How did sea spiders recolonize the Chilean fjords after glaciation? DNA barcoding of Pycnogonida,with remarks on phylogeography of Achelia assimilis (Haswell, 1885)

The present paper reports on a first attempt at resolving the taxonomy of Chilean Pycnogonida using a combination of DNA sequence and morphological data. In a subproject of the Marine Barcode of Life (MarBoL) campaign we analysed a fragment (about 657 base pairs) of the mitochondrial protein-coding gene COI (cytochrome c oxidase subunit 1) from 76 Chilean/Subantarctic pycnogonids. Since most molecular data on pycnogonids are from the Antarctic region, the new information constitutes a significant extension. The phylogenetic consensus tree displays 10 distinct, well-supported branches corresponding to the studied species, namely Achelia assimilis (Haswell, 1885), Ammothea spinosa (Hodgson, 1907), Tanystylum cavidorsum Stock, 1957, T. neorhetum Marcus, 1940 Marcus, E. 1940. Os Pantopoda brasileiros e os demais sul-americanos. Boletin da Faculdade de Filosofia, Ciencias e Letras da Universidade de São Paulo, Ser. 4, Zoologie, 19: 1–179.  [Google Scholar], Colossendeis macerrima Wilson, 1881, C. megalonyx Hoek, 1881, C. scoresbii Gordon, 1932 Gordon, I. 1932. Pycnogonida. – Discovery Reports Vol. VI., 1–137. London: Cambridge University Press.  [Google Scholar] , Callipallene margarita (Gordon, 1932 Gordon, I. 1932. Pycnogonida. – Discovery Reports Vol. VI., 1–137. London: Cambridge University Press.  [Google Scholar]), Pallenopsis patagonica (Hoek, 1881), and Anoplodactylus californicus Hall, 1912. These represent four superfamilies, and five of the 11 existing pycnogonid families (Bamber & El Nagar, 2011 Bamber, R. N. and El Nagar, A., eds. 2011. Pycnobase: World Pycnogonida Database http://www.marinespecies.org/pycnobase, accessed June 2012 [Google Scholar]): Ammotheidae Dohrn, 1881, Colossendeidae Hoek, 1881, Callipallenidae Hilton, 1942, Pallenopsidae Fry, 1978 and Phoxichilidiidae Sars, 1891. Within Achelia assimilis, four distinct subbranches correspond to the different geographic regions represented in our samples. While these include a total of 11 distinct haplotypes, the morphological differences among the corresponding specimens lie well within the variation described in the literature for this cosmopolitan species. Therefore, the four branches of A. assimilis might represent geographically limited subspecies rather than cryptic species. Repeated drastic glaciation of the fjord region during the Cenozoic resulting in alternating extinction and recolonization phases and the holobenthic lifecycle of sea spiders are discussed as the main factors resulting in the observed phylogeographic pattern. Standard barcoding sequences are confirmed as a suitable tool in addition to morphology for taxonomic analyses in Pycnogonida. The corresponding haplotype distribution patterns allow inferences on the biogeographical history of the relatively unexplored Chilean fjord region.     

On Udvardy's Insulantarctica province: a test from the weevils (Coleoptera: Curculionoidea)

'The scattered islands of the southern, cold ocean have clear affinities with one another . . . They are the basis for my Insulantarctica province.'
Udvardy, 1987, p. 190
Analyses of the distributional patterns of weevils (Coleoptera: Curculionoidea) from several Subantarctic islands, namely, Campbell, Auckland, Snares, Antipodes, Chatham, Falklands, Tierra del Fuego, Tristan da Cunha, Inaccessible, Nightingale, Gough, Marion, Prince Edward, Crozet, Kerguelen, and Heard, as well as South America and New Zealand, were carried out in order to determine their historical relationships, and to test the validity of Udvardy's (1987 ) Insulantarctica province. Three parsimony analyses of endemicity (PAE) considering (a) only species, (b) only supraspecific taxa, and (c) species and supraspecific taxa together, were undertaken. The following four groups emerged from the analyses: (1) New Zealand with the Snares, Auckland, Campbell, and Chatham Is., where New Zealand is the sister area to the Chatham Is., and the Auckland Is. are the sister area to Campbell I.; (2) South America with the Falkland Is. and Tierra del Fuego, where South America and Tierra del Fuego together are the sister area to the Falkland Is.; (3) Tristan da Cunha-Gough group, with the islands following the sequence Gough, Tristan da Cunha, Inaccessible, and Nightingale Is.; and (4) Kerguelen, Heard, Crozet, Marion, and Prince Edward Is., with Kerguelen and Heard Is. being sister areas, and Marion and Prince Edward Is. together being the sister area to the Crozet Is. It is concluded that the weevil fauna does not support the existence of an Insulantarctica province; the similarities among the different Subantarctic islands are due more to similar environmental conditions rather than to a common history.     

Barcoding of Central European Cryptops centipedes reveals large interspecific distances with ghost lineages and new species records from Germany and Austria (Chilopoda,Scolopendromorpha)

In order to evaluate the diversity of Central European Myriapoda species in the course of the German Barcode of Life project, 61 cytochrome c oxidase I sequences of the genus Cryptops Leach, 1815, a centipede genus of the order Scolopendromorpha, were successfully sequenced and analyzed. One sequence of Scolopendracingulata Latreille, 1829 and one of Theatopserythrocephalus Koch, 1847 were utilized as outgroups. Instead of the expected three species (Cryptopsparisi Brolemann, 1920; Cryptopsanomalans Newport, 1844; Cryptopshortensis (Donovan, 1810)), analyzed samples included eight to ten species. Of the eight clearly distinguishable morphospecies of Cryptops, five (Cryptopsparisi; Cryptopscroaticus Verhoeff, 1931; Cryptopsanomalans; Cryptopsumbricus Verhoeff, 1931; Cryptopshortensis) could be tentatively determined to species level, while a further three remain undetermined (one each from Germany, Austria and Croatia, and Slovenia). Cryptopscroaticus is recorded for the first time from Austria. A single specimen (previously suspected as being Cryptopsanomalans), was redetermined as Cryptopsumbricus Verhoeff, 1931, a first record for Germany. All analyzed Cryptops species are monophyletic and show large genetic distances from one another (p-distances of 13.7–22.2%). Clear barcoding gaps are present in lineages represented by >10 specimens, highlighting the usefulness of the barcoding method for evaluating species diversity in centipedes. German specimens formally assigned to Cryptopsparisi are divided into three clades differing by 8.4–11.3% from one another; their intra-lineage genetic distance is much lower at 0–1.1%. The three clades are geographically separate, indicating that they might represent distinct species. Aside from Cryptopsparisi, intraspecific distances of Cryptops spp. in Central Europe are low (<3.3%).     

Using DNA barcodes for assessing diversity in the?family Hybotidae (Diptera,Empidoidea)

Empidoidea is one of the largest extant lineages of flies, but phylogenetic relationships among species of this group are poorly investigated and global diversity remains scarcely assessed. In this context, one of the most enigmatic empidoid families is Hybotidae. Within the framework of a pilot study, we barcoded 339 specimens of Old World hybotids belonging to 164 species and 22 genera (plus two Empis as outgroups) and attempted to evaluate whether patterns of intra- and interspecific divergences match the current taxonomy. We used a large sampling of diverse Hybotidae. The material came from the Palaearctic (Belgium, France, Portugal and Russian Caucasus), the Afrotropic (Democratic Republic of the Congo) and the Oriental realms (Singapore and Thailand). Thereby, we optimized lab protocols for barcoding hybotids. Although DNA barcodes generally well distinguished recognized taxa, the study also revealed a number of unexpected phenomena: e.g., undescribed taxa found within morphologically very similar or identical specimens, especially when geographic distance was large; some morphologically distinct species showed no genetic divergence; or different pattern of intraspecific divergence between populations or closely related species. Using COI sequences and simple Neighbour-Joining tree reconstructions, the monophyly of many species- and genus-level taxa was well supported, but more inclusive taxonomical levels did not receive significant bootstrap support. We conclude that in hybotids DNA barcoding might be well used to identify species, when two main constraints are considered. First, incomplete barcoding libraries hinder efficient (correct) identification. Therefore, extra efforts are needed to increase the representation of hybotids in these databases. Second, the spatial scale of sampling has to be taken into account, and especially for widespread species or species complexes with unclear taxonomy, an integrative approach has to be used to clarify species boundaries and identities.     

甘肃省鱼类资源现状及DNA条形码在鱼类物种鉴定中的应用

为了摸清甘肃省土著鱼类资源与分布现状, 探索DNA条形码在鱼类辅助物种鉴定中的适用性, 2012年6-9月对甘肃境内黄河水系、嘉陵江水系和河西内陆河水系进行了较全面的鱼类调查。共采集鱼类标本3,087尾, 隶属于5目10科38属64种, 以鲤科种类最多, 为30种, 占总种数的46.88%。物种多样性分析表明, 在黄河水系的夏河和庄浪河多样性指数是所有调查点中最低的, 分别为1.38和1.09。嘉陵江水系各河段的多样性指数较高(H = 2.15-3.27), 其次为河西内陆河水系(H = 2.01-2.83)。在河西内陆河水系中, 疏勒河的均匀度指数最高, 为1.10, 黑河最低(0.68)。庄浪河的优势度指数最高, 为0.34, 而嘉陵江干流两当段的优势度指数在所有调查点中最低, 为0.04。利用DNA条形码分析了49种662尾标本的COI基因部分序列, 大部分种类在neighbor-joining系统树中形成各自的单系, 种内平均遗传距离0.88%, 种间平均遗传距离为9.99%, 在种内和种间COI序列遗传距离之间形成明显的条形码间隙, 斯氏高原鳅(Triplophysa stoliczkae)与达里湖高原鳅(T. dalaica), 甘肃高原鳅(T. robusta)与似鲇高原鳅(T. siluroides), 嘉陵裸裂尻鱼(Schizopygopsis kialingensis)与黄河裸裂尻鱼(S. pylzovi)之间的遗传距离低于2%, 甘肃高原鳅与似鲇高原鳅不能通过COI基因片段区分开, 其他两对物种可以采用核苷酸诊断法来进一步区分。斯氏高原鳅和拉氏鱼岁(Phoxinus lagowskii)种内遗传分歧较大, 揭示种内可能存在隐存种。结果表明, 对某些近缘种和不同地理种群差异较大的物种, 要将分子、形态和地理分布特点结合起来才能准确鉴定。     

Loss of planktotrophy and speciation: geographical fragmentation in the deep-water gastropod genus Bathytoma (Gastropoda,Conoidea) in the western Pacific

Dispersal capabilities are crucial in how speciation patterns are determined in marine invertebrates. Species possessing a long-living planktonic larva apparently have a dispersal advantage over those with non-planktotrophic development, and their distant populations may exchange genetic material, maintaining a broad geographical range for the species. Recent species of the gastropod genus Bathytoma (Conoidea) are all characterized by non-planktotrophic development, having most probably lost a free-swimming larva in the pre-Pliocene, as Miocene fossils have protoconchs indicating planktotrophic larval development. All have a bathyal distribution (100–1500 m), which implies that their capability for direct expansion on the bottom is restricted by both deep-sea basins and shallow-water areas, especially in insular West and South-West Indo-Pacific. Therefore, it can be hypothesized that Bathytoma populations should represent numerous, mostly allopatric taxa restricted to a single or contiguous island groups. We tested this hypothesis using molecular and morphological characters independently. One hundred and thirty-eight specimens from the Philippines, Solomons, Vanuatu, and the Coral Sea were sequenced for one mitochondrial (COI) and one nuclear (ITS2) gene, and 14 operational molecular units were recognized. When these molecular units are overlaid over shell characters, 13 species (11 unnamed) and one form of uncertain status are recognized: three occur in the Philippines, six in the Solomons and one in New Caledonia. Broad distributions (inter-archipelagic) are uncommon (three species). On the whole, the phylogeographic pattern of the diversity in the genus is rather complex and probably also reflects processes of sympatric and fine-scale allopatric speciation, and local extinctions. The eleven new species are described and named.     

DNA barcoding of stygofauna uncovers cryptic amphipod diversity in a calcrete aquifer in Western Australia's arid zone

The arid Yilgarn region of Western Australia contains numerous subterranean calcrete aquifers with unique assemblages of obligate groundwater invertebrates (stygofauna). We aimed to establish a DNA barcoding framework for the macro-invertebrates present in a single calcrete, as a basis for future assessment of biodiversity of the Yilgarn calcretes and for investigating food webs. Intense sampling of a bore field grid in the Sturt Meadows calcrete was undertaken to obtain representatives of the entire macro-invertebrate ecosystem. A 623-bp fragment of the mitochondrial cytochrome c oxidase 1 (COI) gene was used to provide DNA barcodes for stygobiont macro-invertebrates plus terrestrial organisms that are found in the calcrete. Phylogenetic analyses revealed the existence of 12 divergent monophyletic groups of haplotypes. Subterranean amphipods (Chiltoniidae) showed three groups of COI haplotypes with sequence divergences between them of >11%. Allozyme analyses found a large number of fixed allelic differences between these three amphipod groups, indicating that there are three morphologically cryptic species within the Sturt Meadows calcrete. Unlike the sister triplet of dytiscid beetles present, the amphipods are not sister clades and are more closely related to other Yilgarn and non-Yilgarn amphipods than to each other. Our results show that the aquifer contains at least 12 macro-invertebrate species and DNA barcoding provides a useful means for discriminating species in this system.     

COI barcoding of Nebelid testate amoebae (Amoebozoa: Arcellinida): extensive cryptic diversity and redefinition of the Hyalospheniidae Schultze

We used Cytochrome Oxidase Subunit 1 (COI) to assess the phylogenetic relationships and taxonomy of Nebela sensu stricto and similar taxa (Nebela group, Arcellinida) in order to clarify the taxonomic validity of morphological characters. The COI data not only successfully separated all studied morphospecies but also revealed the existence of several potential cryptic species. The taxonomic implications of the results are: (1) Genus Nebela is paraphyletic and will need to be split into at least two monophyletic assemblages when taxon sampling is further expanded. (2) Genus Quadrulella, one of the few arcellinid genera building its shell from self-secreted siliceous elements, and the mixotrophic Hyalosphenia papilio branch within the Nebela group in agreement with the general morphology of their shell and the presence of an organic rim around the aperture (synapomorphy for Hyalospheniidae). We thus synonymise Hyalospheniidae and Nebelidae. Hyalospheniidae takes precedence and now includes Hyalosphenia, Quadrulella (previously in the Lesquereusiidae) and all Nebelidae with the exception of Argynnia and Physochila. Leptochlamys is Arcellinida incertae sedis. We describe a new genus Padaungiella Lara et Todorov and a new species Nebela meisterfeldi n. sp. Heger et Mitchell and revise the taxonomic position (and rank) of several taxa. These results show that the traditional morphology-based taxonomy underestimates the diversity within the Nebela group, and that phylogenetic relationships are best inferred from shell shape rather than from the material used to build the shell.     

Global diversity and biogeography of Skeletonema species (bacillariophyta)

Recent studies have shown that the cosmopolitan diatom Skeletonema costatum sensu lato is composed of several morphologically and genetically distinct species. To assess whether the separate species have a cosmopolitan distribution, we analysed 184 strains from marine and estuarine sites worldwide. We identified the strains using light and electron microscopy, and we sequenced the hyper-variable region of nuclear LSU rDNA. All recently described species were genetically distinct, and all but two were morphologically distinct. Variability was found for the only ultrastructural character used to distinguish Skeletonema dohrnii and S. marinoi, which cannot be identified based on morphology alone. Furthermore, multiple genetically distinct taxa, which may represent cryptic species, were found within the S. menzelii and S. tropicum clades. We found that all currently recognized species of Skeletonema are widespread, however, gaps seem to occur in their geographical ranges. For example, some species are found in both the northern and southern temperate latitudes whereas other species appear to have only subtropical to tropical ranges. Skeletonema pseudocostatum and S. grethae seem to have more restricted geographical ranges because the former was not found along American coasts and the latter was encountered only in US waters. A taxonomic update is provided for Skeletonema strains currently available in several culture collections, which could aid reinterpretation of results obtained in comparative studies using these strains.     

First results of the German Barcode of Life (GBOL) – Myriapoda project: Cryptic lineages in German Stenotaenia linearis (Koch, 1835) (Chilopoda,Geophilomorpha)

As part of the German Barcode of Life (GBOL) Myriapoda program, which aims to sequence the COI barcoding fragment for 2000 specimens of Germany’s 200 myriapod species in the near future, 44 sequences of the centipede order Geophilomorpha are analyzed. The analyses are limited to the genera Geophilus Leach, 1814 and Stenotaenia Koch, 1847 and include a total of six species. A special focus is Stenotaenia, of which 19 specimens from southern, western and eastern Germany could be successfully sequenced. The Stenotaenia data shows the presence of three to four vastly different (13.7–16.7% p-distance) lineages of the genus in Germany. At least two of the three lineages show a wide distribution across Germany, only the lineage including topotypes of Stenotaenialinearis shows a more restricted distribution in southern Germany. In a maximum likelihood phylogenetic analysis the Italian species Stenotaenia ‘sorrentina’ (Attems, 1903) groups with the different German Stenotaenialinearis clades. The strongly different Stenotaenialinearis lineages within Germany, independent of geography, are a strong hint for the presence of additional, cryptic Stenotaenia species in Germany.