Molecular systematics and biodiversity of the Cryptotis mexicanus group (Eulipotyphla: Soricidae): two new species from Honduras supported

Small-eared shrews of the genus Cryptotis (Mammalia: Eulipotyphla: Soricidae) are widespread in the northern Neotropics. Systematic studies of these shrews over the past two decades have revealed previously undocumented morphological and species diversity, resulting in a quadrupling of the number of recognized species. Unfortunately, a small proportion of the species in the genus have been included in molecular phylogenetic studies, and evolutionary relationships within the genus are incompletely known. Traditionally, species have been assigned to four or five morphologically defined ‘species groups’, but tests of the monophyly of some of these groups show weak support and relationships amongst species groups remain somewhat speculative. The largest species group is the C. mexicanus group inhabiting Mexico and northern Central America. We studied sequences from mitochondrial cytochrome-b and 16S genes, as well as nuclear ApoB and BRCA1 genes from 22 species of Cryptotis, including 15 species in the C. mexicanus group. Our combined analysis shows that the C. goldmani subgroup is very weakly supported as monophyletic; however, the C. mexicanus group as a whole is not monophyletic. Our molecular phylogenetic analyses confirm the distinctiveness of two newly described species (C. celaque and C. mccarthyi) from isolated highlands of western Honduras and illustrate their relationship with other species formerly considered part of a widespread C. goodwini.     

Characteristics of the mitochondrial genome of Rana omeimontis and related species in Ranidae: Gene rearrangements and phylogenetic relationships

The Omei wood frog (Rana omeimontis), endemic to central China, belongs to the family Ranidae. In this study, we achieved detail knowledge about the mitogenome of the species. The length of the genome is 20,120 bp, including 13 protein‐coding genes (PCGs), 22 tRNA genes, two rRNA genes, and a noncoding control region. Similar to other amphibians, we found that only nine genes (ND6 and eight tRNA genes) are encoded on the light strand (L) and other genes on the heavy strand (H). Totally, The base composition of the mitochondrial genome included 27.29% A, 28.85% T, 28.87% C, and 15.00% G, respectively. The control regions among the Rana species were found to exhibit rich genetic variability and A + T content. R. omeimontis was clustered together with R. chaochiaoensis in phylogenetic tree. Compared to R. amurensis and R. kunyuensi, it was more closely related to R. chaochiaoensis, and a new way of gene rearrangement (ND6‐trnE‐Cytb‐D‐loop‐trnL2 (CUN)‐ND5‐D‐loop) was also found in the mitogenome of R. amurensis and R. kunyuensi. Our results about the mitochondrial genome of R. omeimontis will contribute to the future studies on phylogenetic relationship and the taxonomic status of Rana and related Ranidae species.     

Time‐dependent estimates of molecular evolutionary rates: evidence and causes

We are writing in response to a recent critique by Emerson & Hickerson ( 2015 ), who challenge the evidence of a time‐dependent bias in molecular rate estimates. This bias takes the form of a negative relationship between inferred evolutionary rates and the ages of the calibrations on which these estimates are based. Here, we present a summary of the evidence obtained from a broad range of taxa that supports a time‐dependent bias in rate estimates, with a consideration of the potential causes of these observed trends. We also describe recent progress in improving the reliability of evolutionary rate estimation and respond to the concerns raised by Emerson & Hickerson ( 2015 ) about the validity of rates estimated from time‐structured sequence data. In doing so, we hope to dispel some misconceptions and to highlight several research directions that will improve our understanding of time‐dependent biases in rate estimates.     

Colonization of the Philippines from Taiwan: a multi‐locus test of the biogeographic and phylogenetic relationships of isolated populations of shrews

Aim Colonization of the Philippines from Taiwan or neighbouring areas of the Asian mainland has been proposed as an important source of diversity for some plant and animal groups in the northern Philippines. Previous inferences, however, were based on taxonomic groupings, which sometimes fail to reflect phylogenetic history. Here, we test for colonization of the Philippines from the north in a group of shrews (Soricomorpha: Crocidura) using explicit inferences of evolutionary history. Location Southeast Asia. Methods We estimate the phylogenetic relationships of populations of shrews from Batan and Sabtang islands in the northern Philippines using DNA sequences from two mitochondrial genes and three nuclear loci. We employ topology tests to evaluate the possible relationships of these shrews to species from throughout Southeast Asia. Results We find conclusive evidence that shrews from Batan and Sabtang are closely related to Crocidura tanakae from Taiwan and additional specimens from the Asian mainland. Bayesian and frequentist topology tests using alignments of individual loci strongly reject any notion that shrews from Batan and Sabtang are part of the main Philippine radiation of Crocidura, indicating that the northernmost Philippine islands were almost certainly colonized by shrews from Taiwan or mainland Asia. Main conclusions Our results provide the first compelling evidence for colonization of the Philippine archipelago by a terrestrial vertebrate via a northern route. Invasion of the northern Philippines by shrews, however, did not lead to further range expansion to more southerly parts of the Philippines. This study, combined with previous results, documents that Crocidura colonized the Philippines at least three times. However, only one of these invasions led to in situ speciation and ubiquity across the archipelago. Our findings are part of a growing body of literature suggesting that oceanic archipelagos are often colonized multiple times by groups of closely related species, and occasionally from multiple sources.     

Phylogenetic relationships and taxonomy of Altiplano populations of Biomphalaria (Gastropoda: Planorbidae): inference from a multilocus approach

Isolated closed basins provide a natural laboratory to study the differentiation among wild populations. Here we examined the phylogenetic relationships of the Southern Altiplano populations of Biomphalaria, a genus with medical importance, using nuclear (ITS1, ITS2) and mitochondrial (16S) ribosomal gene markers and a species of Helisoma as outgroup. Phylogenetic trees based on separate and combined analyses show that these populations form a particular lineage within Biomphalaria along with Biomphalaria peregrina (d'Orbigny, 1835) and Biomphalaria oligoza Paraense, 1974. The origin of this clade was estimated to have occurred in the middle Pleistocene. Molecular analyses showed that the nominal species Biomphalaria crequii ( Courty, 1907 ) from the Salar de Ascotán and Biomphalaria costata (Biese, 1951) from the Salar de Carcote, previously synonymized with Biomphalaria andecola (d'Orbigny, 1835) and B. peregrina, respectively, are distinct taxa. Molecular data did not resolve the relationship of Biomphalaria aymara Valdovinos & Stuardo, 1991 from the Isluga swamps to other Biomphalaria species, but confirm that the populations from the Lauca and Huasco basins may represent a distinct undescribed species of Biomphalaria from the Southern Altiplano. Snails examined for trematodes were found to be positive in some Altiplano localities. © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 165 , 795–808.     

Phylogeny of ground beetles subgenus Nialoe (s. lat.) Tanaka (Coleoptera: Carabidae; genus Pterostichus): A molecular phylogenetic approach

We constructed a phylogeny of the ground beetle subgenus Nialoe ( s. lat. ), genus Pterostichus (Coleoptera: Carabidae) based on two mitochondrial (cytochrome oxidase I and 16S ribosomal DNA) and one nuclear (28S ribosomal DNA) gene sequences. Thirty-three representative species of the group and three outgroup species were analyzed. The resultant trees (maximum parsimonious, maximum likelihood and Bayesian trees of the combined data of the three gene sequences) indicated that there are two large and three small lineages in the group, some of which were supported by a previous morphology-based phylogeny. In all the analyses, the small lineage composed of two Korean species is sister to the rest of the subgenus, but relationships of other four lineages differed among the analyses and remained unresolved. The implications of the present results are discussed in terms of taxonomy and biogeography of the group.     

A phylogenetic approach to gene expression data: evidence for the evolutionary origin of mammalian leukocyte phenotypes

SUMMARY The evolution of multicellular organisms involved the evolution of specialized cell types performing distinct functions; and specialized cell types presumably arose from more generalized ancestral cell types as a result of mutational event, such as gene duplication and changes in gene expression. We used characters based on gene expression data to reconstruct evolutionary relationships among 11 types of lymphocytes by the maximum parsimony method. The resulting phylogenetic tree showed expected patterns including separation of the lymphoid and myeloid lineages; clustering together of granulocyte types; and pairing of phenotypically similar cell types such as T-helper cells type 1 and T-helper cells type 2 (Th1 and Th2). We used phylogenetic analyses of sequence data to determine the time of origin of genes showing significant expression difference between Th1 and Th2 cells. Many such genes, particularly those involved in the regulation of gene expression or activation of proteins, were of ancient origin, having arisen by gene duplication before the most recent common ancestor (MRCA) of tetrapods and teleosts. However, certain other genes with significant expression difference between Th1 and Th2 arose after the tetrapod–teleost MRCA, and some of the latter were specific to eutherian (placental) mammals. This evolutionary pattern is consistent with previous evidence that, while bony fishes possess Th1 and Th2 cells, the latter differ phenotypically in important respects from the corresponding cells of mammals. Our results support a gradualistic model of the evolution of distinctive cellular phenotypes whereby the unique characteristics of a given cell type arise as a result of numerous independent mutational changes over hundreds of millions of years.     

Multilocus phylogeny and Bayesian estimates of species boundaries reveal hidden evolutionary relationships and cryptic diversity in Southeast Asian monitor lizards

Recent conceptual, technological and methodological advances in phylogenetics have enabled increasingly robust statistical species delimitation in studies of biodiversity. As the variety of evidence purporting species diversity has increased, so too have the kinds of tools and inferential power of methods for delimiting species. Here, we showcase an organismal system for a data‐rich, comparative molecular approach to evaluating strategies of species delimitation among monitor lizards of the genus Varanus. The water monitors (Varanus salvator Complex), a widespread group distributed throughout Southeast Asia and southern India, have been the subject of numerous taxonomic treatments, which have drawn recent attention due to the possibility of undocumented species diversity. To date, studies of this group have relied on purportedly diagnostic morphological characters, with no attention given to the genetic underpinnings of species diversity. Using a 5‐gene data set, we estimated phylogeny and used multilocus genetic networks, analysis of population structure and a Bayesian coalescent approach to infer species boundaries. Our results contradict previous systematic hypotheses, reveal surprising relationships between island and mainland lineages and uncover novel, cryptic evolutionary lineages (i.e. new putative species). Our study contributes to a growing body of literature suggesting that, used in concert with other sources of data (e.g. morphology, ecology, biogeography), multilocus genetic data can be highly informative to systematists and biodiversity specialists when attempting to estimate species diversity and identify conservation priorities. We recommend holding in abeyance taxonomic decisions until multiple, converging lines of evidence are available to best inform taxonomists, evolutionary biologists and conservationists.     

Different filtering strategies of genotyping‐by‐sequencing data provide complementary resolutions of species boundaries and relationships in a clade of sexually deceptive orchids

Ongoing hybridization and retained ancestral polymorphism in rapidly radiating lineages could mask recent cladogenetic events. This presents a challenge for the application of molecular phylogenetic methods to resolve differences between closely related taxa. We reanalyzed published genotyping‐by‐sequencing (GBS) data to infer the phylogeny of four species within the Ophrys sphegodes complex, a recently radiated clade of orchids. We used different data filtering approaches to detect different signals contained in the dataset generated by GBS and estimated their effects on maximum likelihood trees, global F_ST and bootstrap support values. We obtained a maximum likelihood tree with high bootstrap support, separating the species by using a large dataset based on loci shared by at least 30% of accessions. Bootstrap and F_ST values progressively decreased when filtering for loci shared by a higher number of accessions. However, when filtering more stringently to retain homozygous and organellar loci, we identified two main clades. These clades group individuals independently from their a priori species assignment, but were associated with two organellar haplotype clusters. We infer that a less stringent filtering preferentially selects for rapidly evolving lineage‐specific loci, which might better delimit lineages. In contrast, when using homozygous/organellar DNA loci the signature of a putative hybridization event in the lineage prevails over the most recent phylogenetic signal. These results show that using differing filtering strategies on GBS data could dissect the organellar and nuclear DNA phylogenetic signal and yield novel insights into relationships between closely related species.     

Sequence capture using AFLP‐generated baits: A cost‐effective method for high‐throughput phylogenetic and phylogeographic analysis

Target sequence capture is an efficient technique to enrich specific genomic regions for high‐throughput sequencing in ecological and evolutionary studies. In recent years, many sequence capture approaches have been proposed, but most of them rely on commercial synthetic baits which make the experiment expensive. Here, we present a novel sequence capture approach called AFLP‐based genome sequence capture (AFLP Capture). This method uses the AFLP (amplified fragment length polymorphism) technique to generate homemade capture baits without the need for prior genome information, thus is applicable to any organisms. In this approach, biotinylated AFLP fragments representing a random fraction of the genome are used as baits to capture the homologous fragments from genomic shotgun sequencing libraries. In a trial study, by using AFLP Capture, we successfully obtained 511 orthologous loci (>700,000 bp in total length) from 11 Odorrana species and more than 100,000 single nucleotide polymorphisms (SNPs) in four analyzed individuals of an Odorrana species. This result shows that our method can be used to address questions of various evolutionary depths (from interspecies level to intraspecies level). We also discuss the flexibility in bait preparation and how the sequencing data are analyzed. In summary, AFLP Capture is a rapid and flexible tool and can significantly reduce the experimental cost for phylogenetic studies that require analyzing genome‐scale data (hundreds or thousands of loci).     

A needle in a haystack: A new metabarcoding approach to survey diversity at the species level of Arcellinida (Amoebozoa: Tubulinea)

Environmental DNA-based diversity studies have increased in popularity with the development of high throughput sequencing technologies. This permits the potential simultaneous retrieval of vast amounts of molecular data from many different organisms and species, thus contributing to a wide range of biological disciplines. Environmental DNA protocols designed for protists often focused on the highly conserved small subunit of the ribosome gene, that does not permit species-level assignments. On the other hand, eDNA protocols aiming at species-level assignments allow a fine level ecological resolution and reproducible results. These protocols are currently applied to organisms living in marine and shallow lotic freshwater ecosystems, often in a bioindication purpose. Therefore, in this study, we present a species-level eDNA protocol designed to explore diversity of Arcellinida (Amoebozoa: Tubulinea) testate amoebae taxa that is based on mitochondrial cytochrome oxidase subunit I (COI). These organisms are widespread in lentic water bodies and soil ecosystems. We applied this protocol to 42 samples from peatlands, estuaries and soil environments, recovering all the infraorders in Glutinoconcha (with COI data), except for Hyalospheniformes. Our results revealed an unsuspected diversity in morphologically homogeneous groups such as Cylindrothecina, Excentrostoma or Sphaerothecina. With this protocol we expect to revolutionize the design of modern distributional Arcellinida surveys. Our approach involves a rapid and cost-effective analysis of testate amoeba diversity living in contrasted ecosystems. Therefore, the order Arcellinida has the potential to be established as a model group for a wide range of theoretical and applied studies.     

Origins of species richness in the Indo‐Malay‐Philippine biodiversity hotspot: evidence for the centre of overlap hypothesis

The Indo‐Malay‐Philippine (IMP) biodiversity hotspot, bounded by the Philippines, the Malay Peninsula and New Guinea, is the epicentre of marine biodiversity. Hypotheses to explain the source of the incredible number of species found there include the centre of overlap hypothesis, which proposes that in this region the distinct faunas of the Pacific and Indian Oceans overlap. Here we review the biogeographical evidence in support of this hypothesis. We examined tropical reef fish distributions, paying particular attention to sister species pairs that overlap in the IMP hotspot. We also review phylogeographical studies of wide‐ranging species for evidence of lineage divergence and overlap in the IMP region. Our synthesis shows that a pattern of isolation between the Pacific and the Indian Ocean faunas is evident across a wide range of taxa. The occurrence of sister species, with one member in each ocean, indicates that the mechanism(s) of isolation has been in effect since at least the Miocene, while phylogeographical studies indicate more recent divergences in the Pleistocene. Divergence in isolation followed by population expansion has led to an overlap of closely related taxa or genetic lineages in the hotspot, contributing to diversity and species richness in the region. These findings are consistent with the centre of overlap hypothesis and highlight the importance of this process in generating biodiversity within the IMP.     

Biogeography of the grasses (Poaceae): a phylogenetic approach to reveal evolutionary history in geographical space and geological time

The grasses (Poaceae) are the fifth most diverse family of angiosperms, including 800 genera and more than 10 000 species. Few phylogenetic studies have tried to investigate palaeo‐biogeographical and palaeo‐ecological scenarios that may have led to present‐day distribution and diversity of grasses at the family level. We produced a dated phylogenetic tree based on combined plastid DNA sequences and a comprehensive sample of Poaceae. Furthermore, we produced an additional tree using a supermatrix of morphological and molecular data that included all 800 grass genera so that ancestral biogeography and ecological habitats could be inferred. We used a likelihood‐based method, which allows the estimation of ancestral polymorphism in both biogeographical and ecological analyses for large data sets. The origin of Poaceae was retrieved as African and shade adapted. The crown node of the BEP + PACCMAD clade was dated at 57 Mya, in the early Eocene. Grasses dispersed to all continents by approximately 60 million years after their Gondwanan origin in the late Cretaceous. PACCMAD taxa adapted to open habitats as early as the late Eocene, a date consistent with recent phytolith fossil data for North America. C₄ photosynthesis first originated in Africa, at least for Chloridoideae in the Eocene at c. 30 Mya. The BEP clade members adapted to open habitats later than PACCMAD members; this was inferred to occur in Eurasia in the Oligocene. © 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 162 , 543–557.     

A stage‐wise approach for the analysis of multi‐environment trials

Plant breeders and variety testing agencies routinely test candidate genotypes (crop varieties, lines, test hybrids) in multiple environments. Such multi‐environment trials can be efficiently analysed by mixed models. A single‐stage analysis models the entire observed data at the level of individual plots. This kind of analysis is usually considered as the gold standard. In practice, however, it is more convenient to use a two‐stage approach, in which experiments are first analysed per environment, yielding adjusted means per genotype, which are then summarised across environments in the second stage. Stage‐wise approaches suggested so far are approximate in that they cannot fully reproduce a single‐stage analysis, except in very simple cases, because the variance–covariance matrix of adjusted means from individual environments needs to be approximated by a diagonal matrix. This paper proposes a fully efficient stage‐wise method, which carries forward the full variance–covariance matrix of adjusted means from the individual environments to the analysis across the series of trials. Provided the variance components are known, this method can fully reproduce the results of a single‐stage analysis. Computations are made efficient by a diagonalisation of the residual variance–covariance matrix, which necessitates a corresponding linear transformation of both the first‐stage estimates (e.g. adjusted means and regression slopes for plot covariates) and the corresponding design matrices for fixed and random effects. We also exemplify the extension of the general approach to a three‐stage analysis. The method is illustrated using two datasets, one real and the other simulated. The proposed approach has close connections with meta‐analysis, where environments correspond to centres and genotypes to medical treatments. We therefore compare our theoretical results with recently published results from a meta‐analysis.     

A combined morphological and molecular phylogeny of the genus Chironius Fitzinger, 1826 (Serpentes: Colubridae)

Chironius is one of the most speciose genera of the South American colubrid snakes. Although the genus represents a well‐known radiation of diurnal racers, its monophyly, affinities with other Neotropical colubrid genera, and intrageneric relationships are open questions. Here, we present a phylogenetic analysis of Chironius based on a data matrix that combines one nuclear (c‐mos) and two mitochondrial (12S and 16S rRNA) genes with 37 morphological characters derived from scutellation, skull, and hemipenial features. Phylogenetic relationships were inferred using maximum parsimony (MP) and maximum likelihood (ML). Our combined morphological and molecular analyses strongly support the monophyly of the genus Chironius and its sister‐group relationship with a clade formed by the genera Dendrophidion and Drymobius. Phylogenetic relationships within the genus Chironius is still controversial, although five clades are retrieved with medium to strong support. © 2014 The Linnean Society of London     

Repeated evolution of sympatric,palaeoendemic species in closely related,co‐distributed lineages of Hemiphyllodactylus Bleeker, 1860 (Squamata: Gekkonidae) across a sky‐island archipelago in Peninsular Malaysia

A time‐calibrated phylogenetic tree indicates that the evolution of sympatric, montane, endemic species from closely related, co‐distributed lineages of the Hemiphyllodactylus harterti group were not the result of rapid, forest‐driven, climatic oscillations of the Last Glacial Maximum, but rather the result of infrequent episodes of environmental fluctuation during the Late Miocene. This hypothesis is supported by genetic divergences (based on the mitochondrial gene ND2) between the three major lineages of the H. harterti group (17.5–25.1%), their constituent species (9.4–14.3%), and the evolution of discrete, diagnostic, morphological, and colour pattern characteristics between each species. Sister species pairs from two of the three lineages occur in sympatry on mountain tops from opposite sides of the Thai–Malay Peninsula, but the lineages to which each pair belongs are not sister lineages. A newly discovered species from Gunung Tebu, Terengganu State, H emiphyllodactylus bintik sp. nov. , is described. © 2015 The Linnean Society of London