other species of Pteris with Microsorium punctatum considered as an out group taxon. For the present study 48 P. vittata (36 tetraploid and 12 pentaploid) and five of other species (four P. cretica, one P. pellucida, one P. tremula, one P. quadriaurita, and two P. ensiformis) accessions were used. The UPGMA (unweighted pair group method with arithmetic mean) dendrograms were generated for each method separately, as well as for all methods cumulatively, after a 1000 replicate bootstrap analysis. In order to determine the utility of each of the method, a comparative statistical assessment was done and marker index (MI), expected average heterozygosity, fraction of polymorphic loci and effective multiplex ratio (EMR) were calculated in case of each of the methods used in the present study. At the level of individual methods highest MI was obtained for directed amplification of minisatellites DNA (DAMD) method. Our findings of the present study concluded that out of the three methods Random Amplified Polymorphic DNA (RAPD), Inter-Simple Sequence Repeat (ISSR), and Directed Amplification of Minisatellite DNA (DAMD), DAMD was the best in term of polymorphism and heterozygosity as scores exhibited highest MI. The different accessions of P. vittata collected from different phytogeographical regions falls into six groups. Out of six clusters, one cluster is of pentaploid cytotype, four clusters are of tetraploid cytotype and one for outgroup taxon (M. punctatum). The result thus showed that within tetraploid, heterozygosity with variable genomic structure exists.

Rewinding the molecular clock in the genus Carabus (Coleoptera: Carabidae) in light of fossil evidence and the Gondwana split: a re-analyses

1AbstractBackgroundMolecular clocks have become powerful tools given increasing sequencing and fossil resources. However, outcome of calibration analyses depend on choosing priors. Here we revisit a seminal dating study of the genus Carabus by Andujar et al. proposing that their prior choices need re-evaluation with the hypothesis that reflecting fossil evidence and the Gondwanan split properly rewinds the molecular clock significantly. We used the same dataset including five mitochondrial and four nuclear DNA fragments with 7888 nt total length. We set the root age based on the fossil evidence of Harpalinae ground beetles in the Upper Cretaceous and introduce the Paleogene divergence of the outgroup taxa Ceroglossus (endemic to South-America) and Pamborus + Maoripamborus (Australia, New Zealand) as a new prior based on current paleontological and geological literature.ResultsThe ultrametric time-calibrated tree of the extended nd5 dataset resulted in a median TMRCA Carabus age of 58.48 Ma (HPD95% 46.61-72.04), roughly 35 Ma older than in the Andujar study. The splits between C. rugosus and C. morbillosus (A), between C. riffensis from the European Mesocarabus (B), and between Eurycarabus and Nesaeocarabus (C) were dated to 19.19 (13.54-25.87), 25.95 (18.8-34.62), and 23.98 (17.28-31.47) Ma and were thus decidedly older than previously reported (7.48, 10.93, and 9.51 Ma). These changes were driven solely by constraining the Carabidae time tree root with Harpalinae amber fossils at ∼99 Ma. Utilizing the nd5 dating results of three well supported Carabus clades as secondary calibration points for the complete MIT-NUC data set lead to a TMRCA of Carabus of 53.56 (41.25-67.05) Ma compared to 25.16 (18.41-33.04) in Andujar’s study.ConclusionTaking into account the Gondwanan split as a new prior, together with the fossil evidence of the outgroup taxon Harpalini in the Late Cretaceous, our new approach supports an origin of the genus Carabus in the Paleocene-Early Eocene. Our results are preliminary due to the heavy reliance on the nd5 gene and thus will have to be tested with sufficient set of nuclear markers. In addition, uncertainties arise from dating the root age of the tree based on a single fossil and outgroup taxon which has a major effect on the results. Improvement of the fossil data base particularly in the supertribe Carabitae is thus strongly needed to reduce the currently large uncertainties in dating Carabus phylogeny.

Molecular and Morphological Variation among Populations of Pediomelum tenuiflorum (Pursh) A.N. Egan (Fabaceae) in Nebraska, USA

Individuals of Pediomelum tenuiflorum, “wild alfalfa”, from disjunct populations in Nebraska vary extensively in their overall gestalt. Those in the western and central part of the state have a very slender growth habit, with thin stems and few, small flowers; whereas, those in the southeast have a very robust growth habit with heavy-looking stems and many tightly clustered flowers. For nearly 200 years, taxonomists have alternated between splitting P. tenuiflorum into two species, with the many-flowered morphotype named P. floribundum, and lumping all the morphological variants into one species as they are now. In this study, we investigated morphological and molecular characters that could be used to clarify taxonomic classifications of these morphotypes. We measured 10 morphological characters on 51 specimens and sequenced nearly 300,000 nucleotide characters on the Illumina platform from three cellular genomes in seven samples of Pediomelum plus an outgroup taxon. Results revealed six significantly different morphological characters but ambiguous evolutionary histories of the plastid and mitochondrial genomes in P. tenuiflorum. Our complete plastid genomes and genes and noncoding regions of the mitochondrial genome may be used as a foundation for studying the evolutionary histories of these genomes. Additionally, we identified seven highly variable genomic regions in the chloroplast genome upon which a molecular phylogenetic investigation on an expanded set of samples from across the species’ geographic distribution can be conducted to further define the taxonomic placements of P. tenuiflorum and P. floribundum.

Genetic equidistance at the nucleotide level

The genetic equidistance phenomenon was first discovered in 1963 by Margoliash and shows complex taxa to be all approximately equidistant to a less complex species in amino acid percentage identity. The result has been mis-interpretated by the ad hoc universal molecular clock hypothesis, and the much overlooked mystery was finally solved by the maximum genetic diversity hypothesis (MGD). Here, we studied 15 proteomes and their coding DNA sequences (CDS) to see if the equidistance phenomenon also holds at the CDS level. We performed DNA alignments for a total of 5 groups with 3 proteomes per group and found that in all cases the outgroup taxon was equidistant to the two more complex taxa species at the DNA level. Also, when two sister taxa (snake and bird) were compared to human as the outgroup, the more complex taxon bird was closer to human, confirming species complexity rather than time to be the primary determinant of MGD. Finally, we found the fraction of overlap sites where coincident substitutions occur to be inversely correlated with CDS conservation, indicating saturation to be more common in less conserved DNAs. These results establish the genetic equidistance phenomenon to be universal at the DNA level and provide additional evidence for the MGD theory.

Implications of a new species of the Oligo-Miocene kangaroo (Marsupialia: Macropodoidea) Nambaroo, from the Riversleigh World Heritage Area, Queensland, Australia

A partial skeleton (including both skull and postcranium) and referred dental material attributable to a new species of Oligo-Miocene kangaroo, Nambaroo gillespieae, are described from the Riversleigh World Heritage Area, northwestern Queensland, Australia. The holotype specimen is one of the oldest articulated fossil kangaroo skeletons yet discovered and includes the first postcranial material definitively attributable to the extinct family Balbaridae. Functional-adaptive analysis (including comparisons with modern taxa) of the hindlimb and pedal elements suggests consistent use of quadrupedal progression rather than true hopping. Robust forelimbs and an opposable first pedal digit (lost in most macropodoids) might also indicate limited climbing ability. Cladistic analysis of 104 discrete cranio-dental and postcranial characters coded for 25 ingroup and one outgroup taxon places N. gillespieae in a plesiomorphic sister clade (also containing other Balbarids and the propleopine Ekaltadeta ima) to all other macropodoids. This result supports recent revisions to the classification of kangaroos, which recognize Balbaridae as the most basal macropodoid family-level taxon.

Chromosome painting among Proboscidea, Hyracoidea and Sirenia: support for Paenungulata (Afrotheria, Mammalia) but not Tethytheria

Despite marked improvements in the interpretation of systematic relationships within Eutheria, particular nodes, including Paenungulata (Hyracoidea, Sirenia and Proboscidea), remain ambiguous. The combination of a rapid radiation, a deep divergence and an extensive morphological diversification has resulted in a limited phylogenetic signal confounding resolution within this clade both at the morphological and nucleotide levels. Cross-species chromosome painting was used to delineate regions of homology between Loxodonta africana (2 n =56), Procavia capensis (2 n =54), Trichechus manatus latirostris (2 n =48) and an outgroup taxon, the aardvark ( Orycteropus afer , 2 n =20). Changes specific to each lineage were identified and although the presence of a minimum of 11 synapomorphies confirmed the monophyly of Paenungulata, no change characterizing intrapaenungulate relationships was evident. The reconstruction of an ancestral paenungulate karyotype and the estimation of rates of chromosomal evolution indicate a reduced rate of genomic repatterning following the paenungulate radiation. In comparison to data available for other mammalian taxa, the paenungulate rate of chromosomal evolution is slow to moderate. As a consequence, the absence of a chromosomal character uniting two paenungulates (at the level of resolution characterized in this study) may be due to a reduced rate of chromosomal change relative to the length of time separating successive divergence events.

Phylogeny of Prosopis (Leguminosae) as shown by morphological and biochemical evidence

Prosopis L. is a rather primitive genus within the Leguminosae–Mimosoideae. This genus has been divided into five sections on the basis especially of the vegetative diversification of the spines. Three of the sections, Algarobia, Monilicarpa and Strombocarpa, are distributed in America. In order to elucidate systematic relationships between the American sections of Prosopis, a morphological and biochemical phylogeny were obtained. Twenty-two morphological characters were scored for 27 species of Prosopis and the outgroup taxon following polyacrilamide gel electrophoresis of seed storage proteins. The results obtained clearly prove that the secction Strombocarpa is a natural taxon. The section Algarobia, on the other hand, seems to comprise an artificial grouping of species. Members of the series Denudantes appear isolated from the remaining species, therefore this taxon should be treated as a new section within Prosopis. The section Algarobia should be circumscribed to series Chilenses, Ruscifoliae and Pallidae, which are always united in a monophyletic clade. Finally, no evidence was found to confirm the existence of the section Monilicarpa.

A molecular phylogenetic approach to Longidoridae (Nematoda: Dorylaimida)

The Longidoridae are a group of ectoparasitic nematodes including two subfamilies and six genera with hundreds of species. Sequences of the D2 and D3 expansion region of the large subunit (LSU) rRNA nuclear gene were amplified and used to reconstruct the phylogeny of longidorids. Phylogenetic analyses with maximum parsimony (MP), maximum likelihood (ML) and Bayesian inference (BI) were performed with one outgroup taxon and 62 longidorid sequences. Confidence of inferred clades was assessed by non-parametric bootstrapping for MP and Bayesian posterior probability for ML. All analyses placed Paralongidorus species as an inner group within the otherwise monophyletic genus Longidorus. The genus Xiphinema, except for X. americanum-group species, was placed as the sister group of Longidorus with strong support from the ML and BI analyses. The X. americanum-group was strongly supported as an exclusive clade to other genus Xiphinema species. The position of the Xiphidorus clade was not well resolved and the phylogenetic analyses did not support it as a sister group to Longidorus as previously inferred from morphology. Secondary structure models were constructed for the D2/D3 region of LSU rRNA for all studied species. It was found that sequence-based and structural morphometric rRNA phylogenies were incongruent.

Molecular phylogeny of Acacia subgenus Phyllodineae (Mimosoideae : Leguminosae) based on DNA sequences of the internal transcribed spacer region

The largest monophyletic group within Acacia is subgenus Phyllodineae, with more than 950 predominately Australian species, the majority characterised by adult foliage consisting of phyllodes. Molecular sequence data from the internal transcribed spacers (ITS) of the nuclear ribosomal DNA repeat were used to investigate the monophyly of seven sections within the subgenus. A nested PCR approach was used to amplify the ITS region. Fifty-one species representative of all sections were sequenced together with one outgroup taxon Lysiloma divaricata (Ingeae).Phylogenetic parsimony analysis suggested that there are two main clades within Phyllodineae but that only one section, Lycopodiifoliae, is apparently monophyletic. In one of the main clades, Lycopodifoliae is related to some taxa in sections Alatae and Pulchellae and some members of section Phyllodineae. In the second main clade, sections Juliflorae, Plurinerves and Botrycephalae cluster with other members of section Phyllodineae. The two sections that are characterised by bipinnate foliage, Botrycephalae and Pulchellae, are nested within phyllodinous clades, indicating that at least two separate reversals to bipinnate leaves have occurred. Botrycephalae is paraphyletic with respect to taxa from section Phyllodineae that have single-nerved phyllodes and racemose inflorescences.