Two Eocene fossil fruits from the Changchang Basin of Hainan Island,China

Two types of fossil fruit, one belonging to Palaeocarya sp. (Juglandaceae) and the other to Acer cf. A. miofranchetii Hu et Chaney (Aceraceae), are found in the Eocene coal-bearing series from the Changchang Basin of Hainan Island, China. This is the first fossil record of Palaeocarya and Acer in a tropical area of China. These fossils provide evidence for an investigation of the phytogeographic history of these two genera. Since their extant relative genera are distributed mostly in northern temperate or tropical–subtropical mountainous regions, I propose that the Changchang Basin of Hainan Island was close to a mountainous region in the Eocene; the plants bearing these fruits were growing at a mid-high altitude with a relatively cool climate, and the fruits were not preserved in situ but transported to the fossil site. The characters of other associated fossil plants and palynological data also support this hypothesis.     

A systematic overview of fossil osmundalean ferns in China: Diversity variation,distribution pattern,and evolutionary implications

The order Osmundales is a unique fern taxon with extensive fossil records in geological past. Diverse osmundalean fossils have been reported from China, ranging in age from the Late Palaeozoic to the Cenozoic. Most of them are based on leaf impressions/compressions, but permineralized rhizomes are also well documented. In this study, we provide a systematic overview on fossil osmundalean ferns in China with special references on diversity variations, distribution patterns, and evolutionary implications. Fossil evidence indicates that this fern lineage first appeared in the Late Palaeozoic in China. The Late Triassic to Middle Jurassic interval was the radiation stage. From the Late Jurassic onward, fossil diversity declined rapidly. Cenozoic osmundalean taxa are represented by the relict species of Osmunda. Geographically, osmundalean fossils are found from both the Northern and Southern phytoprovinces of China, though variations are documented for geographical ranges. The Chinese fossil records cover almost all important stages for the macroevolution of the Osmundales, and contribute to further understanding of evolutionary processes of this peculiar fern lineage.     

Phylogeographic evidence for two mesic refugia in a biodiversity hotspot

Phylogeographic studies of flora in species-rich south-western Australia point to complex evolutionary histories, reflecting patterns of persistence and resilience to climatic changes during the Pleistocene. We asked whether coastal areas of the mid-west and south, as well as granite outcrops and inland ranges, have acted as major refugia within this region during Pleistocene climatic fluctuations by analysing phylogeographic patterns in the shrub Calothamnus quadrifidus R.Br. (Myrtaceae). We determined variation in chloroplast DNA data for 41 populations across the geographic range. Relationships and major clades were resolved using parsimony and Bayesian analyses. We tested for demographic and spatial expansion of the major clades and estimated clade divergence dates using an uncorrelated, lognormal relaxed clock based on two conservative chloroplast mutation rates. Two distinct phylogeographic clades were identified showing divergence during the Pleistocene, consistent with other phylogeographic studies of south-west Australian flora, emphasising the impact of climatic oscillations in driving divergence in this landscape. The southern clade was more diverse, having higher haplotype diversity and greater genetic structure, while the northern clade showed evidence of fluctuation in population size. Regions of high haplotype diversity with adjacent areas of low diversity observed in each clade indicated the locations of two coastal refugia: one on the south coast and another along the mid-west coast. This is the first evidence for major Pleistocene refugia using chloroplast genetic data in a common, widespread species from this region.     

The Puzzle of Italian Rice Origin and Evolution: Determining Genetic Divergence and Affinity of Rice Germplasm from Italy and Asia

The characterization of genetic divergence and relationships of a set of germplasm is essential for its efficient applications in crop breeding and understanding of the origin/evolution of crop varieties from a given geographical region. As the largest rice producing country in Europe, Italy holds rice germplasm with abundant genetic diversity. Although Italian rice varieties and the traditional ones in particular have played important roles in rice production and breeding, knowledge concerning the origin and evolution of Italian traditional varieties is still limited. To solve the puzzle of Italian rice origin, we characterized genetic divergence and relationships of 348 rice varieties from Italy and Asia based on the polymorphisms of microsatellite fingerprints. We also included common wild rice O. rufipogon as a reference in the characterization. Results indicated relatively rich genetic diversity (H _e = 0.63-0.65) in Italian rice varieties. Further analyses revealed a close genetic relationship of the Italian traditional varieties with those from northern China, which provides strong genetic evidence for tracing the possible origin of early established rice varieties in Italy. These findings have significant implications for the rice breeding programs, in which appropriate germplasm can be selected from a given region and utilized for transferring unique genetic traits based on its genetic diversity and evolutionary relationships.     

Differential levels of genetic diversity and divergence among populations of an ancient Australian rainforest conifer, Araucaria cunninghamii

Historical fragmentation and subsequent isolation has affected the levels of genetic diversity in many lineages of ancient plant taxa. This study investigated the effects of historical processes on Araucaria cunninghamii (Hoop Pine), Araucariaceae, using both RAPD and ISSR markers. While most populations maintain moderate levels of diversity, there is some evidence for reduced genetic variation. Most sampled populations are significantly differentiated from each other, thereby emphasizing the high degree of population structuring in A. cunninghamii. Increased divergence among the northern Queensland populations relative to southern Queensland populations suggests historical pressures have impacted differently on extant population distribution. More research is specifically required into the biology and population demographics of Australian Araucariaceae.     

Sequence data reflect the introduction pathways of the Sirex woodwasp parasitoid,Ibalia leucospoides (Ibaliidae,Hymenoptera)

1. The parasitoid wasp Ibalia leucospoides is native to the northern hemisphere and has been introduced to the southern hemisphere as a biological control agent for the invasive woodwasp Sirex noctilio. Two subspecies of the parasitoid, Ibalia leucospoides leucospoides (Palearctic distribution) and Ibalia leucospoides ensiger (Nearctic distribution), were introduced and are reported to have hybridized.
2. Despite extensive records of the numbers and origins of the wasps imported into the southern hemisphere, nothing is known regarding their current population diversity. We investigated the genetic variation of I. leucospoides in its native and introduced ranges using mitochondrial (COI) and nuclear (ITS) markers.
3. Mitochondrial DNA diversity in the introduced range was limited, with only five haplotypes, although sequence divergence between these haplotypes was high. Similarly, the ITS rDNA sequences revealed multiple clades present in the introduced range.
4. These results reflect introductions from a wide geographical range but where genetic bottlenecks have possibly reduced the genetic diversity. The data further reflect the origin of the I. leucospoides populations in South America and South Africa from New Zealand or Australia. We found no evidence of hybridization between the two subspecies of the parasitoid in its introduced range, and no evidence that I. leucospoides ensiger has established outside its native range.

     

Phylogeography of Rhodiola kirilowii (Crassulaceae): A Story of Miocene Divergence and Quaternary Expansion

The evolution and current distribution of the Sino-Tibetan flora have been greatly affected by historical geological events, such as the uplift of the Qinghai-Tibetan Plateau (QTP), and Quaternary climatic oscillations. Rhodiola kirilowii, a perennial herb with its distribution ranging from the southeastern QTP and the Hengduan Mountains (HM) to adjacent northern China and central Asia, provides an excellent model to examine and disentangle the effect of both geological orogeny and climatic oscillation on the evolutionary history of species with such distribution patterns. We here conducted a phylogeographic study using sequences of two chloroplast fragments (trnL-F and trnS-G) and internal transcribed spacers in 29 populations of R. kirilowii. A total of 25 plastid haplotypes and 12 ITS ribotypes were found. Molecular clock estimation revealed deep divergence between the central Asian populations and other populations from the HM and northern China; this split occurred ca. 2.84 million year ago. The majority of populations from the mountains of northern China were dominated by a single haplotype or ribotype, while populations of the HM harbored both high genetic diversity and high haplotype diversity. This distribution pattern indicates that HM was either a diversification center or a refugium for R. kirilowii during the Quaternary climatic oscillations. The present distribution of this species on mountains in northern China may have resulted from a rapid glacial population expansion from the HM. This expansion was confirmed by the mismatch distribution analysis and negative Tajima''s D and Fu''s F _S values, and was dated to ca. 168 thousand years ago. High genetic diversity and population differentiation in both plastid and ITS sequences were revealed; these imply restricted gene flow between populations. A distinct isolation-by-distance pattern was suggested by the Mantel test. Our results show that in old lineages, populations may harbour divergent genetic forms that are sufficient to maintain or even increase overall genetic diversity despite fragmentation and low within-population variation.     

Revisiting tree-migration rates: Abies alba (Mill.), a case study

At northern temperate latitudes trees have adjusted their ranges substantially in response to changing climates during the Holocene. Results from dispersal model simulations suggest that postglacial migration rates may have been over-estimated from fossil pollen data. As a contribution to this debate, we infer the migration rates of Abies alba (Mill.), silver fir, as a case-study species, by using a spatially explicit approach based on fossil pollen but taking into account its modern genetic diversity pattern. Maximum estimates of migration rates from fossil pollen data alone are higher than 700 m yr^?1 during the Holocene. Considering the potential refugia as suggested from all the fossil data but restricting the area over which silver fir expanded from each glacial refugium using data on the current haplotype distribution, the estimated maximum migration rates of silver fir are less than 250 m yr^?1. Genetic information may allow for (1) the exclusion of those refugial areas where the species may have survived during the last glacial period but from which it did not spread or spread only very locally and (2) the delineation of the areas over which the species spread from each glacial refugium. The estimated rates in the present study are generally consistent with rates suggested from modelling approaches. This study shows that integrating fossil pollen records can improve simulations of dispersal processes and, thus, allow for better predictions of future changes in tree species’ ranges.     

Contrasting mitochondrial DNA diversity estimates in two sympatric genera of Arctic lemmings (Dicrostonyx: Lemmus) indicate different responses to Quaternary environmental fluctuations

The fossil records show that since the Middle Pleistocene, lemmings (Dicrostonyx, Lemmus) have been sympatric across their ranges. I compared mitochondrial DNA (mtDNA) diversity estimates between the two genera to infer a difference in demographic history resulting from biotic responses to Quaternary environmental fluctuations. The mtDNA diversity estimates in Lemmus consistently exceed those in Dicrostonyx on regional and continental spatial scales. However, as opposed to the mainland, the diversity estimates in Lemmus are lower than those in Dicrostonyx on Wrangel Island. Under the assumption of equal mutation rates, a difference in diversity estimates reflects a difference in the historical effective size. On a regional scale, the low mtDNA diversity in Dicrostonyx suggests it suffered a reduction in effective population size, probably due to range contractions during warming events in the Holocene. On a continental scale, the low average divergence in Dicrostonyx indicates a dispersion event after the range contraction in Eurasia to a single refugium, also due to warming events in one of the interglacials. In contrast to Dicrostonyx, the high mtDNA diversity in Lemmus gives no indications for a reduction in its effective size during late Quaternary warming events. This implies that the two historically codistributed genera responded differently to Quaternary environmental changes, even if their differences in biotic responses are undetectable in the Arctic fossil record. This study demonstrates that molecular genetic data increase the resolution of palaeoecological analyses at the community level.     

Genetic landscape and landscape connectivity of Ceratopteris thalictroides,an endangered aquatic fern

Global climate change will have great impacts on ecosystems with high biodiversity and landscape connectivity. Here, we employ species distribution models (SDMs) and geospatial analyses to predict future changes in C. thalictroides distribution under the future climate change based on Community Climate System Model (CCSM4). We predict the ranges of C. thalictroides will contract about 11,523 km² from the present to the year 2080. The changes in species distribution present a main range contraction in high latitude regions. We map the patterns of genetic divergence and diversity using the Genetic Landscape GIS Toolbox in ArcGIS v10.2. By visualizing dispersal networks in SDMtoolbox v 1.1, we predict a major decrease in connectivity will occur between YD (Yingde) and NP (Nanping) population. Populations with high diversity and divergence regions were considered to be evolutionary hotspots. Therefore, we suggest the populations CZ(Chengzhou), YD(Yingde), HP(Hepu), SY(Sanya), DH(Dinghu) and NP(Nanping) are in need of protection, concluding that strategically maintained ecological connectivity must be a key component of conservation strategies for C. thalictroides. We believe the creation of genetic landscape based on genetic datasets and connectivity assessment in relation to climate change will provide increasingly useful information and new implications for prioritizing the conservation of the endangered species.     

Community ecology of the Middle Miocene primates of La Venta,Colombia: the relationship between ecological diversity,divergence time,and phylogenetic richness

It has been suggested that the degree of ecological diversity that characterizes a primate community correlates positively with both its phylogenetic richness and the time since the members of that community diverged (Fleagle and Reed in Primate communities. Cambridge University Press, New York, pp 92–115, 1999). It is therefore questionable whether or not a community with a relatively recent divergence time but high phylogenetic richness would be as ecologically variable as a community with similar phylogenetic richness but a more distant divergence time. To address this question, the ecological diversity of a fossil primate community from La Venta, Colombia, a Middle Miocene platyrrhine community with phylogenetic diversity comparable with extant platyrrhine communities but a relatively short time since divergence, was compared with that of modern Neotropical primate communities. Shearing quotients and molar lengths, which together are reliable indicators of diet, for both fossil and extant species were plotted against each other to describe the dietary “ecospace” occupied by each community. Community diversity was calculated as the area of the minimum convex polygon encompassing all community members. The diversity of the fossil community was then compared with that of extant communities to test whether the fossil community was less diverse than extant communities while taking phylogenetic richness into account. Results indicate that the La Ventan community was not significantly less ecologically diverse than modern communities, supporting the idea that ecological diversification occurred along with phylogenetic diversification early in platyrrhine evolution.     

Sympatric diversification vs. immigration: deciphering host‐plant specialization in a polyphagous insect,the stolbur phytoplasma vector Hyalesthes obsoletus (Cixiidae)

The epidemiology of vector transmitted plant diseases is highly influenced by dispersal and the host‐plant range of the vector. Widening the vector's host range may increase transmission potential, whereas specialization may induce specific disease cycles. The process leading to a vector's host shift and its epidemiological outcome is therefore embedded in the frameworks of sympatric evolution vs. immigration of preadapted populations. In this study, we analyse whether a host shift of the stolbur phytoplasma vector, Hyalesthes obsoletus from field bindweed to stinging nettle in its northern distribution range evolved sympatrically or by immigration. The exploitation of stinging nettle has led to outbreaks of the grapevine disease bois noir caused by a stinging nettle‐specific phytoplasma strain. Microsatellite data from populations from northern and ancestral ranges provide strong evidence for sympatric host‐race evolution in the northern range: Host‐plant associated populations were significantly differentiated among syntopic sites (0.054 < F_HT < 0.098) and constant over 5 years. While gene flow was asymmetric from the old into the predicted new host race, which had significantly reduced genetic diversity, the genetic identity between syntopic host‐race populations in the northern range was higher than between these populations and syntopic populations in ancestral ranges, where there was no evidence for genetic host races. Although immigration was detected in the northern field bindweed population, it cannot explain host‐race diversification but suggests the introduction of a stinging nettle‐specific phytoplasma strain by plant‐unspecific vectors. The evolution of host races in the northern range has led to specific vector‐based bois noir disease cycles.     

Influence of sexual selection and feeding functional morphology on diversification rate of parrotfishes (Scaridae)

Scaridae (parrotfishes) is a prominent clade of 96 species that shape coral reef communities worldwide through their actions as grazing herbivores. Phylogenetically nested within Labridae, the profound ecological impact and high species richness of parrotfishes suggest that their diversification and ecological success may be linked. Here, we ask whether parrotfish evolution is characterized by a significant burst of lineage diversification and whether parrotfish diversity is shaped more strongly by sexual selection or modifications of the feeding mechanism. We first examined scarid diversification within the greater context of labrid diversity. We used a supermatrix approach for 252 species to propose the most extensive phylogenetic hypothesis of Labridae to date, and time-calibrated the phylogeny with fossil and biogeographical data. Using divergence date estimates, we find that several parrotfish clades exhibit the highest diversification rates among all labrid lineages. Furthermore, we pinpoint a rate shift at the shared ancestor of Scarus and Chlorurus, a scarid subclade characterized by territorial behaviour and strong sexual dichromatism, suggesting that sexual selection was a major factor in parrotfish diversification. Modifications of the pharyngeal and oral jaws that happened earlier in parrotfish evolution may have contributed to this diversity by establishing parrotfishes as uniquely capable reef herbivores.     

Phylogenomics,molecular evolution,and estimated ages of lineages from the deep phylogeny of Poaceae

The deeply diverging subfamilies of grasses: Anomochlooideae, Pharoideae, and Puelioideae, today inhabit tropical forest floors as sparsely distributed depauperate lineages. The BEP/PACMAD grasses, which make up the majority of the family, are the result of a more recent radiation. Species in the deeply diverging subfamilies were here investigated to better understand molecular evolutionary processes and ages of divergence. Complete chloroplast genomes (plastomes) of Pharus latifolius L., P. lappulaceus Aubl., and Puelia olyriformis (Franch.) Clayton were determined. Four plastome loci from seven species of the deep subfamilies were also sequenced. Phylogenetic and mutation analyses and divergence estimations were conducted on all sequences together with homologous sequences from other Poaceae. Mutation analyses surveyed insertion/deletion mutations across the plastomes, clarified a trend in the molecular evolution of the rpoC2 locus, and indicated unique pseudogenizations in the plastomes of Pharus and Puelia. Phylogenetic analyses largely confirmed earlier multi-gene phylogenies. Phylogenomic and divergence analyses produced estimated origins of the crown nodes of Anomochlooideae at 65–104 Ma, Pharoideae at 44–71 Ma, and Puelioideae at 62–96 Ma. The upper ends of our estimated ranges are in general agreement with previous estimates. However, the lower ends of our ranges are considerably older than previous estimates, reflecting the influence of the less commonly used oldest fossil calibration point. The deeply diverging subfamilies exhibited the accumulation of numerous substitution and indel mutations consistent with a long evolutionary history that predated the radiation of the BEP/PACMAD grasses. We hypothesize that relatively rapid warming and drying in Africa at 55–56.5 Ma may have acted as selective forces stimulating adaptive radiations of grasses from the African tropical forests into diverse habitats.     

Genetic,Ecological and Morphological Divergence between Populations of the Endangered Mexican Sheartail Hummingbird (Doricha eliza)

The Mexican Sheartail (Doricha eliza), an endangered hummingbird, is endemic to Mexico where two populations have a disjunct distribution. One population is distributed along the northern tip of the Yucatan Peninsula whereas the other is mostly restricted to central Veracruz. Despite their disjunct distribution, previous work has failed to detect morphological or behavioral differences between these populations. Here we use variation in morphology, mtDNA and nuDNA sequences to determine the degree of morphological and molecular divergence between populations, their divergence time, and historical demography. We use species distribution modeling and niche divergence tests to infer the relative roles of vicariance and dispersal in driving divergence in the genus. Our Bayesian and maximum likelihood phylogenetic analyses revealed that Doricha eliza populations form a monophyletic clade and support their sister relationship with D. enicura. We found marked genetic differentiation, with reciprocal monophyly of haplotypes and highly restricted gene flow, supporting a history of isolation over the last 120,000 years. Genetic divergence between populations is consistent with the lack of overlap in environmental space and slight morphological differences between males. Our findings indicate that the divergence of the Veracruz and Yucatan populations is best explained by a combination of a short period of isolation exacerbated by subsequent divergence in climate conditions, and that rather than vicariance, the two isolated ranges of D. eliza are the product of recent colonization and divergence in isolation.     

Molecular Phylogeography and Population Genetic Structure of O. longilobus and O. taihangensis (Opisthopappus) on the Taihang Mountains

Historic events such as the uplift of mountains and climatic oscillations in the Quaternary periods greatly affected the evolution and modern distribution of the flora. We sequenced the trnL–trnF, ndhJ-trnL and ITS from populations throughout the known distributions of O. longilobus and O. taihangensis to understand the evolutionary history and the divergence related to the past shifts of habitats in the Taihang Mountains regions. The results showed high genetic diversity and pronounced genetic differentiation among the populations of the two species with a significant phylogeographical pattern (N _ST>G _ST, P<0.05), which imply restricted gene flow among the populations and significant geographical or environmental isolation. Ten chloroplast DNA (cpDNA) and eighteen nucleus ribosome DNA (nrDNA) haplotypes were identified and clustered into two lineages. Two corresponding refuge areas were revealed across the entire distribution ranges of O. longilobus and at least three refuge areas for O. taihangensis. O. longilobus underwent an evolutionary historical process of long-distance dispersal and colonization, whereas O. taihangensis underwent a population expansion before the main uplift of Taihang Mountains. The differentiation time between O. longilobus and O. taihangensis is estimated to have occurred at the early Pleistocene. Physiographic complexity and paleovegetation transition of Taihang Mountains mainly shaped the specific formation and effected the present distribution of these two species. The results therefore support the inference that Quaternary refugial isolation promoted allopatric speciation in Taihang Mountains. This may help to explain the existence of high diversity and endemism of plant species in central/northern China.     

Complete plastome sequences of Equisetum arvense and Isoetes flaccida: implications for phylogeny and plastid genome evolution of early land plant lineages

Background

Understanding the forces that shaped Neotropical diversity is central issue to explain tropical biodiversity and inform conservation action; yet few studies have examined large, widespread species. Lowland tapir (Tapirus terrrestris, Perissodactyla, Tapiridae) is the largest Neotropical herbivore whose ancestors arrived in South America during the Great American Biotic Interchange. A Pleistocene diversification is inferred for the genus Tapirus from the fossil record, but only two species survived the Pleistocene megafauna extinction. Here, we investigate the history of lowland tapir as revealed by variation at the mitochondrial gene Cytochrome b, compare it to the fossil data, and explore mechanisms that could have shaped the observed structure of current populations.

Results

Separate methodological approaches found mutually exclusive divergence times for lowland tapir, either in the late or in the early Pleistocene, although a late Pleistocene divergence is more in tune with the fossil record. Bayesian analysis favored mountain tapir (T. pinchaque) paraphyly in relation to lowland tapir over reciprocal monophyly, corroborating the inferences from the fossil data these species are sister taxa. A coalescent-based analysis rejected a null hypothesis of allopatric divergence, suggesting a complex history. Based on the geographic distribution of haplotypes we propose (i) a central role for western Amazonia in tapir diversification, with a key role of the ecological gradient along the transition between Andean subcloud forests and Amazon lowland forest, and (ii) that the Amazon river acted as an barrier to gene flow. Finally, the branching patterns and estimates based on nucleotide diversity indicate a population expansion after the Last Glacial Maximum.

Conclusions

This study is the first examining lowland tapir phylogeography. Climatic events at the end of the Pleistocene, parapatric speciation, divergence along the Andean foothill, and role of the Amazon river, have similarly shaped the history of other taxa. Nevertheless further work with additional samples and loci is needed to improve our initial assessment. From a conservation perspective, we did not find a correspondence between genetic structure in lowland tapir and ecogeographic regions proposed to define conservation priorities in the Neotropics. This discrepancy sheds doubt into this scheme's ability to generate effective conservation planning for vagile species.     

Speciation of two desert poplar species triggered by Pleistocene climatic oscillations

Despite the evidence that the Pleistocene climatic fluctuations have seriously affected the distribution of intraspecific diversity, less is known on its impact on interspecific divergence. In this study, we aimed to test the hypothesis that the divergence of two desert poplar species Populus euphratica Oliv. and P. pruinosa Schrenk. occurred during the Pleistocene. We sequenced 11 nuclear loci in 60 individuals from the two species to estimate the divergence time between them and to test whether gene flow occurred after species separation. Divergence time between the two species was estimated to be 0.66–1.37 million years ago (Ma), a time at which glaciation was at its maximum in China and deserts developed widely in central Asia. Isolation-with-Migration model also indicated that the two species had diverged in the presence of gene flow. We also detected evidence of selection at GO in P. euphratica and to a lesser extent at PhyB2. Together, these results underscore the importance of Pleistocene climate oscillations in triggering plant speciation as a result of habitats divergence.     

Calibration uncertainty in molecular dating analyses: there is no substitute for the prior evaluation of time priors

Calibration is the rate-determining step in every molecular clock analysis and, hence, considerable effort has been expended in the development of approaches to distinguish good from bad calibrations. These can be categorized into a priori evaluation of the intrinsic fossil evidence, and a posteriori evaluation of congruence through cross-validation. We contrasted these competing approaches and explored the impact of different interpretations of the fossil evidence upon Bayesian divergence time estimation. The results demonstrate that a posteriori approaches can lead to the selection of erroneous calibrations. Bayesian posterior estimates are also shown to be extremely sensitive to the probabilistic interpretation of temporal constraints. Furthermore, the effective time priors implemented within an analysis differ for individual calibrations when employed alone and in differing combination with others. This compromises the implicit assumption of all calibration consistency methods, that the impact of an individual calibration is the same when used alone or in unison with others. Thus, the most effective means of establishing the quality of fossil-based calibrations is through a priori evaluation of the intrinsic palaeontological, stratigraphic, geochronological and phylogenetic data. However, effort expended in establishing calibrations will not be rewarded unless they are implemented faithfully in divergence time analyses.