Molecular Analysis of the Freshwater Prawn Macrobrachium olfersii (Decapoda,Palaemonidae) Supports the Existence of a Single Species throughout Its Distribution

Macrobrachium olfersii is an amphidromous freshwater prawn, widespread along the eastern coasts of the Americas. This species shows great morphological modifications during ontogenesis, and several studies have verified the existence of a wide intraspecific variation. Because of this condition, the species is often misidentified, and several synonyms have been documented. To elucidate these aspects, individuals of M. olfersii from different populations along its range of distribution were investigated. The taxonomic limit was established, and the degree of genetic variability of this species was described. We extracted DNA from 53 specimens of M. olfersii, M. americanum, M. digueti and M. faustinum, which resulted in 84 new sequences (22 of 16S mtDNA, 45 of Cythocrome Oxidase I (COI) mtDNA, and 17 of Histone (H3) nDNA). Sequences of three genes (single and concatenated) from these species were used in the Maximum Likelihood and Bayesian Inference phylogenetic analyses and COI sequences from M. olfersii were used in population analysis. The genetic variation was evaluated through the alignment of 554 bp from the 16S, 638 bp from the COI, and 338 bp from the H3. The rates of genetic divergence among populations were lower at the intraspecific level. This was confirmed by the haplotype net, which showed a continuous gene flow among populations. Although a wide distribution and high morphological intraspecific variation often suggest the existence of more than one species, genetic similarity of Caribbean and Brazilian populations of M. olfersii supported them as a single species.     

Morphometrics of the final instar exuviae of five cicada species occurring in urban areas of central Korea

Cicadas produce loud calling songs for mate attraction that can be a nuisance to city dwellers in Korea. Exuviae of final instars can be used to estimate population density. We investigated the morphological characteristics of final instar exuviae for five cicada species that are abundant in central Korea: Cryptotympana atrata, Hyalessa fuscata, Graptopsaltria nigrofuscata, Meimuna opalifera, and Meimuna mongolica. The characters analyzed were pro-mesonotum length, pronotum width, pronotum length, head width, abdominal circumference, body length, wing length, and femoral tooth angle. The results of a general linear model showed that species and sex were significant for morphological characters, and the result of post hoc analyses revealed that the mean values of most of the morphological characters measured were significantly different among the five cicada species. Three groups, Cryptotympana atrata, H. fuscata/G. nigrofuscata, and M. opalifera/M. mongolica, were distinctively identified based on most morphological characters. The distributions of femoral tooth angles did not overlap between H. fuscata and G. nigrofuscata, and the distributions of abdominal circumference did not overlap between M. opalifera and M. mongolica. Thus, the exuviae of all five cicada species could be easily distinguished based on morphological characters. We also provide a morphological key for the exuviae of all five cicada species in Korea.     

Morphological comparison and DNA barcoding of four closely related species in the genera Misgurnus and Paramisgurnus (Cypriniformes: Cobitidae)

To evaluate the feasibility of morphological and genetic identification of the closely related species in the genera Misgurnus and Paramisgurnus, the morphological characters of four species in these genera and DNA barcoding of five loaches (P. dabryanus, M. anguillicaudatus, M. bipartitus, M. mohoity, and Barbatula toni) were investigated. Twelve morphological characters were measured in 542 individuals to perform the comparative analysis. Among these characters, only the caudal peduncle length (L_CP) revealed significant difference (P < 0.05) among these four species. The clustering based on morphological characters formed two clusters (P. dabryanus and M. anguillicaudatus; M. bipartitus and M. mohoity). A total of 186 COI fragments for the five loaches investigated were sequenced and analyzed. The results showed that interspecific K2P distance was much higher than intraspecific distance within the five species. Bayesian inference of phylogeny showed that individuals of these species were divided into five specific clades. Meanwhile, the COI fragments exhibited 22 character attributes for the differentiation of the five loach species based on character-based method. Our results suggested that DNA barcoding based on COI can be used as an efficient identifier of these five loach species; the combination of distance-based method, Bayesian inference and character-based approach provides higher resolution of identification at species level.     

A taxonomic study of Chinese species of the insidiosus group of Metaphycus (Hymenoptera,Encyrtidae)

In this paper, twelve insidiosus-group species of the genus Metaphycus Mercet from China are reviewed. Five species, M. corniae sp. n., M. cylindricus sp. n., M. deltoideus sp. n., M. transversus sp. n. and M. yaanensis sp. n., are described as new to science. A key to the females of these species is given to facilitate species recognition. Photomicrographs are provided to illustrate morphological characters of these species. All specimens, unless otherwise specified, are deposited in the National Zoological Museum of China, Institute of Zoology, Chinese Academy of Sciences, Beijing.     

Myrtlemiris,a new genus and new species of Australian plant bugs (Insecta: Heteroptera: Miridae): systematics,phylogeny and host associations

A new Australian genus of Orthotylinae, Myrtlemiris Cheng, Mututantri & Cassis gen.n. , is described, with nine included species described as new to science (M. agnew sp.n. , M. astartephila sp.n. , M. meanarra sp.n. , M. newmanensis sp.n. , M. rubrocuneatus sp.n. , M. russulatus sp.n. , M. silveirae sp.n. , M. tesselatus sp.n. , M. yalgoo sp.n. ). A phylogenetic analysis based on 39 morphological characters is presented for all Myrtlemiris species and 6 outgroup taxa. This analysis establishes Myrtlemiris as monophyletic, defined by a broad apophysis on the left paramere. A key to species is provided and diagnostic characters are illustrated. Myrtlemiris is endemic to southwest Western Australia. Host plants for Myrtlemiris species are near restricted to the myrtaceous tribe Chamelaucieae.     

Morphometric studies and taxonomic delimitation in Menonvillea scapigera and related species (Cremolobeae: Brassicaceae)

Previous systematic treatments of Menonvillea recognized a group of six morphologically related species, the ‘M. scapigera group,' distinguished by having pinnately lobed leaves and united filaments of median staminal pairs. The number of recognized species ranged from six to two, one with three subspecies. In order to clarify the taxonomy of this group, multivariate and univariate analyses of qualitative and quantitative characters from herbarium specimens were performed. Additionally, to test the morphological results, the phylogenetic relationships and degree of exclusive ancestry were studied using ITS sequences and the genealogical sorting index. Our results support the recognition of three species (M. famatinensis, M. hirsuta, M. scapigera) delimited by a unique combination of qualitative characters. Quantitative characters support the division of the latter species into two subspecies: scapigera and longipes. The molecular data are congruent with the morphology and support the inclusion of M. hookeri within M. scapigera. A key to taxa of the M. scapigera group is presented.     

Russula velenovskyi new to Japan,with phylogenetic implications of Russula species between Japanese subalpine forests and Northern Europe

To compare morphological characters and phylogenetic placement between Japanese and European Russula, 32 specimens of 12 species were collected from Japanese subalpine forests and Northern Europe. Several sequences of nrDNA ITS region (ITS) of these Russula species were obtained. High homological similarities were shown between ITS sequences of several Russula samples collected from Japanese subalpine forests, Europe and North America. These facts show distribution of the same Russula species among these areas. From morphological observations and phylogenetic analyses, two same Russula species, R. velenovskyi, and R. decolorans are found in Japan, Europe and North America. Of these, R. velenovskyi collected from Mt. Fuji, Mt. Nyukasa and Mt. Tateshina in mountainous area of central Honshu is reported as a new Japanese record.     

Two new Truncatelloidea species from Melissotrypa Cave in Greece (Caenogastropoda)

In the small lake located in the cave Melissotrypa in Thessalia, Greece, truncatelloidean gastropods representing two species were found, new to science. One of them, represented by two specimens only, has been described based on the shell characters only; with its cytochrome oxidase sequence it has been assigned to the genus Iglica, and to the family Moitessieriidae, Iglica hellenica sp. n. For the other species, represented by 30 collected specimens, the shell, protoconch, radula, head, penis and female reproductive organs have been described; all the morphological characters and cytochrome oxidase sequences have confirmed its assignment to the genus Daphniola (Hydrobiidae: Sadlerianinae), Daphniola magdalenae Falniowski, sp. n.     

Genetic and morphological variation of bee-parasitic <Emphasis Type="Italic">Tropilaelaps</Emphasis> mites (Acari: Laelapidae): new and re-defined species

Mites in the genus Tropilaelaps are parasites of social honeybees. Two species, Tropilaelaps clareae and T. koenigerum, have been recorded and their primary hosts are presumed to be the giant honeybees of Asia, Apis dorsata and A. laboriosa. The most common species, T. clareae, is also an economically important pest of the introduced Western honeybee (A. mellifera) throughout Asia and is considered an emerging threat to world apiculture. In the studies reported here, genetic (mtDNA CO-I and nuclear ITS1-5.8S-ITS2 gene sequence) and morphological variation and host associations were examined among Tropilaelaps isolates collected from A. dorsata, A. laboriosa and A. mellifera throughout Asia and neighbouring regions. The results clearly indicate that the genus contains at least four species. Tropilaelaps clareae, previously assumed to be ubiquitous in Asia, was found to be two species, and it is here redefined as encompassing haplotypes (mites with distinct mtDNA gene sequences) that parasitise native A. dorsata breviligula and introduced A. mellifera in the Philippines and also native A. d. binghami on Sulawesi Island in Indonesia. Tropilaelaps mercedesae n. sp., which until now has been mistaken for T. clareae, encompasses haplotypes that, together with haplotypes of T. koenigerum, parasitise native A. d. dorsata in mainland Asia and Indonesia (except Sulawesi Island). It also parasitises introduced A. mellifera in these and surrounding regions and, with another new species, T. thaii n. sp., also parasitises A. laboriosa in mountainous Himalayan regions. Methods are described for identifying each species. These studies help to clarify the emerging threat of Tropilaelaps to world apiculture and will necessitate a revision of quarantine protocols for countries that import and export honeybees.     

Evidence of gene flow between Sulawesi macaques

A pilot field study was conducted in Sulawesi (Indonesia) to assess the status of macaque populations on the island. Wild and captive animals were sampled, mainly in border areas between presumed different species. The five species investigated were Macaca maurus, M. tonkeana, M. hecki, M. nigrescens, and M. nigra, for which morphological and gene frequency data suggested the presence of hybridization zones. Some individuals within these zones showed intermediate or mosaic morphology between parental forms. These individuals also had intermediate gene frequencies for most of the polymorphic systems investigated. Karyotypes were identical in all species, and no cytogenetic barrier to hybridization existed between species. A review of the recent literature also provided evidence for hybridization between Sulawesi macaques. Clinical frequencies in both morphological and biomolecular traits perhaps can be best explained by the operation of gene flow between the various forms of macaques on the island. However, additional data are necessary before current classification schemes are revised. The unique opportunity and need of further study of Sulawesi macaques for a range of evolutionary questions is emphasized.     

Phylogeny and Classification of the Trapdoor Spider Genus Myrmekiaphila: An Integrative Approach to Evaluating Taxonomic Hypotheses

Background

Revised by Bond and Platnick in 2007, the trapdoor spider genus Myrmekiaphila comprises 11 species. Species delimitation and placement within one of three species groups was based on modifications of the male copulatory device. Because a phylogeny of the group was not available these species groups might not represent monophyletic lineages; species definitions likewise were untested hypotheses. The purpose of this study is to reconstruct the phylogeny of Myrmekiaphila species using molecular data to formally test the delimitation of species and species-groups. We seek to refine a set of established systematic hypotheses by integrating across molecular and morphological data sets.

Methods and Findings

Phylogenetic analyses comprising Bayesian searches were conducted for a mtDNA matrix composed of contiguous 12S rRNA, tRNA-val, and 16S rRNA genes and a nuclear DNA matrix comprising the glutamyl and prolyl tRNA synthetase gene each consisting of 1348 and 481 bp, respectively. Separate analyses of the mitochondrial and nuclear genome data and a concatenated data set yield M. torreya and M. millerae paraphyletic with respect to M. coreyi and M. howelli and polyphyletic fluviatilis and foliata species groups.

Conclusions

Despite the perception that molecular data present a solution to a crisis in taxonomy, studies like this demonstrate the efficacy of an approach that considers data from multiple sources. A DNA barcoding approach during the species discovery process would fail to recognize at least two species (M. coreyi and M. howelli) whereas a combined approach more accurately assesses species diversity and illuminates speciation pattern and process. Concomitantly these data also demonstrate that morphological characters likewise fail in their ability to recover monophyletic species groups and result in an unnatural classification. Optimizations of these characters demonstrate a pattern of “Dollo evolution” wherein a complex character evolves only once but is lost multiple times throughout the group''s history.     

Variability and relationships of the Far Eastern species of sculpins Myoxocephalus and Megalocottus (Cottidae) based on mtDNA markers and karyological data

The phylogenetic relationships among five species of sculpins, including Myoxocephalus stelleri, M. brantii, M. jaok, M. ochotensis, and Megalocottus platycephalus, were estimated from the sequence variability of the mtDNA cytochrome b (cytb) and cytochrome oxidase 1 (CO1) genes. Analysis of the topologies of combined phylogenetic trees showed that all of the morphologically described species from different genera represented monophyletic groups with high support of branch robustness. Haplotypes with different karyotypes from different geographical localities with an intragroup nucleotide diversity of 0.42% were combined into the M. stelleri clade. All of the species (except for M. stelleri from the Sea of Japan and the Okhotsk Sea coast of the Hokkaido Island) were characterized by relatively low values of the intragroup variation, along with high values of interspecific variation of mtDNA-encoded markers. Studies of the M. stelleri karyotypes using Ag-banding showed that karyotypes of the individuals from the Sea of Japan and Okhotsk differed not only in the number of chromosomes (2n) but also in the number of active nucleolus organizers (NO) and stained NO blocks, calling into question whether they belong to a single species. The observed discrepancy between the phylogenetic topologies and karyological data is discussed in relation to the informative capacity of mtDNA fragments and the phenomenon of intraspecific chromosomal polymorphism.     

基于线粒体CO Ⅰ基因的闪蛱蝶亚科系统发育关系研究

测定了重要林业害虫闪蛱蝶亚科Apaturinae 11属17种蝶类的线粒体细胞色素氧化酶Ⅰ的部分序列,并结合由GenBank下载的该亚科4种蝶类的相应序列进行分析,探讨了闪蛱蝶亚科各属间的系统发育关系。以玉杵带蛱蝶Athymajina,白斑眼蝶Penthema adelma,忘忧尾蛱蝶Polyura nepenthes和白带螯蛱蝶Charaxes bernardus作为外群,采用PAUP4.0b4a软件构建了闪蛱蝶亚科的MP和NJ分子系统树。虽然COI基因数据中第3位点的转换替换已达饱和,但由于这些位点含有大量系统发育信息,因而在数据统计分析时并没有将这些位点删除。同时通过对各分枝稳定性的比较,研究在简约分析中不同转换/颠换加权方式对假定所有特征具有相同权重的影响。分子系统树显示:NJ和不同转换/颠换加权方式下构建的MP系统树中闪蛱蝶亚科均有4个主要的聚类簇,该亚科系统树中存在许多置信度高且稳定的分枝,同时也存在一些因分枝置信度低且不稳定而使其分类地位不能确定的类群。在所构建的系统树中,由分子数据得到的蛱蝶亚科系统发育关系与传统分类学的基本一致,其中迷蛱蝶属Mimathyma为单系群;累积蛱蝶Lelecella limenitoides为明窗蛱蝶Dilipa fenestra的姐妹群且支持二者关系的置信度很高;支持将白斑迷蛱蝶Mimathyma schrenckii,迷蛱蝶M.chevana,夜迷蛱蝶M.nycteis,栗铠蛱蝶Chitoria subcaerulea,黄带铠蛱蝶C.fasciola,铂铠蛱蝶C.pallas,and银白蛱蝶Helcyra subalba等物种由闪蛱蝶属中移出的修订。     

An unrecorded species in Korea:Monochasma sheareri (S. Moore) maxim, ex Franch. Et Sav. (Scrophulariaceae)

Plants of a genus and species previously unrecorded in Korea were collected from the hillside along a forest road at the Gwaneum peak in the Byeonsanbando National Park, Buan-gun, Jeollabuk-do, together with plants belonging to Gramineae and Cyperaceae.Monochasma sheareri (S. Moore) Maxim, ex Franch. et Sav. (Scrophulariaceae) is most similar toM. savatieri Franch. ex Maxim., but differs distinctively in several morphological characters. WhereasM. sheareri exhibits a slender stem, and a calyx that is longer than the corolla, plants ofM. savatieri, which is distributed in Japan and China, have stout stems with glandular pilose trichomes, along with calyxes that are shorter than their corollas. In addition, seeds ofM. sheareri are elliptical, with falcate trichomes on their surfaces. This new plant has been given a Korean name, “Byeon-san-but-sal-i-pul”, based on its parasitic habit and features of its natural habitat. Here, we describe its morphological characters and provide a taxonomic key and illustrations, plus photographs of the flower, fruit, and roots.     

Morphological identification of Lucilia sericata,Lucilia cuprina and their hybrids (Diptera,Calliphoridae)

Hybrids of Lucilia sericata and Lucilia cuprina have been shown to exist in previous studies using molecular methods, but no study has shown explicitly that these hybrids can be identified morphologically. Published morphological characters used to identify L. sericata and L. cuprina were reviewed, and then scored and tested using specimens of both species and known hybrids. Ordination by multi-dimensional scaling indicated that the species were separable, and that hybrids resembled L. cuprina, whatever their origin. Discriminant function analysis of the characters successfully separated the specimens into three unambiguous groups – L. sericata, L. cuprina and hybrids. The hybrids were morphologically similar irrespective of whether they were from an ancient introgressed lineage or more modern. This is the first evidence that hybrids of these two species can be identified from their morphology. The usefulness of the morphological characters is also discussed and photographs of several characters are included to facilitate their assessment.     

Morphological and molecular tools in identifying the Mediterranean limpets Patella caerulea, Patella aspera and Patella rustica

Allozyme electrophoresis, a partial nucleotide sequence of a mitochondrial gene for cytochrome oxidase I (COI) and discriminant analysis of shell morphometric characters were used to study the relationships among the Sicilian marine gastropods of the Patella genus. Allozyme and mtDNA markers unequivocally distinguished the species and were very useful markers in correctly classifying the different species when morphological characters overlapped each other. Several allozyme loci and many nucleotide positions were diagnostic of species. In contrast, the discriminant analysis of simple morphometric shell characters failed to adequately discriminate the species, suggesting that environmental factors influence colouration and morphological patterns in the Patella species. Our results underline the importance of a genetic approach, as compared to a morphological approach, in discriminating the Mediterranean Patella species.