Effects of taxon sampling on molecular dating for within-genus divergence events, when deep fossils are used for calibration

A universal method of molecular dating that can be applied to all families and genera regardless of their fossil records, or lack thereof, is highly desirable. A possible method for eudicots is to use a large phylogeny calibrated using deep fossils including tricolpate pollen as a fixed （124 mya） calibration point. This method was used to calculate node ages within three species-poor disjunct basal eudicot genera, Caulophyllum, Podophyllum and Pachysandra, and sensitivity of these ages to effects such as taxon sampling were then quantified. By deleting from one to three accessions related to each genus in 112 different combinations, a confidence range describing variation due only to taxon sampling was generated. Ranges for Caulophyllum, Podophyllum and Pachysandra were 8.4-10.6, 7.6-20.0, and 17.6-25.0 mya, respectively. However, the confidence ranges calculated using bootstrapping were much wider, at 3-19, 0-32 and 11-32 mya, respectively. Furthermore, deleting 10 adjacent taxa had a large effect in Pachysandra only, indicating that undersampling effects are significant among Buxales. Changes to sampling density in neighboring clades, or to the position of the fixed fossil calibration point had small to negligible effects. Non-parametric rate smoothing was more sensitive to taxon sampling effects than was penalized likelihood. The wide range for Podophyllum, compared to the other two genera, was probably due to a high degree of rate heterogeneity within this genus. Confidence ranges calculated by this method could be narrowed by sampling more individuals within the genus of interest, and by sequencing multiple DNA regions from all species in the phylogeny.     

Historical biogeography of the widespread macroalga Sargassum (Fucales,Phaeophyceae)

Sargassum is a cosmopolitan brown algal genus spanning the three ocean basins of the Atlantic, Pacific and Indian Oceans, inhabiting temperate, subtropical and tropical habitats. Sargassum has been postulated to have originated in the Oligocene epoch approximately 30 mya according to a broad phylogenetic analysis of brown macroalgae, but its diversification to become one of the most widespread and speciose macroalgal genera remains unclear. Here, we present a Bayesian molecular clock study, which analyzed data from the order Fucales of the brown algal crown radiation (BACR) group to reconstruct a time-calibrated phylogeny of the Sargassum clade. Our phylogeny included a total of 120 taxa with 99 Sargassum species sampled for three molecular markers – ITS-2, cox3 and rbcLS – calibrated with an unambiguous Sargassaceae fossil from between the lower and middle Miocene. The analysis revealed a much later origin of Sargassum than expected at about 6.7 mya, with the genus diversifying since approximately 4.3 mya. Current geographic distributions of Sargassum species were then analyzed in conjunction with the time-calibrated phylogeny using the dispersal-extinction-cladogenesis (DEC) model to estimate ancestral ranges of clades in the genus. Results strongly support origination of Sargassum in the Central Indo-Pacific (CIP) region with subsequent independent dispersal events into other marine realms. The longer history of diversification in the ancestral CIP range could explain the much greater diversity there relative to other marine areas today. Analyses of these dynamic processes, when fine-tuned to a higher spatial resolution, enable the identification of evolutionary hotspots and provide insights into long-term dispersal patterns.     

Timing of the evolutionary history of Corallinaceae (Corallinales,Rhodophyta)

The temporal dimension of the most recent Corallinaceae (order Corallinales) phylogeny was presented here, based on first occurrence time estimates from the fossil record. Calibration of the molecular clock of the genetic marker SSU entailed a separation of Corallinales from Hapalidiales in the Albian (Early Cretaceous ~105 mya). Neither the calibration nor the fossil record resolved the succession of appearance of the first three emerging subfamilies: Mastophoroideae, Corallinoideae, and Neogoniolithoideae. The development of the tetra/bisporangial conceptacle roofs by filaments surrounding and interspersed among the sporangial initials was an evolutionary novelty emerging at the Cretaceous–Paleogene boundary (~66 mya). This novelty was shared by the subfamilies Hydrolithoideae, Metagoniolithoideae, and Lithophylloideae, which diverged in the early Paleogene. Subclades within the Metagoniolithoideae and Lithophylloideae diversified in the late Oligocene–middle Miocene (~28–12 mya). The most common reef corallinaceans (Hydrolithon, Porolithon, Harveylithon, “Pneophyllum” conicum, and subclades within Lithophylloideae) appeared in this interval in the Indo‐Australian Archipelago.     

Divergence times and origin of neotropical sheath-tailed bats (tribe Diclidurini) in South America

Times of divergence and origin of sheath-tailed bats (family Emballonuridae) in the New World were approximated with a relaxed molecular clock approach using Bayesian analysis of introns from the three nuclear genetic transmission systems in mammals (autosomal, X and Y sex chromosomes). An upper constraint of 30 mya for the oldest known Neotropical emballonurid fossil and a lower constraint of 13 mya for the only pre-Pleistocene fossil of an extant genus were used as calibration points. Differentiation began in the Late Oligocene with the appearance of two subtribes as independently corroborated by each gene. Following an explosive model of evolution, the genera diversified relatively suddenly in the Early Miocene with seven of the eight genera radiating within 1.4 myr and most intrageneric speciation occurring before the Pliocene. Optimization of ancestral areas onto the phylogeny suggests that the ancestor of New World emballonurid bats has its origin in Africa and this is the third report of placental mammals colonizing South America by trans-Atlantic dispersal and subsequent speciation in allopatry.     

Phylogenetics and biogeography of eastern Asian–North American disjunct genus Pachysandra (Buxaceae) inferred from nucleotide sequences

Abstract Pachysandra is an eastern Asian–North American disjunct genus with three species, two in eastern Asia (Pachysandra axillaris and Pachysandra terminalis) and one in eastern North America (Pachysandra procumbens). Although morphological and cytological studies suggest a close affinity of P. procumbens with P. axillaris, molecular data from nuclear and chloroplast DNA regions have provided conflicting signals. In this study, we tested previous phylogenetic hypotheses using sequences of nuclear ribosomal DNA internal transcribed spacers and chloroplast ndhF gene from multiple individuals of each of the three species. We also estimated the time of divergence between eastern Asia and eastern North America. Our results support the morphological and cytological conclusion that P. procumbens is more closely related to P. axillaris than to P. terminalis. The estimated time of divergence of P. axillaris and P. procumbens was 14.6±5.5 mya, consistent with estimates from many other eastern Asian–North American disjunct genera. The migration of Pachysandra populations from eastern Asia to North America might have occurred by way of the North Atlantic land bridge.     

Molecular and morphological phylogeny of Diplazontinae (Hymenoptera,Ichneumonidae)

Klopfstein, S., Quicke, D. L. J., Kropf, C. & Frick, H. (2011) Molecular and morphological phylogeny of Diplazontinae (Hymenoptera, Ichneumonidae). —Zoologica Scripta, 40, 379–402. Parasitoid wasps are among the most species rich and at the same time most understudied of all metazoan taxa. To understand their diversification and test hypotheses about their evolution, we need robust phylogenetic hypotheses. Here, we reconstruct the phylogeny of the subfamily Diplazontinae using four genes and 66 morphological characters both in separate analyses and in a total evidence approach. The resulting phylogeny is highly resolved, with most clades supported by multiple independent data partitions. It contains three highly supported genus groups, for which we suggest morphological and behavioural synapomorphies. The placement of some of the genera, especially Xestopelta Dasch, is unexpected, but also supported by morphology. Most of the genera are retrieved as monophyletic, with the exception of the morphologically diverse genus Syrphoctonus Förster. We split this genus into three genera, including Fossatyloides gen. n., to restore the phylogeny–classification link. Conflict between the morphological and the molecular topology was mostly resolved in favour of the molecular partition in the total evidence approach. We discuss reasons for this finding, and suggest strategies for future taxon and character sampling in Diplazontinae.     

A fossil‐calibrated relaxed clock for Ephedra indicates an Oligocene age for the divergence of Asian and New World clades and Miocene dispersal into South America

Abstract Ephedra comprises approximately 50 species, which are roughly equally distributed between the Old and New World deserts, but not in the intervening regions (amphitropical range). Great heterogeneity in the substitution rates of Gnetales (Ephedra, Gnetum, and Welwitschia) has made it difficult to infer the ages of the major divergence events in Ephedra, such as the timing of the Beringian disjunction in the genus and the entry into South America. Here, we use data from as many Gnetales species and genes as available from GenBank and from a recent study to investigate the timing of the major divergence events. Because of the tradeoff between the amount of missing data and taxon/gene sampling, we reduced the initial matrix of 265 accessions and 12 loci to 95 accessions and 10 loci, and further to 42 species (and 7736 aligned nucleotides) to achieve stationary distributions in the Bayesian molecular clock runs. Results from a relaxed clock with an uncorrelated rates model and fossil‐based calibration reveal that New World species are monophyletic and diverged from their mostly Asian sister clade some 30 mya, fitting with many other Beringian disjunctions. The split between the single North American and the single South American clade occurred approximately 25 mya, well before the closure of the Panamanian Isthmus. Overall, the biogeographic history of Ephedra appears dominated by long‐distance dispersal, but finer‐scale studies are needed to test this hypothesis.     

The timing of diversification within the most divergent parrot clade

The Strigopidae are an ancient parrot (Psittaciformes) family consisting of three extant species placed in two genera (Nestor, Strigops) and restricted to New Zealand. Their evolutionary history is clouded because the timing of divergence events within this family has variously been attributed to Pleistocene climate change or much earlier earth‐historic events. Here we examine new psittaciform DNA sequence data, and combine them with previously published sequences, to shed light on the poorly understood timing of diversification within the Strigopidae. Using calibrations indirectly derived from both psittaciform and non‐psittaciform fossils, our data indicate a Late Pliocene or Early Pleistocene (ca 1.2–3.6 mya) differentiation between the two Nestor species (kea and kaka), possibly in response to shifts in habitat distribution associated with sea level fluctuations. The unique, monotypic, nocturnal and flightless genus Strigops (kakapo) is shown to have diverged from the Nestor lineage probably ca 28–29 mya, coinciding with the potential Oligocene submergence of Zealandia when much of its landmass may have been fragmented into smaller islands, providing a setting for allopatric diversification.     

Phylogeny of the family Sialidae (Insecta: Megaloptera) inferred from morphological data,with implications for generic classification and historical biogeography

Sialidae (alderflies) is a family of the holometabolous insect order Megaloptera, with ca. 75 extant species in eight genera distributed worldwide. Alderflies are a group of “living fossils” with a long evolutionary history. The oldest fossil attributed to Sialidae dates back to the Early Jurassic period. Further, the global distribution of modern‐day species shows a remarkably disjunctive pattern. However, due to the rareness of most species and scarcity of comprehensive taxonomic revisions, the phylogeny of Sialidae remains largely unexplored, and the present classification system is in great need of renewal. Here we reconstruct the first phylogeny for Sialidae worldwide based on the most comprehensive sampling and broadest morphological data ever presented for this group of insects. All Cenozoic alderflies belong to a monophyletic clade, which may also include the Early Jurassic genus ?Dobbertinia, and the Late Jurassic genus ?Sharasialis is their putative sister taxon. Two subfamilies of Sialidae are proposed, namely ?Sharasialinae subfam. nov. and Sialidinae. Austrosialis is the sister of all other extant genera, an assemblage which comprises three monophyletic lineages: the Stenosialis lineage, the Ilyobius lineage, and the Sialis lineage. The revised classification of Sialidae is composed of 12 valid genera and 87 valid species. Ilyobius and Protosialis are recognized as valid generic names, while Nipponosialis is treated as a synonym of Sialis. Reconstruction of the ancestral area proposes a global distribution of alderflies in Pangaea before their diversification. The generic diversification of alderflies might have occurred before the breakup of Pangaea, but the divergence of some lineages or genera was probably promoted by the splitting of this supercontinent.     

The phylogenetic placement and biogeographical origins of the New Zealand stick insects (Phasmatodea)

The Lanceocercata are a clade of stick insects (Phasmatodea) that have undergone an impressive evolutionary radiation in Australia, New Caledonia, the Mascarene Islands and areas of the Pacific. Previous research showed that this clade also contained at least two of the nine New Zealand stick insect genera. We have constructed a phylogeny of the Lanceocercata using 2277 bp of mitochondrial and nuclear DNA sequence data to determine whether all nine New Zealand genera are indeed Lanceocercata and whether the New Zealand fauna is monophyletic. DNA sequence data were obtained from mitochondrial cytochrome oxidase subunits I and II and the nuclear large subunit ribosomal RNA and histone subunit 3. These data were subjected to Bayesian phylogenetic inference under a partitioned model and maximum parsimony. The resulting trees show that all the New Zealand genera are nested within a large New Caledonian radiation. The New Zealand genera do not form a monophyletic group, with the genus Spinotectarchus Salmon forming an independent lineage from the remaining eight genera. We analysed Lanceocercata apomorphies to confirm the molecular placement of the New Zealand genera and to identify characters that confirm the polyphyly of the fauna. Molecular dating analyses under a relaxed clock coupled with a Bayesian extension to dispersal‐vicariance analysis was used to reconstruct the biogeographical history for the Lanceocercata. These analyses show that Lanceocercata and their sister group, the Stephanacridini, probably diverged from their South American relatives, the Cladomorphinae, as a result of the separation of Australia, Antarctica and South America. The radiation of the New Caledonian and New Zealand clade began 41.06 million years ago (mya, 29.05–55.40 mya), which corresponds to a period of uplift in New Caledonia. The main New Zealand lineage and Spinotectarchus split from their New Caledonian sister groups 33.72 (23.9–45.62 mya) and 29.9 mya (19.79–41.16 mya) and began to radiate during the late Oligocene and early Miocene, probably in response to a reduction in land area and subsequent uplift in the late Oligocene and early Miocene. We discuss briefly shared host plant patterns between New Zealand and New Caledonia. Because Acrophylla sensu Brock & Hasenpusch is polyphyletic, we have removed Vetilia Stål from synonymy with Acrophylla Gray.     

Biogeographical history of cuckoo‐shrikes (Aves: Passeriformes): transoceanic colonization of Africa from Australo‐Papua

Aim Cuckoo‐shrikes and allies (Campephagidae) form a radiation of birds widely distributed in the Indo‐Pacific and Africa. Recent studies on the group have been hampered by poor taxon sampling, causing inferences about systematics and biogeography to be rather speculative. With improved taxon sampling and analyses within an explicit spatiotemporal framework, we elucidate biogeographical patterns of dispersal and diversification within this diverse clade of passerine birds. Location Africa, Asia, Australo‐Papua, the Pacific, the Philippines and Wallacea. Methods We use model‐based phylogenetic methods (Mr Bayes and garli ) to construct a phylogenetic hypothesis of the core Campephagidae (Campephagidae with the exclusion of Pericrocotus). The phylogeny is used to assess the biogeographical history of the group with a newly developed Bayesian approach to dispersal–vicariance analysis (Bayes‐diva) . We also made use of a partitioned beast analysis, with several calibration points taken from island ages, passerine mitochondrial substitution rates and secondary calibration points for passerine birds, to assess the timing of diversification and dispersal. Results We present a robust molecular phylogeny that includes all genera and 84% of the species within the core Campephagidae. Furthermore, we estimate divergence dates and ancestral area relationships. We demonstrate that Campephagidae originated in Australo‐Papua with a single lineage (Pericrocotus) dispersing to Asia early. Later, there was further extensive transoceanic dispersal from Australo‐Papua to Africa involving lineages within the core Campephagidae radiation. Main conclusions The phylogenetic relationships, along with the results of the ancestral area analysis and the timing of dispersal events, support a transoceanic dispersal scenario from Australo‐Papua to Africa by the core Campephagidae. The sister group to core Campephagidae, Pericrocotus, dispersed to mainland Asia in the late Oligocene. Asia remained uncolonized by the core Campephagidae until the Pliocene. Transoceanic dispersal is by no means an unknown phenomenon, but our results represent a convincing case of colonization over a significant water gap of thousands of kilometres from Australo‐Papua to Africa.     

Molecular phylogeny of the ant tribe Myrmicini (Hymenoptera: Formicidae)

The interrelationships within ant subfamilies remain elusive, despite the recent establishment of the phylogeny of the major ant lineages. The tribe Myrmicini belongs to the subfamily Myrmicinae, and groups morphologically unspecialized genera. Previous research has struggled with defining Myrmicini, leading to considerable taxonomic instability. Earlier molecular phylogenetic studies have suggested the nonmonophyly of Myrmicini, but were based on limited taxon sampling. We investigated the composition of Myrmicini with phylogenetic analyses of an enlarged set of taxa, using DNA sequences of eight gene fragments taken from 37 representatives of six of the seven genera (Eutetramorium, Huberia, Hylomyrma, Manica, Myrmica, and Pogonomyrmex), and eight outgroups. Our results demonstrate the invalidity of Myrmicini as currently defined. We recovered sister‐group relationships between the genera Myrmica and Manica, and between Pogonomyrmex and Hylomyrma. This study illustrates that to understand the phylogeny of over 6000 myrmicine species, comprehensive taxon sampling and DNA sequencing are an absolute requisite. © 2010 The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 160 , 482–495.     

Integrating fossils in a molecular‐based phylogeny and testing them as calibration points for divergence time estimates in Menispermaceae

Abstract The phylogeny of extant Menispermaceae (Ranunculales) is reconstructed based on DNA sequences of two chloroplast genes (rbcL and atpB) from 94 species belonging to 56 genera. Fossilized endocarps represent 34 genera. The positions of these are inferred using 30 morphological characters and the molecular phylogeny as a backbone constraint. Nine of the thirteen nodes that are each dated by a fossil are used as calibration points for the estimates of molecular divergence times. BEAST is used to estimate stem age (121.2 Myr) and crown age (105.4 Myr) for Menispermaceae. This method does not require an input tree topology and can also account for rate heterogeneity among lineages. The sensitivity of these estimates to fossil constraints is then evaluated by a cross‐validation procedure. The estimated origin for Menispermaceae is dated to the mid‐Jurassic if the customary maximum age of 125 Myr for eudicots is not implemented. All constraints when used alone failed to estimate node ages in some parts of the tree. Fossils from the Palaeocene and Eocene impose strict constraints. Likewise, the use of Prototinomiscium as a dating constraint for Menispermaceae appears to be a conservative approach.     

Phylogeny and systematics of the Orycteropodidae (Mammalia,Tubulidentata)

The systematics of the order Tubulidentata is poorly known. Its phylogeny has never been thoroughly analysed and only a single review has ever been performed, which was over 30 years ago. This situation has hampered palaeoecological and palaeobiogeographical studies of these Neogene mammals. The present revision of the Orycteropodidae deals with the phylogeny and systematics of all African and Eurasian species over the last 20 Myr. The first comprehensive cladistic analysis of the family is presented here. The results of this analysis, based on 39 coded morphological characters, supplemented by non‐coded features taken from all over the skeleton, was used to reconstruct the phylogeny of the order Tubulidentata. Two distinct lineages within the genus Orycteropus are recognized and characterized. The new genus Amphiorycteropus is subsequently created, in order to harmonize taxonomy and phylogeny. The fossil genera Leptorycteropus and Myorycteropus are validated, bringing the number of genera in the order Tubulidentata to four. Moreover, within the family Orycteropodidae, the number of confirmed species is now 14. The outcome of this study allows us to propose a consistent palaeobiogeographical scenario for aardvarks. Finally, this revision represents the most comprehensive work on the evolutionary history of the order Tubulidentata to date, and provides a new framework for future studies. © 2009 The Linnean Society of London, Zoological Journal of the Linnean Society, 2009, 155 , 649–702.     

Molecular dating and diversification of the South American lizard genus Liolaemus (subgenus Eulaemus) based on nuclear and mitochondrial DNA sequences

The temperate South American lizard genus Liolaemus is the one of the most widely distributed and species‐rich genera of lizards on earth. The genus is divided into two subgenera, Liolaemus sensu stricto (the ‘Chilean group’) and Eulaemus (the ‘Argentino group’), a division that is supported by recent molecular and morphological data. Owing to a lack of reliable fossil data, previous studies have been forced to use either global molecular clocks, a standardized mutation rate adopted from previous studies, or the use of geological events as calibration points. However, simulations indicate that these types of assumptions may result in less accurate estimates of divergence times when clock‐like models or mutation rates are violated. We used a multilocus data set combined with a newly described fossil to provide the first calibrated phylogeny for the crown groups of the clade Eulaemus, and derive new fossil‐calibrated substitution rates (with error) of both nuclear and mtDNA gene regions for Eulaemus specifically. Divergence date estimates for each of the crown groups and appropriate rate estimates will provide the foundation for understanding rates of speciation, historical biogeography, and phylogeographical history for various clades in one of the most diverse lizard genera in the poorly studied Patagonian region. © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 164 , 825–835.     

Phylogeny of the North American catfish family Ictaluridae (Teleostei: Siluriformes) combining morphology,genes and fossils

We performed the first combined‐data phylogenetic analysis of ictalurids including most living and fossil species. We sampled 56 extant species and 16 fossil species representing outgroups, the seven living genera, and the extinct genus ?Astephus long thought to be an ictalurid. In total, 209 morphological characters were curated and illustrated in MorphoBank from published and original work, and standardized using reductive coding. Molecular sequences harvested from GenBank for one nuclear and four mitochondrial genes were combined with the morphological data for total evidence analysis. Parsimony analysis recovers a crown clade Ictaluridae composed of seven living genera and numerous extinct species. The oldest ictalurid fossils are the Late Eocene members of Ameiurus and Ictalurus. The fossil clade ?Astephus placed outside of Ictaluridae and not as its sister taxon. Previous morphological phylogenetic studies of Ictaluridae hypothesized convergent evolution of troglobitic features among the subterranean species. In contrast, we found morphological evidence to support a single clade of the four troglobitic species, the sister taxon of all ictalurids. This result holds whether fossils are included or not. Some previously published clock‐based age estimates closely approximate our minimum ages of clades.     

INVESTIGATIONS OF ANGIOSPERMS FROM THE EOCENE OF NORTH AMERICA: RHAMNUS MARGINATUS (RHAMNACEAE) REEXAMINED

Leaf compressions, previously assigned to Rhamnus marginatus Lesquereux, were collected from the Middle Eocene Claiborne Formation of western Kentucky and Tennessee. The leaf architecture and cuticular features of over 40 compressions were carefully examined and compared to those of many extant species of Rhamnaceae and related families as well as fossil specimens previously assigned to this taxon. This leaf type appears to belong to the Rhamnaceae, however, it conforms more closely to species of several genera in the tribe Zizypheae than to those of Rhamnus or other genera in the tribe Rhamneae. Confident assignment to any specific genus within this complex of genera cannot be made on the basis of leaf characteristics alone and would require discovery and analysis of additional vegetative and reproductive organs. Because this fossil leaf form cannot be confidently assigned to any modern genus and earlier classifications appear to be improper, this leaf type has been reassigned to the taxon Berhamniphyllum claibornense gen. et sp. nov. The transfer of this leaf form at the tribe level reaffirms the need for close examination of taxonomic determinations made by early workers.     

COLEOFASCICULUS GEN. NOV. (CYANOBACTERIA): MORPHOLOGICAL AND MOLECULAR CRITERIA FOR REVISION OF THE GENUS MICROCOLEUS GOMONT1

Species currently classified within the cyanobacterial genus Microcoleus were determined to fall into two distinct clades in a 16S rDNA phylogeny, one containing taxa within the Oscillatoriaceae, the other containing taxa within the Phormidiaceae. The two lineages were confirmed in an analysis of the 16S–23S internal transcribed spacer (ITS) region sequences and secondary structures. The type species for Microcoleus is M. vaginatus Gomont, and this taxon belongs in the Oscillatoriaceae. Consequently, Microcoleus taxa in the Phormidiaceae must be placed in separate genera, and we propose the new genus Coleofasciculus to contain marine taxa currently placed in Microcoleus. The type species for Coleofasciculus is the well‐studied and widespread marine mat‐forming species Microcoleus chthonoplastes (Mert.) Zanardini ex Gomont. Other characters separating the two families include type of cell division and thylakoid structure.