DNA‐based species delimitation separates highly divergent populations within morphologically coherent clades of poorly dispersing beetles

We compared species‐level entities recovered using distance, tree‐based, and DNA‐character based methods with morphologically defined species in poorly dispersing lyponiine net‐winged beetles. The phylogenetic relationships were investigated using the cytochrome c oxidase 1–2 mtDNA fragment. We identified 31 species using a morphology‐based concept and additional candidate species were delimited by the general mixed Yule‐coalescent method and barcoding threshold within the morphologically coherent lineages owing to high genetic divergence (up to 10.97% within morphologically defined species, highest density 2.5–6.5%). Genetic divergence was positively correlated with geographical distance: lower in continental China where no apparent dispersal barriers are present (r² = 0.172, P ≤ 0.001, average increase of genetic distance of 0.32% per 100 km) and much higher in Japan (r² = 0.490, P ≤ 0.001, 1.81% per 100 km). We hypothesize that low dispersal propensity contributed to the high level of intraspecific, geographically structured divergence. DNA‐based methods suggested a high number of morphologically undistinguishable species. The observed patterns agree with the model of neutral evolution. The poor dispersers produce gradually divergent populations across the range of the morphologically coherent lineages. We pose the question of what size of range and level of genetic difference justify formal acceptance of a species without morphological divergence from both the taxonomical and conservation management view points. © 2015 The Linnean Society of London     

Molecular evolution and SSRs analysis based on the chloroplast genome of Callitropsis funebris

Chloroplast genome sequences have been used to understand evolutionary events and to infer efficiently phylogenetic relationships. Callitropsis funebris (Cupressaceae) is an endemic species in China. Its phylogenetic position is controversial due to morphological characters similar to those of Cupressus, Callitropsis, and Chamaecyparis. This study used next‐generation sequencing technology to sequence the complete chloroplast genome of Ca. funebris and then constructed the phylogenetic relationship between Ca. funebris and its related species based on a variety of data sets and methods. Simple sequence repeats (SSRs) and adaptive evolution analysis were also conducted. Our results showed that the monophyletic branch consisting of Ca. funebris and Cupressus tonkinensis is a sister to Cupressus, while Callitropsis is not monophyletic; Ca. nootkatensis and Ca. vietnamensis are nested in turn at the base of the monophyletic group Hesperocyparis. The statistical results of SSRs supported the closest relationship between Ca. funebris and Cupressus. By performing adaptive evolution analysis under the phylogenetic background of Cupressales, the Branch model detected three genes and the Site model detected 10 genes under positive selection; and the Branch‐Site model uncovered that rpoA has experienced positive selection in the Ca. funebries branch. Molecular analysis from the chloroplast genome highly supported that Ca. funebris is at the base of Cupressus. Of note, SSR features were found to be able to shed some light on phylogenetic relationships. In short, this chloroplast genomic study has provided new insights into the phylogeny of Ca. funebris and revealed multiple chloroplast genes possibly undergoing adaptive evolution.     

Noncoding plastid trnT-trnF sequences reveal a well resolved phylogeny of basal angiosperms

Recent contributions from DNA sequences have revolutionized our concept of systematic relationships in angiosperms. However, parts of the angiosperm tree remain unclear. Previous studies have been based on coding or rDNA regions of relatively conserved genes. A phylogeny for basal angiosperms based on noncoding, fast-evolving sequences of the chloroplast genome region trnT-trnF is presented. The recognition of simple direct repeats allowed a robust alignment. Mutational hot spots appear to be confined to certain sectors, as in two stem-loop regions of the trnL intron secondary structure. Our highly resolved and well-supported phylogeny depicts the New Caledonian Amborella as the sister to all other angiosperms, followed by Nymphaeaceae and an Austrobaileya-Illicium-Schisandra clade. Ceratophyllum is substantiated as a close relative of monocots, as is a monophyletic eumagnoliid clade consisting of Piperales plus Winterales sister to Laurales plus Magnoliales. Possible reasons for the striking congruence between the trnT-trnF based phylogeny and phylogenies generated from combined multi-gene, multi-genome data are discussed.     

Phylogeny and species delimitation of the C‐genome diploid species in Oryza

Abstract The diploid Oryza species with C‐genome type possesses abundant genes useful for rice improvement and provides parental donors of many tetraploid species with the C‐genome (BBCC, CCDD). Despite extensive studies, the phylogenetic relationship among the C‐genome species and the taxonomic status of some taxa remain controversial. In this study, we reconstructed the phylogeny of three diploid species with C‐genome (Oryza officinalis, O. rhizomatis, and O. eichingeri) based on sequences of 68 nuclear single‐copy genes. We obtained a fully resolved phylogenetic tree, clearly indicating the sister relationship of O. officinalis and O. rhizomatis, with O. eichingeri being the more divergent lineage. Incongruent phylogenies of the C‐genome species found in previous studies might result from lineage sorting, introgression/hybridization and limited number of genetic markers used. We further applied a recently developed Bayesian species delimitation method to investigate the species status of the Sri Lankan and African O. eichingeri. Analyses of two datasets (68 genes with a single sample, and 10 genes with multiple samples) support the distinct species status of the Sri Lankan and African O. eichingeri. In addition, we evaluated the impact of the number of sampled individuals and loci on species delimitation. Our simulation suggests that sampling multiple individuals is critically important for species delimitation, particularly for closely related species.     

Rapid and accurate species identification for ecological studies and monitoring using CRISPR‐based SHERLOCK

One of the most fundamental aspects of ecological research and monitoring is accurate species identification, but cryptic speciation and observer error can confound phenotype‐based identification. The CRISPR‐Cas toolkit has facilitated remarkable advances in many scientific disciplines, but the fields of ecology and conservation biology have yet to fully embrace this powerful technology. The recently developed CRISPR‐Cas13a platform SHERLOCK (Specific High‐sensitivity Enzymatic Reporter unLOCKing) enables highly accurate taxonomic identification and has all the characteristics needed to transition to ecological and environmental disciplines. Here we conducted a series of “proof of principle” experiments to characterize SHERLOCK’s ability to accurately, sensitively and rapidly distinguish three fish species of management interest co‐occurring in the San Francisco Estuary that are easily misidentified in the field. We improved SHERLOCK’s ease of field deployment by combining the previously demonstrated rapid isothermal amplification and CRISPR genetic identification with a minimally invasive and extraction‐free DNA collection protocol, as well as the option of instrument‐free lateral flow detection. This approach opens the door for redefining how, where and by whom genetic identifications occur in the future.     

Multi-locus species delimitation in closely related animals and fungi: one marker is not enough

Despite taxonomy’s 250‐year history, the past 20 years have borne witness to remarkable advances in technology and techniques, as well as debate. DNA barcoding has generated a substantial proportion of this debate, with its proposition that a single mitochondrial sequence will consistently identify and delimit species, replacing more evidence‐rich and time‐intensive methods. Although mitochondrial DNA (mtDNA) has since been the focus of voluminous discussion and case studies, little effort has been made to comprehensively evaluate its success in delimiting closely related species. We have conducted the first broadly comparative literature review addressing the efficacy of molecular markers for delimiting such species over a broad taxonomic range. By considering only closely related species, we sought to avoid confusion of success rates with those due to deeply divergent taxa. We also address whether increased population‐level or geographic sampling affects delimitation success. Based on the results from 101 studies, we found that all marker groups had approximately equal success rates (～70%) in delimiting closely related species and that the use of additional loci increased average delimitation success. We also found no relationship between increased sampling of intraspecific variability and delimitation success. Ultimately, our results support a multi‐locus integrative approach to species delimitation and taxonomy.     

Congruent species delimitation of two controversial gold‐thread nanmu tree species based on morphological and restriction site‐associated DNA sequencing data

Species delimitation is fundamental to conservation and sustainable use of economically important forest tree species. However, the delimitation of two highly valued gold‐thread nanmu species (Phoebe bournei (Hemsl.) Yang and P. zhennan S. K. Lee & F. N. Wei) has been confusing and debated. To address this problem, we integrated morphology and restriction site‐associated DNA sequencing (RAD‐seq) to define their species boundaries. We obtained consistent results from the two datasets, supporting two distinct lineages corresponding to P. bournei and P. zhennan. In P. bournei, higher order leaf venation is more prominent, petioles are thicker, and leaf apex angle is narrower, compared to P. zhennan. Both datasets also revealed that the former putative P. bournei populations from northeastern Guizhou belong to P. zhennan. The two species are now distinct in distributions except in the Wuling Mountains, where they overlap. Phoebe bournei occurs mainly in central Fujian, southern Jiangxi, the Nanling Mountains, and the Wuling Mountains, whereas P. zhennan is found in the adjoining eastern regions of the Qionglai Mountains, the southern Sichuan hills, and the Wuling Mountains. The re‐delimitation of P. bournei and P. zhennan and clarification of their ranges provide a better scientific basis guiding the conservation and sustainable utilization of these tree species.     

Design of character‐based DNA barcode motif for species identification: A computational approach and its validation in fishes

The DNA barcodes are generally interpreted using distance‐based and character‐based methods. The former uses clustering of comparable groups, based on the relative genetic distance, while the latter is based on the presence or absence of discrete nucleotide substitutions. The distance‐based approach has a limitation in defining a universal species boundary across the taxa as the rate of mtDNA evolution is not constant throughout the taxa. However, character‐based approach more accurately defines this using a unique set of nucleotide characters. The character‐based analysis of full‐length barcode has some inherent limitations, like sequencing of the full‐length barcode, use of a sparse‐data matrix and lack of a uniform diagnostic position for each group. A short continuous stretch of a fragment can be used to resolve the limitations. Here, we observe that a 154‐bp fragment, from the transversion‐rich domain of 1367 COI barcode sequences can successfully delimit species in the three most diverse orders of freshwater fishes. This fragment is used to design species‐specific barcode motifs for 109 species by the character‐based method, which successfully identifies the correct species using a pattern‐matching program. The motifs also correctly identify geographically isolated population of the Cypriniformes species. Further, this region is validated as a species‐specific mini‐barcode for freshwater fishes by successful PCR amplification and sequencing of the motif (154 bp) using the designed primers. We anticipate that use of such motifs will enhance the diagnostic power of DNA barcode, and the mini‐barcode approach will greatly benefit the field‐based system of rapid species identification.     

Molecular phylogeny of Osmanthus (Oleaceae) based on non‐coding chloroplast and nuclear ribosomal internal transcribed spacer regions

Abstract The phylogenetic relationships of Osmanthus Lour. were investigated using the nuclear ribosomal internal transcribed spacer (ITS) regions and non‐coding chloroplast regions (psbA‐trnH, trnL‐F). The two datasets support the conclusion that Osmanthus is polyphyletic, with some species of the subtribe Oleinae nested within Osmanthus. Osmanthus didymopetalus P. S. Green is nested within the clade formed by species of section Osmanthus in two trees. Osmanthus attenuatus P. S. Green, O. yunnanensis P. S. Green, and O. gracilinervis R. L. Lu of traditional section Osmanthus are clearly divergent from other accessions, and do not form a monophyletic group with other Osmanthus accessions. Osmanthus marginatus Hemsl. is embedded in the clade formed by species of section Osmanthus in the ITS tree. In cpDNA trees all species of section Osmanthus are placed in the large clade and all species of section Leiolea formed a group. The taxonomic incongruence among trees for ITS and cpDNA indicate hybridization, as introgression may have occurred among some species of sections Osmanthus and Leiolea. Phylogeny of Osmanthus is discussed in light of molecular and morphological data, and a revised infrageneric classification with three sections (Leiolea, Siphosmanthu, and Osmanthus) is presented. The section Linocieroides is abandoned and united with section Osmanthus.     

Development of high‐resolution DNA barcodes for Dioscorea species discrimination and phylogenetic analysis

The genus Dioscorea is widely distributed in tropical and subtropical regions, and is economically important in terms of food supply and pharmaceutical applications. However, DNA barcodes are relatively unsuccessful in discriminating between Dioscorea species, with the highest discrimination rate (23.26%) derived from matK sequences. In this study, we compared genic and intergenic regions of three Dioscorea chloroplast genomes and found that the density of SNPs and indels in intergenic sites was about twice and seven times higher than that of SNPs and indels in the genic regions, respectively. A total of 52 primer pairs covering highly variable regions were designed and seven pairs of primers had 80%–100% PCR success rate. PCR amplicons of 73 Dioscorea individuals and assembled sequences of 47 Dioscorea SRAs were used for estimating intraspecific and interspecific divergence for the seven loci: The rpoB‐trnC locus had the highest interspecific divergence. Automatic barcoding gap discovery (ABGD), Poisson tree processes (PTP), and generalized mixed Yule coalescence (GMYC) analysis were applied for species delimitation based on the seven loci and successfully identified the majority of species, except for species in the Enantiophyllum section. Phylogenetic analysis of 51 Dioscorea individuals (28 species) showed that most individuals belonging to the same species tended to cluster in the same group. Our results suggest that the variable loci derived from comparative analysis of plastid genome sequences could be good DNA barcode candidates for taxonomic analysis and species delimitation.     

Towards resolving the systematics of Cerastium subsection Cerastium (Caryophyllaceae): a cytogenetic approach

Analyses of mitotic chromosome numbers and nuclear DNA content were performed for 39 populations of 17 perennial Cerastium taxa from south‐eastern Europe. The DNA content ranged from 2C = 2.43 to 8.78 pg, revealing four ploidy levels corresponding to 4x (2n = 36), 8x, 12x and 16x. High‐polyploid cytotypes with a greater range of ploidy (up to 2n = 144) occur mostly in the central mountainous parts of the Balkan Peninsula. The chromosome number was determined for the first time for C. dinaricum (2n = 36 + 1B), C. decalvans subsp. orbelicum (2n = 36), C. decalvans subsp. glutinosum (2n = 36), C. neoscardicum (2n = 144), C. malyi subsp. serpentini (2n = 144) and C. moesiacum s.s. (2n = 144). New chromosome counts were recorded for C. arvense subsp. strictum (2n = 108), C. banaticum subsp. kosaninii (2n = 36) and C. grandiflorum (2n = 36). For the first time, flow cytometry was used to estimate C values for six species (15 taxonomic entities). The intraspecific variation quotient of C values is high, ranging from 1.003 in C. malyi to 1.306 in C. decalvans subsp. decalvans. The variation in chromosome size among both tetra‐ and octoploid members of Cerastium is much more prominent than in most other angiosperm polyploid series. Significant genome downsizing after polyploidization was observed in some investigated taxa. Differences in ploidy levels and monoploid genome size values confirm the taxonomic status of C. decalvans subsp. glutinosum and C. decalvans subsp. leontopodium. The results obtained indicate a possible close relationship between C. banaticum and C. grandiflorum, but not C. arvense. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 182 , 205–224.     

Multilocus phylogeny and species delimitation within the genus Glauconycteris (Chiroptera,Vespertilionidae), with the description of a new bat species from the Tshopo Province of the Democratic Republic of the Congo

The genus Glauconycteris Dobson, 1875 currently contains 12 species of butterfly bats, all endemic to sub‐Saharan Africa. Most species are rarely recorded, with half of the species known from less than six geographic localities. The taxonomic status of several species remains problematic. Here, we studied the systematics of butterfly bats using both morphological and molecular approaches. We examined 45 adult specimens for external anatomy and skull morphology, and investigated the phylogeny of Glauconycteris using DNA sequences from three mitochondrial genes and 116 individuals, which in addition to outgroup taxa, included nine of the twelve butterfly bat species currently recognized. Four additional nuclear genes were sequenced on a reduced sample of 69 individuals, covering the outgroup and Glauconycteris species. Our molecular results show that the genus Glauconycteris is monophyletic, and that it is the sister‐group of the Asian genus Hesperoptenus. Molecular dating estimates based on either Cytb or RAG2 data sets suggest that the ancestor of Glauconycteris migrated into Africa from Asia during the Tortonian age of the Late Miocene (11.6–7.2 Mya), while the basal diversification of the crown group occurred in Africa at around 6 ± 2 Mya. The species G. superba is found to be the sister‐group of G. variegata, questioning its placement in the recently described genus Niumbaha. The small species living in tropical rainforests constitute a robust clade, which contains three divergent lineages: (i) the “poensis” group, which is composed of G. poensis, G. alboguttata, G. argentata, and G. egeria; (ii) the “beatrix” group, which contains G. beatrix and G. curryae; and (iii) the “humeralis” group, which includes G. humeralis and a new species described herein. In the “poensis” group, G. egeria is found to be monophyletic in the nuclear tree, but polyphyletic in the mitochondrial tree. The reasons for this mito‐nuclear discordance are discussed.     

Identifying footprints of selection in stocked brown trout populations: a spatio‐temporal approach

Studies of interactions between farmed and wild salmonid fishes have suggested reduced fitness of farmed strains in the wild, but evidence for selection at the genic level is lacking. We studied three brown trout populations in Denmark which have been significantly admixed with stocked hatchery trout (19–64%), along with two hatchery strains used for stocking. The wild populations were represented by contemporary samples (2000–2006) and two of them by historical samples (1943–1956). We analysed 61 microsatellite loci, nine of which showed putative functional relationships [expressed sequence tag (EST)‐linked or quantitative trait loci]. F_ST‐based outlier tests provided support for diversifying selection at chromosome regions marked by three loci, two anonymous and one EST‐linked. Patterns of differentiation suggested that the loci were candidates for being under diversifying hitch‐hiking selection in hatchery vs. wild environments. Analysis of hatchery strain admixture proportions showed that in one wild population, two of the loci showed significantly lower admixture proportions than the putatively neutral loci, implying contemporary selection against alleles introduced by hatchery strain trout. In the most strongly admixed population, however, there was no evidence for selection, possibly because of immigration by stocked trout overcoming selection against hatchery‐derived alleles or supportive breeding practices allowing hatchery strain trout to escape natural selection. To our knowledge, this is the first study demonstrating footprints of selection in wild salmonid populations subject to spawning intrusion by farmed fish.     

Molecular phylogeny of the extinct Steller's sea cow and other Sirenia species based on their complete mitochondrial genomes

The Steller's sea cow – Hydrodamalis gigas (Dugongidae: Sirenia) – is an extinct herbivorous marine mammal which inhabited the North Pacific Ocean during the Pleistocene and Holocene. H. gigas was the largest member of the Sirenia order and disappeared in the middle of the 18th century. Here, we present the complete sequence of the mitochondrial genome of this extinct animal. The Steller's sea cow mitochondrial DNA (mtDNA) is 16,872 base pairs (bp) in length and contains a set of mitochondrial genes typical for mammals. Phylogenetic analysis based on complete mitochondrial genomes of the sirenian species allows accurate assessment of the degree of their mitogenomic diversification during millions of years of evolution.     

A molecular phylogeny of historical and contemporary specimens of an under‐studied micro‐invertebrate group

Resolution of relationships at lower taxonomic levels is crucial for answering many evolutionary questions, and as such, sufficiently varied species representation is vital. This latter goal is not always achievable with relatively fresh samples. To alleviate the difficulties in procuring rarer taxa, we have seen increasing utilization of historical specimens in building molecular phylogenies using high throughput sequencing. This effort, however, has mainly focused on large‐bodied or well‐studied groups, with small‐bodied and under‐studied taxa under‐prioritized. Here, we utilize both historical and contemporary specimens, to increase the resolution of phylogenetic relationships among a group of under‐studied and small‐bodied metazoans, namely, cheilostome bryozoans. In this study, we pioneer the sequencing of air‐dried cheilostomes, utilizing a recently developed library preparation method for low DNA input. We evaluate a de novo mitogenome assembly and two iterative methods, using the sequenced target specimen as a reference for mapping, for our sequences. In doing so, we present mitochondrial and ribosomal RNA sequences of 43 cheilostomes representing 37 species, including 14 from historical samples ranging from 50 to 149 years old. The inferred phylogenetic relationships of these samples, analyzed together with publicly available sequence data, are shown in a statistically well‐supported 65 taxa and 17 genes cheilostome tree, which is also the most broadly sampled and largest to date. The robust phylogenetic placement of historical samples whose contemporary conspecifics and/or congenerics have been sequenced verifies the appropriateness of our workflow and gives confidence in the phylogenetic placement of those historical samples for which there are no close relatives sequenced. The success of our workflow is highlighted by the circularization of a total of 27 mitogenomes, seven from historical cheilostome samples. Our study highlights the potential of utilizing DNA from micro‐invertebrate specimens stored in natural history collections for resolving phylogenetic relationships among species.