Rediscovery of Celes akitanus (Orthoptera: Acrididae) from semi‐natural grasslands in Japan

Celes akitanus was rediscovered in semi‐natural grasslands in Japan. This species is one of the most endangered insects in Japan; most local populations were thought to be extinct. Reliable records of this rare species had been obtained only from Yamagata, Niigata, Tokyo and Nagano Prefectures. We compared the morphology of the holotype of Oedipoda akitana (=C. akitanus) and newly collected specimens and found that they matched with each other. The species was redescribed based on the holotype and new specimens. A molecular phylogenetic analysis involving other acridid species indicated that our samples were practically identical to C. akitanus from China. We conclude that the specimens we collected are C. akitanus, which has been rediscovered in Japan after about 30 years. Because the discovered populations were restricted to narrow and scattered areas, conservation of these populations should be a high priority. Population monitoring and habitat assessment are required. Because C. akitanus and many other endangered species are found in the grassland areas, these habitats should be protected and maintained.     

Morphological and genetic evidence for two evolutionarily significant units (ESUs) in the South American fur seal, <Emphasis Type="Italic">Arctocephalus gazella</Emphasis>

The South American fur seal (Arctocephalus australis) is widely distributed, occurring along both the Atlantic and the Pacific coasts of South America. Previous work suggests there may be more than one subspecies, highlighting the need for further study. Here, we combine traditional and geometric morphometric analysis of skull shape and size with genetic data to compare two populations of South American fur seals, one from Uruguay and one from Peru. As a control group we used material from the closely related species Arctocephalus gazella. Both techniques of morphometric analysis reveal pronounced geographic variation in size and shape of the skull, with Peruvian specimens (n = 102) being larger than Uruguayan skulls (n = 133) and significant shape differences concentrated in the rostral region. Similarly, seven highly polymorphic microsatellite loci reveal highly significant differences in allele frequency. Moreover, Bayesian analysis implemented using the program structure reveals two separate clusters corresponding perfectly to the two populations, with an assignment test correctly placing over 98% of specimens in their population of origin. This degree of differentiation for both genetic and morphological traits suggests complete and possibly prolonged isolation to the extent that we believe these populations should be considered distinct evolutionarily significant units.     

Genetic structure of the rare and endangered moss <Emphasis Type="Italic">Campylopus oerstedianus</Emphasis> (Dicranaceae) in Europe

The genetic structure, diversity and phylogeography of the moss species Campylopus oerstedianus in Europe was studied, based on the ITS region of the nrDNA of nine selected European populations. Although this species is only known in sterile stage, long-distance dispersal and gene flow among populations seem to be present within Europe. High levels of genetic differentiation between the investigated Greek population and the western European populations indicate a long lasting isolation. The populations from France and Switzerland root together and supposedly have a common origin. From the French populations, the one from the Pyrenees is the most basal one and the populations in the Massif Central and the Vosges Mts can be derived from it. This indicates relatively recent dispersal of the species from the Mediterranean to the north, in spite of the lack of sporophytes and the fact that the type of the dispersed propagules and their vectors are not obvious.     

On the Taxonomy and Morphology of the Pine Wood Nematode, Bursaphelenchus xylophilus (Steiner &Buhrer 1934) Nickle 1970

During the past 3 yr, nematologists in the United States have found specimens of Bursaphelenchus sp. in the wood of dead and dying pine trees. This nematode-host association resembles a similar interaction reported from Japan where pine trees are being killed by the pine wood nematode. This taxonomic research was conducted to determine if the Japanese pine wood nematode and similar populations in the United States are of the same species. Based upon typical morphological characters of original specimens of Bursaphelenchus xylophilus (Steiner and Buhrer 1934) Nickle 1970 that were rediscovered in the USDA Nematode Collection and genetic crosses among the Japanese and American nematode populations, it was concluded that they are all the same species, B. xylophilus.     

Genetic variation and population structure of the endangered Hyacinth Macaw (<Emphasis Type="Italic">Anodorhynchus hyacinthinus</Emphasis>): implications for conservation

The Hyacinth Macaw (Anodorhynchus hyacinthinus) is one of 14 endangered species in the family Psittacidae occurring in Brazil, with an estimated total population of 6,500 specimens. We used nuclear molecular markers (single locus minisatellites and microsatellites) and 472 bp of the mitochondrial DNA control region to characterize levels of genetic variability in this species and to assess the degree of gene flow among three nesting sites in Brazil (Pantanal do Abobral, Pantanal de Miranda and Piauí). The origin of five apprehended specimens was also investigated. The results suggest that, in comparison to other species of parrots, Hyacinth Macaws possess relatively lower genetic variation and that individuals from two different localities within the Pantanal (Abobral and Miranda) belong to a unique interbreeding population and are genetically distinct at nuclear level from birds from the state of Piauí. The analyses of the five apprehended birds suggest that the Pantanal is not the source of birds for illegal trade, but their precise origin could not be assigned. The low genetic variability detected in the Hyacinth Macaw does not seem to pose a threat to the survival of this species. Nevertheless, habitat destruction and nest poaching are the most important factors negatively affecting their populations in the wild. The observed genetic structure emphasizes the need of protection of Hyacinth Macaws from different regions in order to maintain the genetic diversity of this species.     

Dental anomalies in the Japanese mole <Emphasis Type="Italic">Mogera wogura</Emphasis> from northeast China and the Primorsky region of Russia

Dental anomalies in the Japanese mole, Mogera wogura Temminck, 1842, from northeast China and the Primorsky region of Russia were examined based on 241 specimens. The most frequent dental anomaly was oligodonty, i.e., missing P² (18 cases) or P₃ (one case). Supernumerary teeth were observed in three cases, two of which were characterized by abnormal shapes. Morphological abnormalities in teeth (six cases) and an asymmetrically curved rostrum (one case) were also observed. Dental anomalies were found at higher frequencies in populations near the northern range limit of the species. This was not caused by size effects. We suggest that the high incidence of dental anomalies was the result of genetic drift, which increases in marginal populations. Considering the nature of subterranean mammals, our results suggest that the high frequency of dental anomalies in a marginal population could have initiated the evolution of dental formulae if parapatric or peripatric speciation occurs in such populations.     

DNA barcoding in Tardigrada: the first case study on Macrobiotus macrocalix Bertolani & Rebecchi 1993 (Eutardigrada, Macrobiotidae)

Morphological and molecular studies on a tardigrade species have been carried out to verify the possibility of using a DNA barcoding approach for species identification in this phylum. Macrobiotus macrocalix Bertolani & Rebecchi, 1993 was chosen as the test species since it belongs to a group of species in which the taxonomy is quite problematic. Animals and eggs belonging to three Italian and one Swedish populations have been investigated. Both morphological and molecular analyses show that all the populations belong to the same species. The low genetic distances recorded among the studied populations (0.3-1.0%) and the high genetic distance (15.9-16.3%) between these populations and a closely related species confirm the possibility of identifying a specimen of this species by its cytochrome oxidase subunit I sequence. Data from other authors support our results indicating that DNA barcoding can be applied to tardigrades. With our protocols, we have obtained voucher specimens that enable us to show a correspondence between morphology and molecular data.     

Are the meiofaunal polychaetes Hesionides arenaria and Stygocapitella subterranea true cosmopolitan species? — results of RAPD‐PCR investigations

The interstitial meiofauna of sand beaches includes many species that are regarded as cosmopolites, on the basis of records from various sites throughout the world. There is a long-standing debate about the causes of such a distribution, but for most of these species it is still questionable whether they are in fact true species or 'merely' complexes of cryptic species. Here genetic differentiation is examined within and between two species considered typically cosmopolitan, Hesionides arenaria arenaria (Hesionidae) and Stygocapitella subterranea (Parergodrilidae), by RAPD-PCR analyses of specimens from diverse marine regions on two continents. For S. subterranea at three sites, on the North Sea coast (Sylt) and the US Atlantic (Massachusetts) and Pacific (Washington) coasts, with 14 primers 335 different DNA fragments were found: 20 diagnostic ones for the European animals, 17 for the animals from the American East Coast and 14 for those from the American West Coast. Five cluster procedures were used, all of which significantly distinguished the three populations as separate genetic clades; it is recommended that each be given species status. In contrast, the individual specimens of H. a. arenaria from eight European sites, between Skagerrak and the eastern Mediterranean (including the Canary Islands), and the North American Pacific coast, for which 468 different DNA fragments were amplified with 12 primers, do not form separate genetic clades. For no single population was it possible to demonstrate even one diagnostic character. Therefore the cosmopolitan nature of H. a. arenaria has been confirmed at the DNA level.     

光肩星天牛种群间及其近缘种遗传关系的RAPD研究

利用RAPD技术对采自中国和美国的星天牛属Anoplophora 5个种及8个光肩星天牛Anoplophora glabripennis (Motschulsky)地理种群共13个样品进行了遗传相似性分析。选用了Operon公司生产的引物H系列20个,L系列20个,Q系列11个共51个引物,最后从40个引物中筛选出26个具有多态性的引物作为第一组用于星天牛属种间和光肩星天牛种群间分析,从31个引物中筛选出19个具有多态性的引物作为第二组单独用于光肩星天牛种群分析。根据第一组引物实验获得的RAPD聚类图及遗传距离表明,各个地理种群的光肩星天牛和黄斑星天牛A. nobilis都聚在一起,形成一个大的分枝,而四川星天牛A. freyi、楝星天牛A. horsfieldi和星天牛A. chinensis均在此分枝之外。来自美国纽约和芝加哥的光肩星天牛种群聚于中国光肩星天牛种群之外的另一个独立的分枝上。分布在我国宁夏、内蒙古和河北的光肩星天牛以及宁夏黄斑星天牛和山东、陕西的光肩星天牛分别聚在一起,而甘肃的光肩星天牛与甘肃的黄斑星天牛则聚于另一枝,且它们之间的遗传距离很近,仅为0.1324,说明这两者之间有着极其相近的亲缘关系,由此推断光肩星天牛和黄斑星天牛的差异很小,遗传关系难以区分,进一步证实了它们很可能是同一个种下的两个不同的型。第二组引物实验得到了相似的结果,来自中国的6个光肩星天牛种群全部聚于同一枝中并分成两小枝： 分布于我国宁夏、河北、山东、甘肃的光肩星天牛聚在一起,内蒙古和陕西的光肩星天牛则成另一枝,而分布于美国纽约和芝加哥的光肩星天牛仍聚于中国光肩星天牛种群之外的一个单独的分枝上。但是美国光肩星天牛与中国光肩星天牛之间的遗传距离最近的为0.4578, 最远的为0.5960。由此认为,本研究中采自美国的两个光肩星天牛种群的样本和采自中国的光肩星天牛种群的样本之间存在显著差异,遗传关系较远。有必要从中国和世界其他天牛分布地采集更多样本做进一步DNA 分析。     

重庆市城口县大巴山国家级自然保护区发现宁陕线形蛇与该物种的遗传分化分析

宁陕线形蛇Stichophanes ningshaanensis Yuan,1983是游蛇科Colubridae的单型属单型种,为中国特有种,至今仅被报道分布于陕西省宁陕县火地塘(模式产地)、湖北省神农架国家级自然保护区和重庆市五里坡国家级自然保护区。目前对该物种的研究和野外采集记录甚少。2017年7月8日,在重庆市城口县大巴山国家级自然保护区采到1号疑似宁陕线形蛇的雌性成体标本。经形态比较和分子系统发育分析,鉴定为宁陕线形蛇。基于线粒体细胞色素b(cyt b)基因和核基因片段cmos的系统发育分析表明,城口大巴山标本嵌入宁陕线形蛇支系中,与该物种其他种群在cyt b基因上的遗传距离(0.001~0.009)远小于与其近缘种的(0.174~0.292)。本研究揭示宁陕线形蛇种群间存在形态和遗传上的分化。形态上,城口大巴山标本上唇鳞数量为5/5,而神农架和火地塘种群均为6/6,前者尾下鳞数量为61对,少于后者(64~73对)。在cyt b基因上,3个种群各自拥有1个单倍型。城口大巴山单倍型与湖北神农架单倍型之间有1个突变、与宁陕火地塘单倍型之间有9个突变,而分子系统树支持宁陕火地塘种群与湖北神农架种群关系更近。研究结果为宁陕线形蛇的地理分布和系统学研究提供了重要信息。     

Molecular and morphological analysis of endangered species <Emphasis Type="Italic">Gymnodiptychus integrigymnatus</Emphasis> (Teleostei: Cyprinidae)

Gymnodiptychus integrigymnatus is a critically endangered species endemic to the Gaoligongshan Mountains. It was thought to be only distributed in several headwater-streams of the Longchuanjiang River (west slope of the Gaoligongshan Mountains, belonging to the Irrawaddy River drainage). In recent years, dozens of G. integrigymnatus specimens have been collected in some streams on the east slope of the Gaoligongshan Mountains (the Salween drainage). We performed a morphological and genetic analyses (based on cytochrome b and D-loop) of the newly discovered populations of G. integrigymnatus to determine whether the degree of separation of these populations warrants species status. Our analysis from the cytochrome b gene revealed that nine individuals from the Irrawaddy drainage area and seven individuals from the Salween drainage area each have only one unique haplotype. The genetic distance between the two haplotypes is 1.97%. Our phylogenetic analysis revealed that G. integrigymnatus is closely related to highly specialized schizothoracine fishes. Analysis from the mitochondrial control region revealed that G. integrigymnatus has relatively high genetic diversity (π was 0.00891 and h was 0.8714), and individuals from different river drainages do not share the same haplotypes. The AMOVA results indicated 87.27% genetic variability between the Salween and Irrawaddy populations. Phylogenetic trees show two major geographic groups corresponding to the river systems. We recommend that G. integrigymnatus should be considered as a high priority for protected species status in the Gaoligongshan Mountains National Nature Reserve, and that the area of the Gaoligongshan Mountains National Nature Reserve should be expanded to cover the entire distribution of G. integrigymnatus. Populations of G. integrigymnatus from different river systems should be treated as evolutionarily significant units.     

Phylogenetic revision of the Strophomenida,a diverse and ecologically important Palaeozoic brachiopod order

The order Strophomenida was an ecologically abundant and taxonomically diverse group of Palaeozoic brachiopods that originated in the earliest Ordovician and went extinct in the Carboniferous. During their long geological range, the Strophomenida survived two of the ‘Big Five’ mass extinction events, the Late Ordovician and the Late Devonian, suggesting that they are potentially informative taxa for studying the evolutionary effects of these two distinct mass extinctions, each with drastically different forcing mechanisms. However, while there have been previous phylogenetic studies on smaller groups within the Strophomenida, the phylogenetic relationships of the whole group are still largely unknown. The group has been divided into two major superfamilies, the Strophomenoidea (strophomenoids) and the Plectambonitoidea (plectambonitoids). Despite being treated as separate clades, the plectambonitoids may form a paraphyletic grade into the strophomenoids. We present a detailed higher‐level parsimony‐based phylogenetic analysis of the Strophomenida, consisting of 69 characters and 62 exemplar species sampled from the majority of the taxonomically defined families/subfamilies. Several species of basal chonetids (strophochonetids) were also included in this analysis, as they may be closely related to the Strophomenida and share several characters with both the plectambonitoids and strophomenoids. The phylogenetic analysis suggests the plectambonitoids, as originally defined, are paraphyletic to the monophyletic strophomenoids. The basal chonetids are reconstructed as a monophyletic group that is sister to the strophomenoids, suggesting that their proper placement might be within the Strophomenida. The topology also suggests that at least 17 of the taxonomically defined strophomenoid and plectambonitoid families are likely to be monophyletic. The Plectambonitidae and the Taffiidae as defined are paraphyletic, and the Grorudiidae and Leptostrophiidae are polyphyletic. Furthermore, subfamilies Leptodontellinae, Dicoelostrophiinae, Palaeostrophomeninae and Aegiromeninae are raised to the level of family. When analysed within this phylogenetic context, the Late Ordovician mass extinction event had little effect on the large‐scale evolution of the group.     

Pollination of the Lady's slipper orchid (Cypripedium calceolus) in Scandinavia – taxonomic and conservational aspects

The Lady's slipper orchid Cypripedium calceolus L. is considered one of the most beautiful orchids of Europe. Consequently, the species has suffered from over-collecting and is now critically endangered in many countries. Although pollination success is suspected to influence the long-term survival of Cypripedium calceolus , relatively little is known about the identity of its pollinators in mainland Sweden – a region that comprises the largest European populations. In order to identify which species pollinate eight representative populations in mainland Sweden, we observed and sampled visitors to flowers using a standardized protocol. Specimens were identified and any pollen smear found on their body was examined for the presence of Cypripedium pollen. Nine species were recognized as effective pollen vectors ( Andrena cineraria, A. carantonica, A. haemorrohoa, A. helvola, A. nigroaenea, A. praecox, Colletes cunicularius, Lasioglossum fratellum and L. fulvicorne ), four of them for the first time in Scandinavia. This is the first time that a species of Colletes is reported to carry pollen of Cypripedium in this region. All but one specimens were females. Our results suggest a taxonomically heterogeneous pollinator fauna for Cypripedium calceolus and are discussed in light of the management of this species.     

Branchinella maduraiensis Raj (Crustacea,Branchiopoda, Anostraca) shown by new evidence to be a valid species

The fairy shrimp Branchinella kugenumaensis (Ishikawa) was long considered a widely, though disjunctly, distributed species occurring from Japan through eastern Asia to southern India. Attempts by Raj (1951, 1961) to make the Indian populations a new variety (B. k. var. madurai) on the basis of antennal and frontal appendage morphology and on its distribution pattern, were considered unconvincing by later authors. Our new comparison of Japanese and Indian specimens has revealed several differences. The resting egg shells of B. kugenumaensis from Japan have irregular polygonal fields; whereas, the shells of the Indian taxon have lip-like units covered with spinules. Furthermore, there are lobes lateral to the basal penes in the Indian specimens that are lacking in the Japanese taxon. As well as differences in antennal and frontal appendage morphology. These differences correspond with previous illustrations of populations from both geographic regions and lead us to consider Raj's proposed subspecies as a valid species. This brings the number of accepted species in the genus Branchinella to 34. More specimens will need to be evaluated from the area between India and Japan to determine if B. maduraiensis is endemic to the Indian subcontinent or is more broadly distributed on the Asian mainland. This revised version was published online in August 2006 with corrections to the Cover Date.     

Molecular identification of the exotic slug <Emphasis Type="Italic">Arion subfuscus</Emphasis> sensu stricto (Gastropoda: Pulmonata) in California,with comments on the source location of introduced populations

Arion subfuscus sensu lato (s. l.) refers to a cryptic slug species complex that is native to Europe and which, from there, has been introduced into the northeastern U.S. The species complex was detected in California for the first time in 2005 and recently 12 specimens were collected during statewide surveys. The genital morphology of these specimens suggested that only A. subfuscus sensu stricto (s. s.) was present and partial sequences of mitochondrial 16S rDNA (443 bp) showed that they all belonged to a single haplotype of the mitochondrial lineage, S1 sensu Pinceel et al. (Genetica 125: 155–171, 2005). This result was corroborated by a parallel analysis of a 655 bp COI DNA barcode. The 16S rDNA S1 haplotype (S1-03) of the Californian specimens is hitherto known only from the northeastern U.S. Hence, it is likely that populations may have been introduced to California from the northeastern states, rather than directly from the native area of the S1 haplogroup in Europe.     

Phylogeography of loaches of the genus lefua (balitoridae, cypriniformes) inferred from mitochondrial DNA sequences

In order to elucidate phylogenetic relationships and intraspecific variations and to infer the evolutionary process of loaches of the genus Lefua, we analyzed nucleotide sequences of the mitochondrial D-loop region of 100 specimens obtained from 97 localities in Japan and Korea. The genus Lefua includes three described species, L. nikkonis, L. echigonia, and L. costata and an undescribed species, Lefua sp. Our results showed that each species of Lefua formed a monophyletic group, indicating clearly that Lefua species can be genetically distinguished from one another. Lefua nikkonis was the most closely related to L. costata, while L. sp. was the most closely related to L. echigonia. Specimens of L. sp. were grouped into two intraspecific populations and specimens of L. echigonia were grouped into six populations. These populations were well separated geographically from one another by mountain ranges and highlands. We estimated the evolutionary time for splitting of the species and intraspecific populations, and speculated on the evolutionary process of the genus Lefua. Species of Lefua are severely threatened. Fundamental genetic information is indispensable for conservation. We presented genetic background in order to protect these threatened loaches.     

Redescription and new data on <Emphasis Type="Italic">Microsomacanthus diorchis</Emphasis> (Fuhrmann, 1913) (Cestoda: Hymenolepididae)

Microsomacanthus diorchis (Fuhrmann, 1913) is redescribed and illustrated on the basis of the type-material and new findings from common eider Somateria mollissima captured in Iceland and specimens from the same host species from the Barents, White and Bering Seas. A lectotype is designated and an amended diagnosis is provided. The main differentiating features of M. diorchis are the size and shape of rostellar hooks and the cirrus, the well-marked delay in the antiporal testis development and the bow-shaped uterus. This parasite is shown to be specific to S. mollissima for both Atlantic and Pacific populations of the host. Hymenolepis (Microsomacanthus) somateriae of Bishop & Threlfall (1974) [nec M. somateriae Ryzhikov, 1965] is recognised as a synonym of M. diorchis (Fuhrmann, 1913). The taxonomic position of the species described as Aploparaksis murmanica Baylis, 1919 from common eider is discussed.     

Spatial and temporal continuity of kangaroo rat populations shown by sequencing mitochondrial DNA from museum specimens

Summary The advent of direct sequencing via the polymerase chain reaction (PCR) has opened up the possibility of molecular studies on museum specimens. Here we analyze genetic variation in populations over time by applying PCR to DNA extracted from museum specimens sampled from populations of one species over the last 78 years. Included in this study were 43 museum specimens of the Panamint kangaroo ratDipodomys panamintinus from localities representing each of three geographically distinct subspecies. These specimens were originally collected and prepared as dried skins in 1911, 1917, or 1937. For each specimen, a 225-bp segment of the mitochondrial genome was sequenced. These mitochondrial DNA sequences were compared to those of 63 specimens collected at the same localities in 1988. The three subspecies were nearly completely distinct. Only 2 of the 106 individuals shared mitochondrial types between subspecies. For all three localities, the diversity levels were maintained between the two temporal samples. The concordance observed between the two temporally separate phylogenies supports the use of museum specimens for phylogenetic inference. This study demonstrates the accuracy and routine nature of the use of museum specimens in the analysis of mitochondrial sequence variation in natural populations and, importantly, that a temporal aspect can now be added to such studies.     

Cryptic speciation in liverworts – a case study in the Aneura pinguis complex

Bryophytes are amongst the most ancestral terrestrial plants and often have large distribution ranges across continents. Recent biochemical and molecular studies have suggested that many worldwide morphological species of bryophyte may represent genetically divergent and reproductively isolated cryptic species. We tested the cryptic species hypothesis in the thalloid liverwort Aneura pinguis complex. We applied analyses of chloroplast DNA (cpDNA) sequence variation and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods to discriminate between specimens of A. pinguis derived from various habitats in five distant geographical regions in Poland. Of the 19 specimens sequence characterized for the cpDNA tRNA^Leu region, seven haplotypes were identified divided into three nonmonophyletic clusters. The application of developed PCR-RFLP markers confirmed the existence of three tRNA^Leu types of A. pinguis (A–C) within the specimens derived from 21 populations. Sympatric populations of different tRNA^Leu types were found in lowland and mountain regions. No clear correlation between stand type and the presence of two tRNA^Leu types (A, B) was observed, as both were growing on soil, humus, and rocks. The tRNA^Leu type C was found only on humus and its distribution was restricted to low-lying northern populations. The above results indicate that the A. pinguis complex is highly differentiated at the molecular level and may represent three cryptic species.  © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society , 2007, 155 , 273–282.