A workflow with R: Phylogenetic analyses and visualizations using mitochondrial cytochrome b gene sequences.

Phylogenetic analyses can provide a wealth of information about the past demography of a population and the level of genetic diversity within and between species. By using special computer programs developed in recent years, large amounts of data have been produced in the molecular genetics area. To analyze these data, powerful new methods based on large computations have been applied in various software packages and programs. But these programs have their own specific input and output formats, and users need to create different input formats for almost every program. R is an open source software environment, and it supports open contribution and modification to its libraries. Furthermore, it is also possible to perform several analyses using a single input file format. In this article, by using the multiple sequences FASTA format file (.fas extension) we demonstrate and share a workflow of how to extract haplotypes and perform phylogenetic analyses and visualizations in R. As an example dataset, we used 120 Bombus terrestris dalmatinus mitochondrial cytochrome b gene (cyt b) sequences (373 bp) collected from eight different beehives in Antalya. This article presents a short guide on how to perform phylogenetic analyses using R and RStudio.

The roles of vicariance and dispersal in the differentiation of two species of the Rhinella marina species complex.

The high levels of Neotropical biodiversity are commonly associated with the intense Neogene-Quaternary geological events and climate dynamics. Here, we investigate the evolutionary history of two species of Neotropical closely related amphibians (R. horribilis and R. marina). We combine published data with new mitochondrial DNA sequences and multiple nuclear markers, including 12 microsatellites. The phylogenetic analyses showed support for grouping the samples in two main clades; R. horribilis (Central America and Mexico) and R. marina (South America east of the Andes). However, the phylogenetic inferences also show an evident mito-nuclear discordance. We use Approximate Bayesian Computation (ABC) to test the role of different events in the diversification between the two groups recovered. We found that both species were affected primarily by a recent Pleistocene divergence, which was similar to the divergence estimate revealed by the Isolation-with-Migration model, under persistent bidirectional gene flow through time. We provide the first evidence that R. horribilis is differentiated from the South American R. marina at the nuclear level supporting the taxonomic status of R. horribilis, which has been controversial for more than a century.

From taxonomic deflation to newly detected cryptic species: Hidden diversity in a widespread African squeaker catfish.

Cryptic genetic diversity and erroneous morphological species determination represent frequent problems in biodiversity research. Here, examination of 138 specimens of Synodontis (Mochokidae, Siluriformes) from the Nile River and Lake Turkana revealed the presence of both S. schall-like and S. frontosus-like morphotypes, with a phenotypic gradient between them. We concluded phylogenetic and population genetic analyses based on two mitochondrial and one nuclear marker including 131 coxI (565 bp), 96 cytb (973 bp) and 19 RAG2 (896 bp) sequences from the Nile-Turkana population, plus additional GenBank data of Synodontis spp. Whilst nuclear data were inconclusive, mitochondrial sequences suggested that both morphotypes and intermediate forms are conspecific. The results imply probable synonymy of S. frontosus with S. schall. Conversely, a strong biogeographical signal was revealed among widely distributed and supposedly conspecific S. schall-like catfish of the Nilo-Sudanian ichthyological province. Synodontis schall sensu stricto (=Eastern clade), as defined by type locality in the Nile, is apparently restricted to the eastern part of the Nilo-Sudanian ichthyological province (e.g. Nile, Turkana, Chad). Synodontis schall Western clade (Senegambia, Niger, Chad) most probably represents a cryptic taxon, unrecognized thus far due to the absence of distinctive morphological differences.

Cryptic diversity in Brazilian endemic monkey frogs (Hylidae, Phyllomedusinae, Pithecopus) revealed by multispecies coalescent and integrative approaches.

Even though Brazil is the world leader in amphibian diversity, a significant part of its richness remains unknown or hidden under cryptic taxa. Here, we used model-based species delimitation in an integrative taxonomic approach, by gathering molecular and morphometric data to assess cryptic taxa within the monkey frogs Pithecopus rohdei, from the Atlantic Forest, and P. megacephalus, from campos rupestres ecosystem. We sampled one mitochondrial, five nuclear loci, and 18 morphometric variables. Using species-delimitation methods with genetic and morphometric data, we recovered five divergent lineages within P. rohdei and no cryptic lineages were recovered for P. megacephalus. Morphometric data show differentiation only for one of the candidate species revealed by the delimitation approaches, suggesting that individuals from Doce River basin constitute a putative species for formal taxonomic description. The time-calibrated mtDNA tree shows that P. rohdei complex lineages began to diverge in late Miocene. However, dates from the multilocus species tree are more recent, occurring in Pleistocene, and suggesting their persistence in refuges of forest and sky islands within the Atlantic Forest biome. The existence of cryptic taxa within P. rohdei is, therefore, relevant for planning conservation strategies for this species complex in the Atlantic Forest biodiversity hotspot.

Molecular systematics of the Triplophysa robusta (Cobitoidea) complex: Extensive gene flow in a depauperate lineage.

Gene flow between populations assumed to be isolated frequently leads to incorrect inferences of evolutionary history. Understanding gene flow and its causes has long been a key topic in evolutionary biology. In this study, we explored the evolutionary history of the Triplophysa robusta complex, using a combination of multilocus analyses and coalescent simulation. Our multilocus approach detected conspicuous mitonuclear discordances in the T. robusta complex. Mitochondrial results showed reticular clades, whereas the nuclear results corresponded with the morphological data. Coalescent simulation indicated that gene flow was the source of these discordances. Molecular clock analysis combined with geological processes suggest that intense geological upheavals have shaped a complicated evolutionary history for the T. robusta complex since the late Miocene, causing extensive gene flow which has distorted the molecular systematics of the T. robusta complex. We suggest that frequent gene flow may restrict speciation in the T. robusta complex, leading to such a depauperate lineage. Based on this comprehensive understanding, we provide our proposals for taxonomic revision of the T. robusta complex.

Diversification of the genus Apogon (Lacepède, 1801) (Apogonidae: Perciformes) in the tropical eastern Pacific.

We examined the role of geographic barriers and historical processes on the diversification of Apogon species within the tropical eastern Pacific (TEP). Mitochondrial and nuclear DNA sequences were used in Bayesian and Maximum likelihood analyses to generate a phylogenetic hypothesis for Apogon species. Bayesian inferences were used to date the cladogenetic events. Analyses with BioGeoBEARS were conducted to reconstruct the biogeographic history and ancestral ranges. The phylogenetic results show a monophyletic clade of TEP Apogon species with A. imberbis from the eastern Atlantic as sister species. The two lineages diverged during the Miocene. Within the TEP clade, two subclades diverged at around 11.1 million years ago (Mya): one clusters the coastal continental species (A. pacificus, A. retrosella and A. dovii), and the second clusters the oceanic island species (A. atradorsatus, A. atricaudus and A. guadalupensis). The estimated diversification times of these subclades were 9.8 and 7.1 Mya, respectively. Within each subclade, species divergences occurred during the Pliocene-Pleistocene epochs. The divergent event between the Atlantic A. imberbis and Apogon TEP clade corresponds to the first closure event of the Central American Seaway. The biogeographic history of Apogon within the TEP appears to be the result of vicariant, dispersal and founder events that occurred during the last 11 million years. The vicariant and dispersal events occurred along the mainland and were associated with the origin of the Central American Gap. The founder events could have allowed the invasion of Apogon to TEP island areas and could have been driven by ancient warming oceanic waters, changes in circulation of marine currents, and the presence of seamounts in ancient marine ridges that allowed the settlement of marine biota. These factors may have allowed Apogon lineages to cross the TEP biogeographic barriers at different times, with subsequent genetic isolation.

Reticulate Pleistocene evolution of Ethiopian rodent genus along remarkable altitudinal gradient.

The Ethiopian highlands are the most extensive complex of mountainous habitats in Africa. The presence of the Great Rift Valley (GRV) and the striking elevational ecological gradients inhabited by recently radiated Ethiopian endemics, provide a wide spectrum of model situations for evolutionary studies. The extant species of endemic rodents, often markedly phenotypically differentiated, are expected to possess complex genetic features which evolved asa consequence of the interplay between geomorphology and past climatic changes. In this study, we used the largest available multi-locus genetic dataset of the murid genus Stenocephalemys (347 specimens from ca 40 localities across the known distributional area of all taxa) to investigate the relative importance of disruptive selection, temporary geographic isolation and introgression in their adaptive radiations in the Pleistocene. We confirmed the four main highly supported mitochondrial (mtDNA) clades that were proposed as four species in a previous pilot study: S. albipes is a sister species of S. griseicauda (both lineages are present on both sides of the GRV), while the second clade is formed by two Afro-alpine species, S. albocaudata (east of GRV) and the undescribed Stenocephalemys sp. A (west of GRV). There is a clear elevational gradient in the distribution of the Stenocephalemys taxa with two to three species present at different elevations of the same mountain range. Surprisingly, the nuclear species tree corresponded only a little to the mtDNA tree. Multispecies coalescent models based on six nuclear markers revealed the presence of six separate gene pools (i.e. candidate species), with different topology. Phylogenetic analysis, together with the geographic distribution of the genetic groups, suggests a complex reticulate evolution. We propose a scenario that involves (besides classical allopatric speciation) two cases of disruptive selection along the elevational ecological gradient, multiple crosses of GRV in dry and cold periods of the Pleistocene, followed by hybridization and mtDNA introgression on imperfect reproductive barriers. Spatial expansion of the currently most widespread "albipes" mtDNA clade was followed by population fragmentation, lineage sorting and again by hybridization and mtDNA introgression. Comparison of this genetic structure to other Ethiopian endemic taxa highlight the geographical areas of special conservation concern, where more detailed biodiversity studies should be carried out to prevent many endemic taxa from going extinct even before they are recognized.

Alternative methods of phylogenetic inference for the Patagonian lizard group Liolaemus elongatus-kriegi (Iguania: Liolaemini) based on mitochondrial and nuclear markers.

We present different approaches to a multi-locus phylogeny for the Liolaemus elongatus-kriegi group, including almost all species and recognized lineages. We sequenced two mitochondrial and five nuclear gene regions for 123 individuals from 35 taxa, and compared relationships resolved from concatenated and species tree methods. The L. elongatus-kriegi group was inferred as monophyletic in three of the five analyses (concatenated mitochondrial, concatenated mitochondrial + nuclear gene trees, and SVD quartet species tree). The mitochondrial gene tree resolved four haploclades, three corresponding to the previously recognized complexes: L. elongatus, L. kriegi and L. petrophilus complexes, and the L. punmahuida group. The BEAST species tree approach included the L. punmahuida group within the L. kriegi complex, but the SVD quartet method placed it as sister to the L. elongatus-kriegi group. BEAST inferred species of the L. elongatus and L. petrophilus complexes as one clade, while SVDquartet inferred these two complexes as monophyletic (although with no statistical support for the L. petrophilus complex). The species tree approach also included the L. punmahuida group as part of the L. elongatus-kriegi group. Our study provides detailed multilocus phylogenetic hypotheses for the L. elongatus-kriegi group, and we discuss possible reasons for differences in the concatenation and species tree methods.

How many pygmy marmoset (Cebuella Gray, 1870) species are there? A taxonomic re-appraisal based on new molecular evidence.

The pygmy marmoset, Cebuella pygmaea, the smallest of the New World monkeys, has one of the largest geographical distributions of the Amazonian primates. Two forms have been recognized: Cebuella pygmaea pygmaea (Spix, 1823), and C. p. niveiventris Lönnberg, 1940. In this study, we investigated if the separation of pygmy marmosets into these two clades can be corroborated by molecular data. We also examine and compare coloration of the pelage in light of the new molecular results. We analyzed the mtDNA cytochrome b gene and, for the first time for any Neotropical primate, we used a reduced representation genome sequencing approach (ddRADseq) to obtain data for recently collected, geographically representative samples from the Rio Japurá, a northern tributary of the Rio Solimões and from the Javarí, Jutaí, Juruá, Madeira and Purus river basins, all tributaries south of the Solimões. We estimated phylogenies and diversification times under both maximum likelihood and Bayesian inference criteria. Our analysis showed two highly supported clades, with intraclade divergences much smaller than interclade divergences, indicating two species of Cebuella: one from the Rio Japurá and one to the south of Solimões. The interpretation of our results in light of the current taxonomy is not trivial however. Lönnberg stated that the type of Spix's pygmy marmoset (type locality 'near Tabatinga') was obtained from the south of the Solimões, and his description of the distinct niveiventris from Lago Ipixuna, south of the Solimões and several hundred kilometres east of Tabatinga, was based on a comparison with specimens that he determined as typical pygmaea that were from the upper Rio Juruá (south of the Solimões). As such it remains uncertain whether the name pygmaea should be applicable to the pygmy marmosets north of the Rio Solimões (Tabatinga type locality) or south (near Tabatinga but across the Solimões). Finally, our analysis of pelage coloration revealed three phenotypic forms: (1) south of the Rio Solimoes, (2) Eirunepé-Acre, upper Juruá basin; and (3) Japurá. More samples from both sides of Solimões in the region of Tabatinga will be necessary to ascertain the exact type locality for Spix's pygmaea and to resolve the current uncertainties surrounding pygmy marmoset taxonomy.

Refining the biogeographical scenario of the land snail Cornu aspersum aspersum: Natural spatial expansion and human-mediated dispersal in the Mediterranean basin.

The land snail Cornu aspersum aspersum, native to the Mediterranean region, has been the subject of several anatomical and molecular studies leading to recognize two divergent lineages, named "East" and "West" according to their geographical distribution in North Africa. The first biogeographical scenario proposed the role of Oligocene paleogeographic events and Quaternary glacial refugia to explain spatial patterns of genetic variation. The aim of this study was to refine this scenario using molecular and morphometric data from 169 populations sampled across Mediterranean islands and continents. The two previously described lineages no longer correspond to distinct biogeographical entities. Phylogenetic relationships reveal the existence of seven clades, do not support the Tyrrhenian vicariance hypothesis, and suggest that C. a. aspersum most likely originates from North Africa. We found two contrasted patterns with the seven clades defining spatially well-structured populations in the southern Mediterranean whereas one clade is distributed across the basin. High genetic diversities and rates of endemism in North Africa support the role of this region for the diversification of C. a. aspersum. In referring to divergence times previously estimated, we suggest allopatric differentiation due to geological changes of the Atlas system and multiple refugial areas during Pleistocene glaciations. The new biogeographical scenario implies an initial range expansion from North Africa to the Iberian Peninsula and the peri-Tyrrhenian regions through land bridges connections during the Messinian Salinity Crisis and Pleistocene glaciations. Historical events appear to have also structured morphometric variation but recent dispersal events favored the emergence of secondary contacts between clades. Southern Mediterranean clades are limited to their initial distribution and populations of the recent clade would have rapidly recolonized the whole Mediterranean in the Holocene due to greater adaptive potential and the influence of human transportations.

Updated molecular phylogeny of the squid family Ommastrephidae: Insights into the evolution of spawning strategies.

Two types of spawning strategy have been described for ommastrephid squids: coastal and oceanic. It has been suggested that ancestral ommastrephids inhabited coastal waters and expanded their distribution into the open ocean during global changes in ocean circulation in the Oligocene. This hypothesis could explain the different reproductive strategies in oceanic squids, but has never been tested in a phylogenetic context. In the present study, we assess the coastal-to-open-ocean hypothesis through inferring the evolution of reproductive traits (spawning type) of ommastrephid squids using the phylogenetic comparative method to estimate ancestral states and divergence times. This analysis was performed using a robust molecular phylogeny with three mitochondrial genes (COI, CYTB and 16S) and two nuclear genes (RHO and 18S) for nearly all species of ommastrephid squid. Our results support dividing the Ommastrephidae into the three traditional subfamilies, plus the monotypic subfamily Todaropsinae as proposed previously. Divergence times were found to be older than those suggested. Our analyses strongly suggest that early ommastrephid squids spawned in coastal areas, with some species subsequently switching to spawn in oceanic areas, supporting previous non-tested hypotheses. We found evidence of gradual evolution change of spawning type in ommastrephid squids estimated to have occurred since the Cretaceous.

A genomic perspective of the pink-headed duck Rhodonessa caryophyllacea suggests a long history of low effective population size.

The first molecular phylogenetic hypothesis for the possibly extinct pink-headed duck Rhodonessa caryophyllacea unambiguously shows that it belongs to the pochard radiation that also includes the genera Aythya and Netta. It is the sister to all modern-day pochards and belongs to a lineage that branched off from the others more than 2.8 million years ago. Rhodonessa caryophyllacea is believed to never have been common in modern time and we show this has probably been the situation for as long as 100,000 years. Our results suggest that their effective population size varied between 15,000 and 25,000 individuals during the last 150,000 years of the Pleistocene. The reasons behind this are largely unknown as very little is known about the life-history and biology of this species. Presumably it is due to factors related to feeding or to breeding, but we may never know this for sure.

Speciation, range contraction and extinction in the endemic New Zealand King Shag complex.

New Zealand's endemic King Shag (Leucocarbo carunculatus) has occupied only a narrow portion of the northeastern South Island for at least the past 240years. However, pre-human Holocene fossil and archaeological remains have suggested a far more widespread distribution of the three Leucocarbo species (King, Otago, Foveaux) on mainland New Zealand at the time of Polynesian settlement in the late 13th Century CE. We use modern and ancient DNA, and morphometric and osteological analyses, of modern King Shags and Holocene fossil Leucocarbo remains to assess the pre-human distribution and taxonomic status of the King Shag on mainland New Zealand, and the resultant conservation implications. Our analyses show that the King Shag was formerly widespread around southern coasts of the North Island and the northern parts of the South Island but experienced population and lineage extinctions, and range contraction, probably after Polynesian arrival. This history parallels range contractions of other New Zealand seabirds. Conservation management of the King Shag should take into account this species narrow distribution and probable reduced genetic diversity. Moreover, combined genetic, morphometric and osteological analyses of prehistoric material from mainland New Zealand suggest that the now extinct northern New Zealand Leucocarbo populations comprised a unique lineage. Although these distinctive populations were previously assigned to the King Shag (based on morphological similarities and geographic proximity to modern Leucocarbo populations), we herein describe them as a new species, the Kohatu Shag (Leucocarbo septentrionalis). The extinction of this species further highlights the dramatic impacts Polynesians and introduced predators had on New Zealand's coastal and marine biodiversity. The prehistoric presence of at least four species of Leucocarbo shag on mainland NZ further highlights its status as a biodiversity hotspot for Phalacrocoracidae.

An integrative approach to untangling species delimitation in the Cataglyphis bicolor desert ant complex in Israel.

Although extensive research has been carried out on the desert ants in the genus Cataglyphis in recent years, some of the specific intra- and interspecific relationships remain elusive. The present study disentangles the phylogenetic relationships among the C. bicolor complex in Israel using an integrative approach based on genetic markers, morphometric measurements, and chemical analyses (cuticular hydrocarbons). Several species delimitation approaches based on four nuclear, two mitochondrial, and eleven microsatellite markers, as well as 16 body measurements and 56 chemical variables, were employed to deciphering the occurrence of cryptic species in our data set. Our findings support the occurrence of at least four distinct species in the C. bicolor group in Israel, one of which may be a complex of three more recent species. The findings confirm the distinctiveness of C. isis and C. holgerseni. They attest the presence of a recently discovered species, C. israelensis, in the central mountain ridge and the occurrence of another clade distributed from the Negev to the Mediterranean coast, comprising the species C. niger, C. savignyi, and C. drusus. Although these three species are separated on the basis of mtDNA, this subgrouping was not supported by any of the nuclear sequence markers nor by the microsatellite analysis. This genetic structure may thus either reflect a possible recent speciation, or a geographical structuring of a single species. Overall, using these different sources of evidence we locate our samples within a global phylogeny of the bicolor group and discuss the processes that underlie speciation in this group.

Molecular systematics, species delimitation and diversification patterns of the Phyllodactylus lanei complex (Gekkota: Phyllodactylidae) in Mexico.

The description of cryptic gecko species worldwide has revealed both that many putative species are, in fact, conformed by a complex of morphologically conserved species that are genetically distinct and highly divergent, and that gecko species diversity could be underestimated. The taxonomy and species delimitation of geckos belonging to the genus Phyllodactylus is still controversial, 16 of which are distributed in Mexico and 13 are endemic. Although the large morphological variation shown by the Phyllodactylus species from Mexico has been amply documented, little is known about their genetic diversity and evolutionary relationships, and much less regarding cryptic speciation. Here, we included the most comprehensive sampling of populations and species of the Phyllodactylus lanei complex distributed in Mexico, and applied an analytical approach that included probabilistic phylogenetic analyses, jointly with species delimitation methods and Bayesian putative species validation analysis. Our results suggest the existence of 10 lineages within the complex, supporting the existence of cryptic species, and in great contrast with the current taxonomic proposal that includes only four subspecies. The most recent common ancestor (MRCA) for the P. lanei clade originated on the Early Eocene (∼54Mya), along the southern coasts of Mexico, followed by the highest diversification of the complex MRCA during the Eocene (34-56Mya). Lineages subsequently dispersed and diversified towards the northwest, and the diversification process ended with the most recent lineages inhabiting two islands on the coasts of Nayarit (Miocene; 5.5-23Mya). Our results highlight three vicariant events associated with the evolution of the lineages, two of them intimately related to the formation of the Sierra Madre del Sur and the Transmexican Volcanic Belt mountain ranges, main geographic barriers that isolated and facilitated the divergence and speciation in this group of geckos. Finally, we propose that there are 10 species in the P. lanei complex, from which four represent taxonomic changes and six are new species and require a formal description. We acknowledge that more analyses, including a detailed evaluation of morphological characters and use of more unlinked nuclear loci with enough variability, are needed to further support their taxonomic description.

Striking pseudogenization in avian phylogenetics: Numts are large and common in falcons.

Nuclear copies of mitochondrial genes (numts) are a well-known feature of eukaryotic genomes and a concern in systematics, as they can mislead phylogenetic inferences when inadvertently used. Studies on avian numts initially based on the chicken genome suggest that numts may be uncommon and relatively short among birds. Here we ask how common numts are in falcons, based on recently sequenced genomes of the Saker falcon (Falco cherrug) and Peregrine falcon (F. peregrinus). We identified numts by BLASTN searches and then extracted CYTB, ND2 and COI sequences from them, which were then used for phylogeny inference along with several sequences from other species in Falconiformes. Our results indicate that avian numts may be much more frequent and longer than previously thought. Phylogenetic inferences revealed multiple independent nuclear insertions throughout the history of the Falconiformes, including cases of sequences available in public databases and wrongly identified as authentic mtDNA. New sequencing technologies and ongoing efforts for whole genome sequencing will provide exciting opportunities for avian numt research in the near future.

Multilocus analysis of the catfish family Trichomycteridae (Teleostei: Ostariophysi: Siluriformes) supporting a monophyletic Trichomycterinae.

Trichomycteridae is the second most diverse family of the order Siluriformes, its members are widely distributed through the freshwaters of Central and South America, exhibiting an exceptional ecological and phenotypic disparity. The most diverse subfamily, Trichomycterinae, represented mainly by the genus Trichomycterus, historically has been recognized as non-monophyletic and various characters used to unite or divide its constituents are repeatedly called into question. No comprehensive molecular phylogenetic hypothesis regarding relationships of trichomycterids has been produced, and the present study is the first extensive phylogeny for the family Trichomycteridae, based on a multilocus dataset of three mitochondrial loci and two nuclear markers (3284bp total). Our analysis has the most comprehensive taxon-sampling of the Trichomycteridae published so far, including members of all subfamilies and a vast representation of Trichomycterus diversity. Analysis of these data showed a phylogenetic hypothesis with broad agreement between the Bayesian (BI) and maximum-likelihood (ML) trees. The results provided overwhelming support for the monophyletic status of Copionodontinae, Stegophilinae, Trichomycterinae, and Vandelliinae, but not Sarcoglanidinae and Glanapteryginae. A major feature of our results is the support to the current conceptualization of Trichomycterinae, which includes Ituglanis and Scleronema and excludes the "Trichomycterus" hasemani group. Divergence time analysis based on DNA substitution rates suggested a Lower Cretaceous origin of the family and the divergence events at subfamilial level shaped by Paleogene events in the geohistory of South America. This hypothesis lays a foundation for an array of future studies of evolution and biogeography of the family.

Molecular phylogeny of Miltogramminae (Diptera: Sarcophagidae): Implications for classification, systematics and evolution of larval feeding strategies.

Miltogramminae is one of the phylogenetically most poorly studied taxa of the species-rich family Sarcophagidae (Diptera). Most species are kleptoparasites in nests of solitary aculeate wasps and bees, although parasitoids and saprophagous species are also known, and the ancestral miltogrammine life habit remains unsettled. Here, we present for the first time a comprehensive phylogenetic tree consisting of 58 representatives of Miltogramminae, reconstructed using sequence data from three mitochondrial (COI, cytB, ND4) and one nuclear (Ef-1α) genes. Our phylogenetic hypothesis suggests that: (1) Miltogramminae are sister to Paramacronychiinae, (2) Miltogramminae can be divided into the "lower miltogrammines" containing two clades of mainly saprophages and a clade of "higher miltogrammines" with mainly kleptoparasitic species, (3) only three genera turn out to be non-monophyletic: Miltogramma, Senotainia and Pterella and (4) the genus Sarcotachina, which traditionally has been considered as belonging to the Paramacronychiinae, is placed in one of the clades of "lower miltogrammines". Ancestral state reconstruction of larval feeding strategy and five larval characters reveals that the ancestor of Miltogramminae was likely a saprophage retaining plesiomorphic oral ridges and a cephaloskeleton with sclerotized dorsal bridge. Synapomorphies like large pseudocephalic sensory organs and well-developed cuticular sculpture suggest that the ancestral first instar larva actively searched for a buried food supply.

Comparison of different strategies for using fossil calibrations to generate the time prior in Bayesian molecular clock dating.

Fossil calibrations are the utmost source of information for resolving the distances between molecular sequences into estimates of absolute times and absolute rates in molecular clock dating analysis. The quality of calibrations is thus expected to have a major impact on divergence time estimates even if a huge amount of molecular data is available. In Bayesian molecular clock dating, fossil calibration information is incorporated in the analysis through the prior on divergence times (the time prior). Here, we evaluate three strategies for converting fossil calibrations (in the form of minimum- and maximum-age bounds) into the prior on times, which differ according to whether they borrow information from the maximum age of ancestral nodes and minimum age of descendent nodes to form constraints for any given node on the phylogeny. We study a simple example that is analytically tractable, and analyze two real datasets (one of 10 primate species and another of 48 seed plant species) using three Bayesian dating programs: MCMCTree, MrBayes and BEAST2. We examine how different calibration strategies, the birth-death process, and automatic truncation (to enforce the constraint that ancestral nodes are older than descendent nodes) interact to determine the time prior. In general, truncation has a great impact on calibrations so that the effective priors on the calibration node ages after the truncation can be very different from the user-specified calibration densities. The different strategies for generating the effective prior also had considerable impact, leading to very different marginal effective priors. Arbitrary parameters used to implement minimum-bound calibrations were found to have a strong impact upon the prior and posterior of the divergence times. Our results highlight the importance of inspecting the joint time prior used by the dating program before any Bayesian dating analysis.

Integrative species delimitation of the widespread North American jumping mice (Zapodinae).

Delimiting species can be challenging, but is a key step for the critical examination of evolutionary history and for prioritizing conservation efforts. Because systematic relationships are often determined iteratively using tests based on taxonomy, such methods can fail to detect cryptic variation and result in biased conclusions. Conversely, discovery-based approaches provide a powerful way to define operational taxonomic units and test species boundaries. We compare both approaches (taxonomy-based delimitation - TBD and discovery-based delimitation - DBD) within North American jumping mice (Zapodinae) using broad sampling, multilocus analyses, and ecological tests. This group diversified through the dynamic glacial-interglacial periods of the Quaternary and phylogeographic tests reveal 28 lineages that correspond poorly with current taxonomy (4 species, 32 nominal subspecies). However, neither the 4-species or 28-lineage hypotheses are optimal for species-level classification. Rather, information theoretic approaches (Bayes Factors) indicate a 15-species hypothesis is best for characterizing genetic variation in this group, with subsequent iterative pairwise ecological tests failing to confirm four species pairs. Taken together, evolutionary and ecological tests capture divergence among 11 putative species that, if upheld by additional tests, will lead to taxonomic revision and reevaluation of conservation plans.