Molecular phylogeny and species delimitation of Stachyuraceae: Advocating a herbarium specimen‐based phylogenomic approach in resolving species boundaries

Species concept and delimitation are fundamental to taxonomic and evolutionary studies. Both inadequate informative sites in the molecular data and limited taxon sampling have often led to poor phylogenetic resolution and incorrect species delineation. Recently, the whole chloroplast genome sequences from extensive herbarium specimen samples have been shown to be effective to amend the problem. Stachyuraceae are a small family consisting of only one genus Stachyurus of six to 16 species. However, species delimitation in Stachyurus has been highly controversial because of few and generally unstable morphological characters used for classification. In this study, we sampled 69 individuals of seven species (each with at least three individuals) covering the entire taxonomic diversity, geographic range, and morphological variation of Stachyurus from herbarium specimens for genome‐wide plastid gene sequencing to address species delineation in the genus. We obtained high‐quality DNAs from specimens using a recently developed DNA reconstruction technique. We first assembled four whole chloroplast genome sequences. Based on the chloroplast genome and one nuclear ribosomal DNA sequence of Stachyurus, we designed primers for multiplex polymerase chain reaction and high throughput sequencing of 44 plastid loci for species of Stachyurus. Data of these chloroplast DNA and nuclear ribosomal DNA internal transcribed spacer sequences were used for phylogenetic analyses. The phylogenetic results showed that the Japanese species Stachyurus praecox Siebold & Zucc. was sister to the rest in mainland China, which indicated a typical Sino‐Japanese distribution pattern. Based on diagnostic morphological characters, distinct distributional range, and monophyly of each clade, we redefined seven species for Stachyurus following an integrative species concept, and revised the taxonomy of the family based on previous reports and specimens, in particular the type specimens. Furthermore, our divergence time estimation results suggested that Stachyuraceae split from its sister group Crossosomataceae from the New World at ca. 54.29 Mya, but extant species of Stachyuraceae started their diversification only recently at ca. 6.85 Mya. Diversification time of Stachyurus in mainland China was estimated to be ca. 4.45 Mya. This research has provided an example of using the herbarium specimen‐based phylogenomic approach in resolving species boundaries in a taxonomically difficult genus.     

Historical herbarium specimens in plant molecular systematics — an example from the fern genus <Emphasis Type="Italic">Lindsaea</Emphasis> (Lindsaeaceae)

We extracted, amplified and sequenced DNA from historical herbarium specimens and silica-dried samples of the fern genus Lindsaea in order to study the sequencing success between the two kinds of samples. High quality sequences were obtained from 57% of the herbarium samples. The specimens age was found to be of little importance for sequencing success when less than 75 years, but the colour of a specimen was found more indicative of sequencing success. Shorter DNA fragments were sequenced successfully twice as often as longer fragments from the herbarium material; in relatively recently collected silica-dried material longer sequences were obtained almost as frequently as short ones. No obvious differences in sequencing success between material originating from different herbaria was observed. We conclude that by using specifically designed DNA extraction protocols and by sequencing short DNA fragments from carefully selected specimens, herbarium material and type specimens can be successfully used in molecular systematics. Typical material or specimens from the type locality (topotypes) should be preferred, when placing a species in a phylogeny.     

Metabarcoding of fungal communities associated with bark beetles

Many species of fungi are closely allied with bark beetles, including many tree pathogens, but their species richness and patterns of distribution remain largely unknown. We established a protocol for metabarcoding of fungal communities directly from total genomic DNA extracted from individual beetles, showing that the ITS3/4 primer pair selectively amplifies the fungal ITS. Using three specimens of bark beetle from different species, we assess the fungal diversity associated with these specimens and the repeatability of these estimates in PCRs conducted with different primer tags. The combined replicates produced 727 fungal Operational Taxonomic Units (OTUs) for the specimen of Hylastes ater, 435 OTUs for Tomicus piniperda, and 294 OTUs for Trypodendron lineatum, while individual PCR reactions produced on average only 229, 54, and 31 OTUs for the three specimens, respectively. Yet, communities from PCR replicates were very similar in pairwise comparisons, in particular when considering species abundance, but differed greatly among the three beetle specimens. Different primer tags or the inclusion of amplicons in separate libraries did not impact the species composition. The ITS2 sequences were identified with the Lowest Common Ancestor approach and correspond to diverse lineages of fungi, including Ophiostomaceae and Leotiomycetes widely found to be tree pathogens. We conclude that Illumina MiSeq metabarcoding reliably captures fungal diversity associated with bark beetles, although numerous PCR replicates are recommended for an exhaustive sample. Direct PCR from beetle DNA extractions provides a rapid method for future surveys of fungal species diversity and their associations with bark beetles and environmental variables.     

Integrative taxonomy at work: DNA barcoding of taeniids harboured by wild and domestic cats

In modern taxonomy, DNA barcoding is particularly useful where biometric parameters are difficult to determine or useless owing to the poor quality of samples. These situations are frequent in parasitology. Here, we present an integrated study, based on both DNA barcoding and morphological analysis, on cestodes belonging to the genus Taenia, for which biodiversity is still largely underestimated. In particular, we characterized cestodes from Italian wildcats (Felis silvestris silvestris), free‐ranging domestic cats (Felis silvestris catus) and hybrids populations. Adult taeniids were collected by post‐mortem examinations of the hosts and morphologically identified as Taenia taeniaeformis. We produced cox1 barcode sequences for all the analysed specimens, and we compared them with reference sequences of individuals belonging to the genus Taenia retrieved from GenBank. In order to evaluate the performance of a DNA barcoding approach to discriminate these parasites, the strength of correlation between species identification based on classical morphology and the molecular divergence of cox1 sequences was measured. Our study provides clear evidence that DNA barcoding is highly efficient to reveal the presence of cryptic lineages within already‐described taeniid species. Indeed, we detected three well‐defined molecular lineages within the whole panel of specimens morphologically identified as T. taeniaeformis. Two of these molecular groups were already identified by other authors and should be ranked at species level. The third molecular group encompasses only samples collected in Italy during this study, and it represents a third candidate species, still morphologically undescribed.     

Next‐generation DNA barcoding: using next‐generation sequencing to enhance and accelerate DNA barcode capture from single specimens

DNA barcoding is an efficient method to identify specimens and to detect undescribed/cryptic species. Sanger sequencing of individual specimens is the standard approach in generating large‐scale DNA barcode libraries and identifying unknowns. However, the Sanger sequencing technology is, in some respects, inferior to next‐generation sequencers, which are capable of producing millions of sequence reads simultaneously. Additionally, direct Sanger sequencing of DNA barcode amplicons, as practiced in most DNA barcoding procedures, is hampered by the need for relatively high‐target amplicon yield, coamplification of nuclear mitochondrial pseudogenes, confusion with sequences from intracellular endosymbiotic bacteria (e.g. Wolbachia) and instances of intraindividual variability (i.e. heteroplasmy). Any of these situations can lead to failed Sanger sequencing attempts or ambiguity of the generated DNA barcodes. Here, we demonstrate the potential application of next‐generation sequencing platforms for parallel acquisition of DNA barcode sequences from hundreds of specimens simultaneously. To facilitate retrieval of sequences obtained from individual specimens, we tag individual specimens during PCR amplification using unique 10‐mer oligonucleotides attached to DNA barcoding PCR primers. We employ 454 pyrosequencing to recover full‐length DNA barcodes of 190 specimens using 12.5% capacity of a 454 sequencing run (i.e. two lanes of a 16 lane run). We obtained an average of 143 sequence reads for each individual specimen. The sequences produced are full‐length DNA barcodes for all but one of the included specimens. In a subset of samples, we also detected Wolbachia, nontarget species, and heteroplasmic sequences. Next‐generation sequencing is of great value because of its protocol simplicity, greatly reduced cost per barcode read, faster throughout and added information content.     

Molecular phylogeny and biogeography of Honey‐buzzards (genera Pernis and Henicopernis)

A partial sequence of the cytb gene (382 bp) was amplified and sequenced from 35 individuals (mainly museum specimens) of the genus Pernis representing all valid taxa (10) and two taxa (P. p. gurneyi, P. p. japonicus) with questionable validity as well as representatives of the Old World Perninae, namely Henicopernis and Aviceda, to assess their relationships to the genus Pernis. Furthermore, Gypaetus barbatus, Neophron percnopterus, and Buteo buteo were included as outgroup taxa. In the trees derived from the sequence data, Aviceda represents the sister group of the genus Pernis. The genus Henicopernis and the Old World vultures Gypaetus andNeophron appear rather distantly related to Pernis. Within the genus Pernis, two of the described species (Pernis apivorus, Pernis ptilorhyncus) form monophyletic groups, whereas the relationships of the two clades representing three subspecies of Pernis celebensis are still uncertain. Although this study is based on comparatively short DNA‐sections, the trees deduced from these sequences can be considered as a first approach for inferring the phylogenetic relationships of the genus Pernis and related genera and for addressing questions concerning the evolutionary history, biogeography, and systematics of this group.     

Pollen beetles are consumed by ground‐ and foliage‐dwelling spiders in winter oilseed rape

Pollen beetles, Meligethes aeneus (Fabricius) (Coleoptera: Nitidulidae), are major pests in oilseed rape (OSR), Brassica napus L. (Brassicaceae). Among the predator species in the generalist predator complex present in OSR fields, wolf spiders (Araneae: Lycosidae) are found on the ground and cobweb spiders (Araneae: Theridiidae) build webs in the foliage. Here we study the incidence of predation of pollen beetles by these two spider groups using DNA‐based molecular analysis. Wolf spiders of the genus Pardosa and the cobweb spider, Theridion impressum L. Koch, were each collected in three winter OSR fields over a period of about 3 weeks. Pollen beetle densities as well as the occurrence of predators and alternative prey were monitored. In total, 13.8% of the collected Pardosa spp. tested positive for pollen beetle DNA in the PCR analyses, whereas 51.7%T. impressum were positive. The likelihood of detecting pollen beetle DNA in the gut contents of both spider groups was positively related to pollen beetle larval density. The implications of these results for conservation biological control and future studies of food webs in OSR are discussed.     

Characterizing DNA preservation in degraded specimens of Amara alpina (Carabidae: Coleoptera)

DNA preserved in degraded beetle (Coleoptera) specimens, including those derived from dry‐stored museum and ancient permafrost‐preserved environments, could provide a valuable resource for researchers interested in species and population histories over timescales from decades to millenia. However, the potential of these samples as genetic resources is currently unassessed. Here, using Sanger and Illumina shotgun sequence data, we explored DNA preservation in specimens of the ground beetle Amara alpina, from both museum and ancient environments. Nearly all museum specimens had amplifiable DNA, with the maximum amplifiable fragment length decreasing with age. Amplification of DNA was only possible in 45% of ancient specimens. Preserved mitochondrial DNA fragments were significantly longer than those of nuclear DNA in both museum and ancient specimens. Metagenomic characterization of extracted DNA demonstrated that parasite‐derived sequences, including Wolbachia and Spiroplasma, are recoverable from museum beetle specimens. Ancient DNA extracts contained beetle DNA in amounts comparable to museum specimens. Overall, our data demonstrate that there is great potential for both museum and ancient specimens of beetles in future genetic studies, and we see no reason why this would not be the case for other orders of insect.     

Morphology and biology of the flower‐visiting water scavenger beetle genus Rygmodus (Coleoptera: Hydrophilidae)

Hydrophilidae (water scavenger beetles) is well known as an aquatic beetle family; however, it contains ca. 1,000 secondarily terrestrial species derived from aquatic ancestors. The New Zealand endemic genus Rygmodus White is a member of the hydrophilid subfamily Cylominae, which is the early‐diverging taxon of the largest terrestrial lineage (Cylominae + Sphaeridiinae) within the Hydrophilidae. In this paper we report that Rygmodus beetles are pollen‐feeding flower visitors as adults, but aquatic predators as larvae. Based on analyses of gut contents and a summary of collecting records reported on museum specimen labels, adult Rygmodus beetles are generalists feeding on pollen of at least 13 plant families. Rygmodus adult mouthparts differ from those of other (saprophagous) hydrophilid beetles in having the simple scoop‐like apex and mola with roughly denticulate surface, resembling the morphology found in pollen‐feeding staphylinid beetles. Larvae were found along the sides of streams, under stones and in algal mats and water‐soaked moss; one collected larval specimen was identified using DNA barcoding of two molecular markers, mitochondrial cytochrome oxidase 1 (cox1) and nuclear histone 3 (H3). Larvae of two species, Rygmodus modestus and Rygmodus sp., are described in detail and illustrated; they closely resemble ambush‐type predatory larvae of the hydrophilid tribe Hydrophilini in the head morphology. Rygmodus is the only known hydrophilid beetle with adults and larvae inhabiting different environments.     

Characterization of <Emphasis Type="Italic">Penicillium</Emphasis> Species by Ribosomal DNA Sequencing and BOX,ERIC and REP-PCR Analysis

The genus Penicillium is one of the largest and widely distributed fungal genera described to date. As a result, its taxonomic classification and species discrimination within this genus has become complicated. In this study, 52 isolates that belonged to the Penicillum genus and other related genera were characterized using two DNA-based methods: (i) analysis of the nucleotide sequences of internal transcribed spacers in ribosomal DNA and (ii) analysis of DNA fingerprints that were generated by polymerase chain reactions with specific primers for enterobacterial repetitive intergenic consensus (ERIC) and repetitive extragenic palindromic (REP) sequences, and BOX elements. Using both methods, Penicillium species were discriminated from other fungal genera. Furthermore, Penicillium species that include strains which are used as biocontrol agents, such as P. glabrum, P. purpurogenum, and P. oxalicum, could be distinguished from other Penicillium species using these techniques. Based on our findings, we propose that a polyphasic approach that includes analysis of the nucleotide sequences of ribosomal DNA and detecting the presence of highly conserved, repeated nucleotide sequences can be used to determine the genetic relationships between different Penicillium species. Furthermore, we propose that our results can be used as a start point to develop a strategy to monitor the environmental presence of particular strains of Penicillium species when they are used as biocontrol agents.     

A large‐scale molecular survey of Clinostomum (Digenea,Clinostomidae)

Members of the genus Clinostomum Leidy, 1856 are parasites that mature in birds, with occasional reports in humans. Because morphological characters for reliable discrimination of species are lacking, the number of species considered valid has varied by an order of magnitude. In this study, sequences from the DNA barcode region of cytochrome c oxidase I (CO1) and/or internal transcribed spacer (ITS) from specimens from Mexico, Bolivia, Peru, Brazil, Kenya, China and Thailand were analysed together with published sequences from Europe, Africa, Indonesia and North America. Although ITS and CO1 distances among specimens were strongly correlated, distance‐based analysis of each marker yielded different groups. Putative species indicated by CO1 distances were consistent with available morphological identifications, while those indicated by ITS conflicted with morphological identifications in three cases. There was little overlap in sequence variation within and between species, particularly for CO1. Although ITS and CO1 distances tended to increase in specimens that were further apart geographically, this did not impair distance‐based species delineation. Phylogenetic analysis suggests a deep division between clades of Clinostomum inhabiting the New World and Old World, which parallels the distribution of their principal definitive hosts, the Ardeidae.     

DNA barcodes from century‐old type specimens using next‐generation sequencing

Type specimens have high scientific importance because they provide the only certain connection between the application of a Linnean name and a physical specimen. Many other individuals may have been identified as a particular species, but their linkage to the taxon concept is inferential. Because type specimens are often more than a century old and have experienced conditions unfavourable for DNA preservation, success in sequence recovery has been uncertain. This study addresses this challenge by employing next‐generation sequencing (NGS) to recover sequences for the barcode region of the cytochrome c oxidase 1 gene from small amounts of template DNA. DNA quality was first screened in more than 1800 century‐old type specimens of Lepidoptera by attempting to recover 164‐bp and 94‐bp reads via Sanger sequencing. This analysis permitted the assignment of each specimen to one of three DNA quality categories – high (164‐bp sequence), medium (94‐bp sequence) or low (no sequence). Ten specimens from each category were subsequently analysed via a PCR‐based NGS protocol requiring very little template DNA. It recovered sequence information from all specimens with average read lengths ranging from 458 bp to 610 bp for the three DNA categories. By sequencing ten specimens in each NGS run, costs were similar to Sanger analysis. Future increases in the number of specimens processed in each run promise substantial reductions in cost, making it possible to anticipate a future where barcode sequences are available from most type specimens.     

Nonlethal sampling of DNA from critically endangered diving beetles (Coleoptera: Dytiscidae) using a single antenna

Nonlethal DNA sampling is highly desirable in molecular genetic studies of protected and endangered species. To develop a demonstrably nonlethal method of obtaining DNA from endangered diving beetles (Dytiscus sharpi sharpi Wehncke, Cybister lewisianus Sharp and Cybister brevis Aubé), we amputated the antennae of these endangered diving beetles and investigated the impact of the amputation on reproductive behaviors, egg‐laying and lifespan. Diving beetles with either one or no antennae copulated without delay and laid eggs, comparable to the pairs of intact beetles under breeding conditions. The lifespan of antennae‐amputated D. sharpi sharpi was the same as that of the intact beetles. A single antenna was sufficient to allow polymerase chain reaction (PCR) detection of a mitochondrial DNA gene, cytochrome‐c oxidase subunit I (COI), and the sequence of the COI gene could be determined directly. The PCR‐ready genomic DNA was available both in fresh antennae isolated from living beetles and in old antennae from whole beetles preserved for at least 5–6 years in pure ethanol. These results suggest that an antenna is a good sampling site for isolating genomic DNA from endangered diving beetles without sacrificing and disturbing reproductive behaviors such as mating and egg‐laying, or lifespan.     

DNA sequencing,anatomy, and calcification patterns support a monophyletic,subarctic, carbonate reef‐forming Clathromorphum (Hapalidiaceae,Corallinales, Rhodophyta)

For the first time, morpho‐anatomical characters that were congruent with DNA sequence data were used to characterize several genera in Hapalidiaceae—the major eco‐engineers of Subarctic carbonate ecosystems. DNA sequencing of three genes (SSU, rbcL, ribulose‐1, 5‐bisphosphate carboxylase/oxygenase large subunit gene and psbA, photosystem II D1 protein gene), along with patterns of cell division, cell elongation, and calcification supported a monophyletic Clathromorphum. Two characters were diagnostic for this genus: (i) cell division, elongation, and primary calcification occurred only in intercalary meristematic cells and in a narrow vertical band (1–2 μm wide) resulting in a “meristem split” and (ii) a secondary calcification of interfilament crystals was also produced. Neopolyporolithon was resurrected for N. reclinatum, the generitype, and Clathromorphum loculosum was transferred to this genus. Like Clathromorphum, cell division, elongation, and calcification occurred only in intercalary meristematic cells, but in a wider vertical band (over 10–20 μm), and a “meristem split” was absent. Callilithophytum gen. nov. was proposed to accommodate Clathromorphum parcum, the obligate epiphyte of the northeast Pacific endemic geniculate coralline, Calliarthron. Diagnostic for this genus were epithallial cells terminating all cell filaments (no dorsi‐ventrality was present), and a distinct “foot” was embedded in the host. Leptophytum, based on its generitype, L. laeve, was shown to be a distinct genus more closely related to Clathromorphum than to Phymatolithon. All names of treated species were applied unequivocally by linking partial rbcL sequences from holotype, isotype, or epitype specimens with field‐collected material. Variation in rbcL and psbA sequences suggested that multiple species may be passing under each currently recognized species of Clathromorphum and Neopolyporolithon.     

Resurrection of Mesoplodon hotaula Deraniyagala 1963: A new species of beaked whale in the tropical Indo‐Pacific

We present genetic and morphological evidence supporting the recognition of a previously synonymized species of Mesoplodon beaked whale in the tropical Indo‐Pacific, Mesoplodon hotaula. Although the new species is closely‐related to the rare ginkgo‐toothed beaked whale M. ginkgodens, we show that these two lineages can be differentiated by maternally (mitochondrial DNA), biparentally (autosomal), and paternally (Y chromosome) inherited DNA sequences, as well as by morphological features. The reciprocal monophyly of the mtDNA genealogies and the largely parapatric distribution of these lineages is consistent with reproductive isolation. The new lineage is currently known from at least seven specimens: Sri Lanka (1), Gilbert Islands, Republic of Kiribati (1+), Palmyra Atoll, Northern Line Islands, U.S.A. (3), Maldives (1), and Seychelles (1). The type specimen (Sri Lanka) was described as a new species, M. hotaula, in 1963, but later synonymized with M. ginkgodens. This discovery brings the total number of Mesoplodon species to 15, making it, by far, the most speciose yet least known genus of cetaceans.     

Characterization of freshwater benthic biofilm‐forming Hydrocoryne (Cyanobacteria) isolates from Antarctica

The aims of this work were to study cyanobacterial isolates resembling the genus Hydrocoryne using a combination of morphology and phylogeny of 16S rRNA and nifH sequences and to investigate genes involved in cyanotoxin and protease inhibitor production. Four new cyanobacterial strains, isolated from biofilm samples collected from King George Island, Antarctica, were studied. In terms of morphology, these new strains share traits similar to true Anabaena morphotypes (benthic ones), whereas phylogenetic analysis of their 16S rRNA gene sequences grouped them with the sequence of the type species Hydrocoryne spongiosa (H. Schwabe ex Bornet and Flahault 1886–1888), but not with sequences of the type species from the genus Anabaena. This cluster is the sister group of Anabaena morphotypes isolated only from the Gulf of Finland. In addition, this cluster is related to two other clusters formed by sequences of Anabaena isolated from different sites. Partial nifH genes were sequenced from two strains and the phylogenetic tree revealed that the Antarctic nifH sequences clustered with sequences from Anabaena. Furthermore, two strains were tested, using PCR with specific primers, for the presence of genes involved in cyanotoxins (microcystin and saxitoxin) and protease inhibitor (aeruginosin, and cyanopeptolin). Only cyanopeptolin was amplified using PCR. These four Hydrocoryne strains are the first to be isolated and sequenced from Antarctica, which improves our knowledge on this poorly defined cyanobacterial genus.     

Molecular identification and first documentation of seven species of Carpophilus Stephens (Nitidulidae: Carpophilinae) in oil palm ecosystem,Peninsular Malaysia

Sap beetles are commonly found in many agricultural systems worldwide. In Malaysia, sap beetles are less documented despite their abundance in oil palm plantations. The similarities in the morphology of the sap beetle makes the identification process difficult from the same genus. This study aims to determine of composition of the sap beetle species from the genus Carpophilus, to identify the species of sap beetles and to document the DNA barcode for those species collected from the oil palm plantation, which has never been done before. DNA of seven species of sap beetles within the subfamily Carpophilinae have been successfully amplified using cytochrome oxidase subunit I marker (COI) namely C. hemipterus (Linnaeus), C. maculatus Murray, C. mutilatus Erichson, C. marginellus Motschulsky, C. sp. 1, C. nepos Murray and C. obsoletus Erichson represented 21, 12, 33, 8, 9, 6 and 11%, respectively from the total number collected. The sequences were analysed and a tree was constructed based on Neighbor-Joining (NJ) analysis with Meligethes thalassophilus chosen as an outgroup. All of the samples showed 90–100% similarities in BOLD and BLAST analyses. NJ trees constructed show that each sap beetle species collected from the study site located at their specific lineages on the tree. Interestingly, the DNA barcoding described in this study is the first to document barcoding data of sap beetles genus Carpophilus in Malaysia and this can be a helpful tool in updating the taxonomic and molecular status of the sap beetles in Malaysia.     

Evaluation of the use of <Emphasis Type="Italic">recN</Emphasis> sequence analysis in the phylogeny of the genus <Emphasis Type="Italic">Amycolatopsis</Emphasis>

Partial recN gene sequences (>1 kb) were obtained from 35 type strains of the genus Amycolatopsis. Phylogenetic trees were constructed to determine the effectiveness of using this gene to predict taxonomic relationships within the genus. The use of recN sequence analysis as an alternative to DNA–DNA hybridization (DDH) for distinguishing closely related species was also assessed. The recN based phylogeny mostly confirmed the conventional 16S rRNA and gyrB gene-based phylogenies and thus provides further support for these phylogenetic groupings. As is the case for the gyrB gene, pairwise recN sequence similarities cannot be used to predict the DNA relatedness between type strains but the recN genetic distance can be used as a means to assess quickly whether an isolate is likely to represent a new species in the genus Amycolatopsis. A recN genetic distance of >0.04 between two Amycolatopsis strains is proposed to provide a good indication that they belong to different species (and that polyphasic taxonomic characterization of the unknown strain is worth undertaking).