New Antarctic holothurians (Echinodermata) – II. Four species of the orders Aspidochirotida, Elasipodida and Apodida

Four new species of antarctic holothurians are described: Bathyplotes rubipunctatus sp.n. Bathyplotes fuscivinculum sp.n. (both Aspidochirotida), Achlyonice violaecuspidata sp.n. (Elasi-podida) and Paradota weddellensis sp.n. (Apodida). The genus, Bathyplotes , is redefined. The material was mainly collected from the continental shelf of the southern and eastern Weddell Sea, at depths between 225 and 840 m. The systematic status of these new species within the genera, as well as relationships to other species and higher taxa are discussed.     

A study of the genetic variability of Rhizoplaca chrysoleuca using DNA sequences and secondary metabolic substances

Specimens of Rhizoplaca chrysoleuca from Mount Wuling can be divided into two distinct groups based on obvious differences in morphological characters. Here we investigated 26 specimens of R. chrysoleuca from Mount Wuling, 10 specimens of this species from other areas and seven specimens of other Rhizoplaca species by analyzing morphology, chemistry and genetics. Nine chemotypes were detected among the specimens of R. chrysoleuca from Mount Wuling, and five of them were reported for the first time. Based on the ITS phylogenetic analysis, the chemotypes and the insertion distribution patterns in SSU rDNA, the samples of R. chrysoleuca from Mount Wuling were grouped in two distinct clades corresponding to two phenotypic groups and no gene flow was detected between these two groups. Our results establish all individuals of Rhizoplaca chrysoleuca are conspecific although some populations have been isolated on Mount Wuling, indicating that they are in the process of speciation. Our study also reveals that the relationships between genotypes and chemotypes are complicated and should be avoided, and we instead recommend using single individuals or few individuals from the same site to represent the population or whole species in systematics study. The results also indicate that Rhizoplaca chrysoleuca might provide a good model for studying the speciation of saxicolous lichenized fungi.     

DNA-based taxonomy for associating adults and larvae in multi-species assemblages of chafers (Coleoptera: Scarabaeidae)

DNA sequences provide a universal character system in taxonomy for associating all developmental stages of organisms, but ambiguity arises due to genetic variation within species. The problem is compounded where target groups are less well studied or incompletely represented in DNA databases. Here we investigate the utility of DNA for larval-adult species associations within chafer (Coleoptera: Scarabaeidae) communities from four sites in the tropical lowlands of Nepal. We sequenced ca. 1600 bp of mitochondrial cox1 and rrnL and 700 bp of nuclear 28S rRNA from 250 larval and adult specimens. Individuals were grouped into putative species using statistical parsimony analysis and population aggregation analysis (PAA), whereby specimens from each locality were grouped according to the presence of diagnostic nucleotides. In addition, species membership was determined based on shifts in branching rates on clock-constrained trees to detect the putative transition from speciation to population coalescence patterns. Using these two methods we delineated between 48 and 56 groups, of which 16-20 were composed of larval and adult individuals. Nuclear and mtDNA-based groups were highly congruent; variation of 28S rRNA within groups was very low, while one widespread 28S rRNA genotype was universally found in a paraphyletic group of five mtDNA clusters. Linnean names could be assigned to 19 groups, and hence between 86.1% and 92.7% of larval specimens could be associated to species by their membership in clearly delineated groups that contained fully identified adults. The remaining larvae were delineated as five species, four of which could be assigned to Anomala or Adoretus based on their phylogenetic position. We conclude that the sequence variation was highly structured in this complex assemblage of chafers and that any given individual (larva or adult) can be readily associated with a particular DNA group using the criterion of diagnos ability. The association of different developmental stages therefore becomes a matter of determining the extent of the DNA-based groups, rather than matching of sequences from adult and larval individuals. This indicates the need for a purely sequence-based taxonomic system when associating different life stages via DNA.     

Sympatric Convergence of the Color Pattern in the Chilean Ceroglossus Ground Beetles Inferred from Sequence Comparisons of the Mitochondrial ND5 Gene

The phylogenetic trees have been constructed using the mitochondrial ND5 gene sequences for 66 specimens of the Chilean Ceroglossus ground beetles collected from various localities of Chile. The trees show that the Ceroglossus specimens examined are composed of four phylogenetic lineages that have diverged 25-30 MYA. The results are consistent with the classification proposed by Jiroux (1996) based on morphology, in which Ceroglossus is divided into four species groups. Despite a remarkable color polymorphism revealed by these ground beetles, the color pattern is geographically linked and is not species-specific, suggesting that some ecological or environmental factors are involved in determining it.     

Identification of sawflies and horntails (Hymenoptera, ‘Symphyta’) through DNA barcodes: successes and caveats

The ‘Symphyta’ is a paraphyletic assemblage at the base of the order Hymenoptera, comprising 14 families and about 8750 species. All have phytophagous larvae, except for the Orussidae, which are parasitoids. This study presents and evaluates the results of DNA barcoding of approximately 5360 specimens of ‘Symphyta’, mainly adults, and 4362 sequences covering 1037 species were deemed of suitable quality for inclusion in the analysis. All extant families are represented, except for the Anaxyelidae. The majority of species and specimens are from Europe, but approximately 38% of the species and 13% of the specimens are of non‐European origin. The utility of barcoding for species identification and taxonomy of ‘Symphyta’ is discussed on the basis of examples from each of the included families. A significant level of cryptic species diversity was apparent in many groups. Other attractive applications include the identification of immature stages without the need to rear them, community analyses based on metabarcoding of bulk samples and association of the sexes of adults.     

A revision of the Encarsia longifasciata species group (Hymenoptera: Aphelinidae)

Abstract.  The Encarsia longifasciata group is established and revised, and its relationship to other species groups of Encarsia is discussed with reference to biological, morphological and molecular evidence. Principal component analysis on all available specimens provided supporting evidence for the existence of five species, two of which are described herein as new: E. dewa Pedata & Polaszek, sp.n and E. prinslooi Pedata & Polaszek, sp.n. All species are either diagnosed or described, and illustrated, and information is given on their distribution and host range. Additional information on the biology and larval stages, and scanning electron micrographs of morphological details, are provided for E. arabica Hayat. A preliminary phylogenetic study using 28S D2 rDNA sequence data for E. arabica was inconclusive, but suggested possible affinities between the longifasciata and parvella species groups. An illustrated dichotomous key to all species of the E. longifasciata group is given.     

Morphological and molecular phylogenetic analysis of <Emphasis Type="Italic">Melampsora</Emphasis> species on poplars in China

Many species of Melampsora on Populus have been reported in China, based on morphological characteristics of both uredial and telial states, and on host species, but their morphology and taxonomy are still poorly defined. In this study, 196 specimens representing Melampsora species on poplars and collected from various areas of China were used for morphological observations. The morphological characteristics of urediniospores and teliospores were examined with light and scanning electron microscopy. The specimens could be classified into five groups based on their morphology. For the sequencing of the nuclear large subunit rDNA (D1/D2), 5.8S rDNA and their internal transcribed spacers, ITS1 and ITS2 region, 54 specimens were selected from the specimens used in morphological observations. These specimens were separated into six clades by phylogenetic analyses of the D1/D2 and ITS regions. Correlations among morphological groups and phylogenetic clades based on these results suggest a revision of these species. In particular, no evidence to discriminate specimens of M. acedioides, M. magnusiana, and M. rostrupii was found from either morphological characteristics or sequence analysis.Contribution no. 185 Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan     

木瓜榕上昆虫群落结构及分布特征

对西双版纳热带雨林木瓜榕上的昆虫群落结构和分布特征进行了研究。结果表明：样地内共采集到木瓜榕树上昆虫48974号,隶属9个目,30个科,56属,70种。主要是鞘翅目、鳞翅目、膜翅目、同翅目、双翅目、等翅目、半翅目、脉翅目和缨翅目的科、属和种类。属和种以鞘翅目和膜翅目类群最丰富;个体数量是膜翅目最多, 高达68.79%, 其中56.58%是传粉类昆虫。从垂直分布规律看：种类和数量是老茎结果部位最高,占71.34%, 在青果期和熟果期表现最丰富;树冠与叶片部位次之,比率为15.68%;根部再次之(9.55%);枝杈和主干最低, 仅占3.42%。在不同季节中,雨季的种类和个体数量最高,为35.96%;干热季节次之(34.22%),干凉季节最低(29.82%)。     

The Identity of Specimens of the Anastrepha fraterculus Complex (Diptera, Tephritidae) with Atypical Aculeus Tip

Several specimens collected in Paraguay along with Anastrepha fraterculus (sensu lato) have an aculeus tip similar to species from the fraterculus complex, but the teeth of the aculeus of these specimens are poorly defined. As Anastrepha species identification is based mostly on subtle differences in the aculeus tip, we studied these specimens with atypical aculeus tips (with poorly defined teeth) that slightly differs from the aculeus tip of species of the fraterculus complex (with well-developed blunt teeth), to determine if this is due to intraspecific variation or if it can characterize a full species. The Paraguayan specimens were separated in six groups under stereomicroscope according to variation in their aculeus tip. Specimens within each group were studied by means of morphometrics (traditional and geometric) and gene sequence analysis (COI and ITS1). Morphometric analyses were significant, but no clear groups were formed by the discriminant analyses of the aculeus and wing, and the COI and ITS1 sequence analysis clustered specimens with all six aculeus variations. Therefore, the subtle morphological differences observed in the aculeus tip of Paraguayan specimens are intraspecific variations and the Paraguayan specimens were more genetically closely related to Anastrepha sp. 3 from the fraterculus complex.     

中国伊洛瓦底江和怒江褶鮡属鱼类的形态差异及分类地位

将采自中国伊洛瓦底江和怒江中115尾褶鮡属(Pseudecheneis)鱼类标本以采集地分为10组,对它们开展多变量形态度量学研究,共测量了25个框架性状和14个常规性状。经两次主成分分析,逐步提取分值散点聚在一起的标本,最终分离出5种不同的类型。第1类仅分布于伊洛瓦底江支流龙川江中下游,以其尾柄细长,区别于其他类型,确认为细尾褶鮡(P.stenura),其余4类均是未被描记的新种。第2类分布于伊洛瓦底江支流龙川江上游,体型极度纤细延长。第3类分布于怒江支流湾甸河,以胸鳍长为主要鉴别特征区别于其他类型,综合解剖学和分子生物学的系统发育研究结果,这类标本应是未被描记的一新种。第4类和第5类分别分布于怒江支流南汀河及伊洛瓦底江支流大盈江,它们以背部色斑的差异相互区分。此外,腾冲曲石部分标本的度量数据无法与大盈江的标本区分,外部形态特征亦无差异。它们是同一种类的不同地理种群,还是不同的种类,或者腾冲曲石的这些标本是另一新种?这些猜测均有待后续采用其他方法研究后进一步确认。     

Molecular taxonomy of members of the Anopheles hyrcanus group from Thailand and Indonesia

During studies of malaria vectors in Indonesia and Thailand, several specimens identified by field staff as members of the Anopheles barbirostris group (Diptera: Culicidae) were found to belong to the Anopheles hyrcanus group, as shown by marked differences in the size of the nuclear rDNA second internal transcribed spacer (ITS2) between the barbirostris (~1500 bp) and hyrcanus (~600 bp) groups. Identification of the species concerned required a more detailed study of ITS2 sequences and subunit I of the mitochondrial DNA cytochrome oxidase gene (COI). A phylogenetic analysis, based on Bayesian methods, revealed that the hyrcanus group specimens comprised five distinct clades, two of which corresponded with known species, Anopheles peditaeniatus and Anopheles sinensis. The remaining specimens formed three additional clades, for which there are no similar sequences in GenBank and which cannot be linked to previously described species. The misidentification of hyrcanus group species has important implications for malaria vector control; more comprehensive studies employing gene sequences are required to clarify the number of species in the group, their distribution and vector status.     

Reappraisal of the "Molecular phylogeny of Western Palaearctic Helicidae s.l. (Gastropoda: Stylommatophora)": when poor science meets GenBank

A paper on the 'Molecular phylogeny of Western Palaearctic Helicidae s.l.', published by Steinke et al. (2004) in this journal, is critically analysed. Several obvious errors are corrected and methodological weaknesses are revealed. BLAST searches on the sequences published in that paper and now in GenBank, showed high percentages of similarity of the alleged species with taxa that are considered only distantly related in the literature. Inspection of the so-called voucher specimens showed that some shells were misidentified, whereas others contained dirt or were bleached, indicating that these had been collected empty. Obviously the sequences published for those species could not have originated from those specimens, which cannot be considered vouchers therefore, even if they are from the same locality. In other instances, spurious sequences were published for correctly identified voucher specimens. For several species for which we collected specimens ourselves, the COI or the 16S sequence, or both, clearly differed from the results published by Steinke et al. The consequences of our results for the molecular data on helicid gastropods and their classification are listed.     

New and improved molecular sexing methods for museum bird specimens

We present two new avian molecular sexing techniques for nonpasserine and passerine birds (Neognathae), which are more suitable for use with museum specimens than earlier methods. The technique for nonpasserines is based on a new primer (M5) which, in combination with the existing P8 primer, targets a smaller amplicon in the CHD1 sex-linked gene than previously. Primers targeting ATP5A1, an avian sex-linked gene not previously used for sex identification, were developed for passerines. Comprehensive testing across species demonstrated that both primer pairs sex a range of different species within their respective taxonomic groups. Rigorous evaluation of each method within species showed that these permitted sexing of specimens dating from the 1850s. For corn bunting museum specimens, the ATP5A1 method sexed 98% of 63 samples (1857-1966). The M5/P8 CHD1 method was similarly successful, sexing 90% of 384 moorhen specimens from six different museum collections (1855-2001). In contrast, the original P2/P8 CHD1 sexing method only identified the sex of less than half of 111 museum moorhen samples. In addition to dried skin samples, these methods may be useful for other types of material that yield degraded or damaged DNA, and are hence potential new sexing tools for avian conservation genetics, population management and wildlife forensics.     

Phyllachora species infecting maize and other grass species in the Americas represents a complex of closely related species

The genus Phyllachora contains numerous obligate fungal parasites that produce raised, melanized structures called stromata on their plant hosts referred to as tar spot. Members of this genus are known to infect many grass species but generally do not cause significant damage or defoliation, with the exception of P. maydis which has emerged as an important pathogen of maize throughout the Americas, but the origin of this pathogen remains unknown. To date, species designations for Phyllachora have been based on host associations and morphology, and most species are assumed to be host specific. We assessed the sequence diversity of 186 single stroma isolates collected from 16 hosts representing 15 countries. Samples included both herbarium and contemporary strains that covered a temporal range from 1905 to 2019. These 186 isolates were grouped into five distinct species with strong bootstrap support. We found three closely related, but genetically distinct groups of Phyllachora are capable of infecting maize in the United States, we refer to these as the P. maydis species complex. Based on herbarium specimens, we hypothesize that these three groups in the P. maydis species complex originated from Central America, Mexico, and the Caribbean. Although two of these groups were only found on maize, the third and largest group contained contemporary strains found on maize and other grass hosts, as well as herbarium specimens from maize and other grasses that include 10 species of Phyllachora. The herbarium specimens were previously identified based on morphology and host association. This work represents the first attempt at molecular characterization of Phyllachora species infecting grass hosts and indicates some Phyllachora species can infect a broad range of host species and there may be significant synonymy in the Phyllachora genus.     

Usefulness and accuracy of MALDI‐TOF mass spectrometry as a supplementary tool to identify mosquito vector species and to invest in development of international database

Arthropod‐borne diseases are important causes of morbidity and mortality. The identification of vector species relies mainly on morphological features and/or molecular biology tools. The first method requires specific technical skills and may result in misidentifications, and the second method is time‐consuming and expensive. The aim of the present study is to assess the usefulness and accuracy of matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) as a supplementary tool with which to identify mosquito vector species and to invest in the creation of an international database. A total of 89 specimens belonging to 10 mosquito species were selected for the extraction of proteins from legs and for the establishment of a reference database. A blind test with 123 mosquitoes was performed to validate the MS method. Results showed that: (a) the spectra obtained in the study with a given species differed from the spectra of the same species collected in another country, which highlights the need for an international database; (b) MALDI‐TOF MS is an accurate method for the rapid identification of mosquito species that are referenced in a database; (c) MALDI‐TOF MS allows the separation of groups or complex species, and (d) laboratory specimens undergo a loss of proteins compared with those isolated in the field. In conclusion, MALDI‐TOF MS is a useful supplementary tool for mosquito identification and can help inform vector control.     

A multivariate analysis of Hyospathe (Palmae)

Previous systematic treatments of the neotropical palm genus Hyospathe have recognized from two to 18 species. An explicit, quantitative, repeatable sequence of operations for delimiting and testing groups of specimens and applying species concepts is carried out. Multivariate statistical analysis of morphological data is used to delimit and test groups of specimens. Cluster analysis is used to distinguish between characters and traits. Analysis of qualitative and quantitative characters reveals six groups of specimens, and the Phylogenetic Species Concept is applied to these groups. Two species, H. peruviana Henderson and H. frontinensis Henderson, are described as new. One of the specimen groups is large and widespread, and six geographically separate subgroups can be recognized within it. These subgroups can be distinguished by one or more significantly different quantitative characters. A Phylogenetic Subspecies Concept is applied to these subgroups. Three subspecies, H. elegans subsp. costaricensis Henderson, H. elegans subsp. sanblasensis Henderson, and H. elegans subsp. tacarcunensis Henderson are described as new, and two new combinations are made: H. elegans subsp. sodiroi (Dammer ex Burret) Henderson and H. elegans subsp. concinna (H. E. Moore) Henderson. One subspecies occurring in the Amazon region is complex morphologically and is not resolved by the methods used here.     

Statistical discrimination of sex in Melanoides tuberculata (Gastropoda: Thiaridae)

Systematists may rely on morphometric differences among samples of specimens for the recognition of living and fossil species, even though morphometric differentiation may be caused by non-genetic factors, such as ecophenotypy, differential growth rates and taphonomic mixing. When genetic differences between sexes or among closely related species are expressed as differences in the morphology of the individual or population, potentially valuable information becomes available to the systematist for a variety of genetic and ecological investigations. We have studied the morphology of the freshwater snail Melanoides tuberculata (Muller, 1774) in Israel, where males occur in what would otherwise be normally parthenogenetic (all female) populations. In modern M. tuberculata, sex may be determined by observation of gonadal tissue; in fossil specimens, any classification according to sex must be accomplished using only preservable features of the mineralized shell. Previous research confirmed that in large samples, mean shell shape of male and female snails differed significantly, but the degree of difference was too small to identify the sex of any individual specimen. We apply a three stage process that results with a high degree of accuracy in the discrimination of individual M. tuberculata specimens by sex on the basis of continuous morphological characters: (1) measurement of many aspects of shell morphology of individuals of known sex, and stepwise discrimination to discover which of the variables, if any, contribute to the morphometric differentiation of males from females (one time only, for the species); (2) use of these selected variables in a clustering procedure to make a preliminary assignment of each specimen to sex; (3) use of cluster assignments in a discrimination procedure to optimally predict sex. For species that exhibit morphometric differences between two groups, and for which continuous morphometric variation precludes the a priori recognition of discrete clusters, this sequential procedure may be of broad applicability. These objective methods may be applied to the discrimination within any set of specimens for which the hypothesis of two, and only two, constituent groups may be entertained.