Molecular and morphometric analyses identify new lineages within a large Eumida (Annelida) species complex

We report on two new lineages of the Eumida sanguinea complex from Great Britain and describe one of them as a new species using a multilocus approach, including the mitochondrial DNA COI-5P and the nuclear markers ITS (ITS1, 5.8S rRNA and ITS2) and 28S rRNA. The molecular analysis placed Eumida mackiei sp. nov. in a monophyletic clade with 19.1% (COI), 10.1% (ITS) and 1.7% (28S) mean distance to its nearest neighbour. Molecular diagnoses were also applied to nine lineages within the E. sanguinea complex. This was complemented with morphometric data employing multivariate statistical analysis and the incorporation of statistical dissimilarities against three other described species from the complex. Eumida mackiei sp. nov. can be distinguished from E. notata and E. maia by the larger distance between the eyes and differences in morphometric proportions mainly in the dorsal and ventral cirri as well as in the prostomial appendages. E. sanguinea sensu stricto failed to produce a cluster of its own in the morphometric analysis, probably due to juvenile bias. Integrative taxonomy provided strong evidence to formally describe a new cryptic species that can now be used in biomonitoring or other relevant ecological research.     

Genetic analyses reveal further cryptic lineages within the Myotis nattereri species complex

In recent years, new cryptic mammalian species have been discovered in Europe, many of which belong to the order Chiroptera. Within this order, some species such as Myotis nattereri contain several cryptic lineages/species, especially in the Mediterranean region. Here we present genetic, phylogenetic and morphological analysis on the Myotis nattereri species complex, focusing on France which is thought to be a contact zone for the different lineages/species. We sequenced the full Cytochrome b gene from individuals from 23 localities and investigated diagnostic morphological characteristics. Our results reveal new phylogenetic relationships within the Myotis nattereri species complex and among closely related species. We discuss morphological characters which are synapomorphic to each lineage and can be used to differentiate them. Our results also demonstrate the presence of a new lineage within the Myotis nattereri species complex. This lineage, endemic to Corsica, possibly represents a new cryptic species for which we present preliminary ecological data. We further identify the presence of three lineages/species in France and detail their distribution with potential contact zones.     

Floral volatiles, pollinator sharing and diversification in the fig-wasp mutualism: insights from Ficus natalensis, and its two wasp pollinators (South Africa)

Combining biogeographic, ecological, morphological, molecular and chemical data, we document departure from strict specialization in the fig-pollinating wasp mutualism. We show that the pollinating wasps Elisabethiella stuckenbergi and Elisabethiella socotrensis form a species complex of five lineages in East and Southern Africa. Up to two morphologically distinct lineages were found to co-occur locally in the southern African region. Wasps belonging to a single lineage were frequently the main regional pollinators of several Ficus species. In South Africa, two sister lineages, E. stuckenbergi and E. socotrensis, pollinate Ficus natalensis but only E. stuckenbergi also regularly pollinates Ficus burkei. The two wasp species co-occur in individual trees of F. natalensis throughout KwaZulu-Natal. Floral volatile blends emitted by F. natalensis in KwaZulu-Natal were similar to those emitted by F. burkei and different from those produced by other African Ficus species. The fig odour similarity suggests evolutionary convergence to attract particular wasp species. The observed pattern may result from selection for pollinator sharing among Ficus species. Such a process, with one wasp species regionally pollinating several hosts, but several wasp species pollinating a given Ficus species across its geographical range could play an important role in the evolutionary dynamics of the Ficus-pollinating wasp association.     

Phylogenetic systematics of the gondwanan Empis macrorrhyncha group (Diptera,Empididae, Empidinae)

The Empis macrorrhyncha group (Diptera: Empididae) from cool to warm temperate areas of South America and Australia is diagnosed and cladistically analysed, and five new species, Empis animosa sp.n. , E. austera sp.n. , E. maculosa sp.n. , E. occidentalis sp.n. and E. pedivillosula sp.n. , are described. Cladistic analysis of 23 adult morphological characters for 14 species of the group generated a single tree of 28 steps (CI = 0.82; RI = 0.93). Monophyly was established on the basis of a single apomorphy, possession of a bilobed cercus of the male hypopygium. Three main clades were inferred: clade 1 included three Patagonian and a single southwestern Australian species; clade 2 included two species from southeastern Australia; clade 3 included a large Patagonian group of five species and a single southeastern Australian species. The E. fulvicollis complex (clade 1) is a sister‐group of the E. macrorrhyncha complex (clades 2 + 3). A provisional historical biogeographic hypothesis is advanced correlating the appearance of the South American and Australian sister lineages with the timing of the break‐up of Gondwana.     

Coalescent species delimitation of a Sumatran parachuting frog

We conduct species delimitation of the widespread parachuting frog species Rhacophorus catamitus using samples from across the island of Sumatra, Indonesia. We use mitochondrial, genomic and morphological data, and find that R. catamitus is composed of three lineages corresponding to northern, central and southern lineages. Mitochondrial and genomic data show admixture or incomplete lineage sorting between the central and southern lineages, but deep divergence from the northern lineage. Coalescent species delimitation supports a three species model for this complex, and we recommend that the northern lineage be described as a new species. Our study highlights the power of coalescent species delimitation in an integrative framework for identifying unrecognised diversity in understudied tropical species complexes. We also emphasise the evolutionary importance of northern Sumatra, a region that harboured montane refugia during Pliocene–Pleistocene climate change, but has also been heavily affected by volcanic activity.     

The Evolutionary History of the Genus Acanthamoeba and the Identification of Eight New 18S rRNA Gene Sequence Types

ABSTRACT The 18S rRNA gene ( Rns ) phylogeny of Acanthamoeba is being investigated as a basis for improvements in the nomenclature and taxonomy of the genus. We previously analyzed Rns sequences from 18 isolates from morphological groups 2 and 3 and found that they fell into four distinct evolutionary lineages we called sequence types T1-T4. Here, we analyzed sequences from 53 isolates representing 16 species and including 35 new strains. Eight additional lineages (sequence types T5-T12) were identified. Four of the 12 sequence types included strains from more than one nominal species. Thus, sequence types could be equated with species in some cases or with complexes of closely related species in others. The largest complex, sequence type T4, which contained six closely related nominal species, included 24 of 25 keratitis isolates. Rns sequence variation was insufficient for full phylogenetic resolution of branching orders within this complex, but the mixing of species observed at terminal nodes confirmed that traditional classification of isolates has been inconsistent. One solution to this problem would be to equate sequence types and single species. Alternatively, additional molecular information will be required to reliably differentiate species within the complexes. Three sequence types of morphological group 1 species represented the earliest divergence in the history of the genus and, based on their genetic distinctiveness, are candidates for reclassification as one or more novel genera.     

Molecular Assessment and Taxonomic Status of the Rapid Racerunner （Eremias velox complex） with Particular Attention to the Populations in Northwestern China

The rapid racerunner,Eremias velox,is a widely distributed lizard from the northern Caucasus across entire Central Asia eastward to China.It is increasingly common to accept E.velox as a species complex in its entire range.To date,published morphological and molecular systematic hypotheses of this complex are only partially congruent,and its taxonomic status and evolutionary history are still far from clear.The mitochondrial cytochrome b gene and12S rRNA sequences were used to evaluate the taxonomy of this complex,with particular attention to the phylogenetic placement of populations in northwestern China.Examination of the phylogenetic analyses recovers seven distinct,biogeographically discrete,and well-supported clades,revealing genetically identifiable populations corresponding to some previously morphology-defined subspecies.Chinese E.v.roborowskii appears to have split from other Central Asian rapid racerunner lizards well before differentiation occurred among the latter taxa.Specifically,we corroborate that there are two subspecies occurring in China,i.e.,E.v.velox and E.v.roborowskii.We recommend a novel subspecific status for the phenotypically and genetically distinct populations in southern Aral Sea region of Uzbekistan previously assigned to E.v.velox.Finally,each of the three independently evolving lineages from Iranian Plateau should be recognized as three species new to science under the general lineage concept.     

Cophylogeny on a fine scale: Geomydoecus chewing lice and their pocket gopher hosts, Pappogeomys bulleri

Many species of pocket gophers and their ectoparasitic chewing lice have broadly congruent phylogenies, indicating a history of frequent codivergence. For a variety of reasons, phylogenies of codiverging hosts and parasites are expected to be less congruent for more recently diverged taxa. This study is the first of its scale in the pocket gopher and chewing louse system, with its focus entirely on comparisons among populations within a single species of host and 3 chewing louse species in the Geomydoecus bulleri species complex. We examined mitochondrial DNA from a total of 46 specimens of Geomydoecus lice collected from 11 populations of the pocket gopher host, Pappogeomys bulleri. We also examined nuclear DNA from a subset of these chewing lice. Louse phylogenies were compared with a published pocket gopher phylogeny. Contrary to expectations, we observed a statistically significant degree of parallel cladogenesis in these closely related hosts and their parasites. We also observed a higher rate of evolution in chewing louse lineages than in their corresponding pocket gopher hosts. In addition, we found that 1 louse species (Geomydoecus burti) may not be a valid species, that subspecies within G. bulleri are not reciprocally monophyletic, and that morphological and genetic evidence support recognition of a new species of louse, Geomydoecus pricei.     

The diversity of Terfezia desert truffles: new species and a highly variable species complex with intrasporocarpic nrDNA ITS heterogeneity

Desert truffles belonging to Terfezia are well known mycorrhizal members of the mycota of the Mediterranean region and the Middle East. We aimed to test (i) whether the morphological criteria of Terfezia species regularly collected in Spain enable their separation and (ii) whether the previously hypothesized edaphic/biotic specificity of one group could be confirmed by study of a larger number of specimens. The species T. arenaria and T. claveryi can be identified unambiguously by morphological characters. We consider T. leptoderma as a distinct species while several lineages of similar spiny spored Terfezia truffles with cellular peridium were detected that have no obvious anatomical differences. Several species treated generally as synonyms of T. olbiensis have been described in this group, and because they cannot be unambiguously assigned to separate lineages we propose to consider the group as the T. olbiensis species complex. A high level of intrasporocarpic variation of the nrDNA ITS was detected in the T. olbiensis species complex, especially in one of its lineages. We detected no exclusive specificity to either plant associates or soil, except in T. leptoderma, which was associated with Quercus spp. and cistaceous plants on acidic soils. Nevertheless the clades showed a tendency either to associate with Quercus/Helianthemum/Cistus or Pinus hosts. Specimens having distinct anatomical features, reticulate spores and cellular peridium formed a separate group in the molecular phylogenetic analyses of nrDNA ITS and LSU regions; for these specimens we propose a new species, Terfezia alsheikhii sp. nov.     

Genetic divergence and geographic variation in the deep-water Conus orbignyi complex (Mollusca: Conoidea)

Puillandre, N. et al. (2010) Genetic divergence and geographic variation in a deep-water cone lineage: molecular and morphological analyses of the Conus orbignyi complex (Mollusca: Conoidea).The cone snails (family Conidae) are a hyperdiverse lineage of venomous gastropods. Two standard markers, COI and ITS2, were used to define six genetically-divergent groups within a subclade of Conidae that includes Conus orbignyi; each of these was then evaluated based on their shell morphology. We conclude that three forms, previously regarded as subspecies of Conus orbignyi are distinct species, now recognized as Conus orbignyi, Conus elokismenos and Conus coriolisi. In addition, three additional species (Conus pseudorbignyi, Conus joliveti and Conus comatosa) belong to this clade. Some of the proposed species (e.g., Conus elokismenos) are possibly in turn complexes comprising multiple species. Groups such as Conidae illustrate the challenges generally faced in species delimitation in biodiverse lineages. In the case of the Conus orbignyi complex, not only are there definable, genetically divergent lineages, but also considerable geographic variation within each group. Our study suggests that an intensive analysis of multiple specimens within a single locality helps to minimize the confounding effects of geographic variation and can be a useful starting point for circumscribing different species within such a confusing complex.     

Cryptic speciation in the Anadenobolus excisus millipede species complex on the Island of Jamaica

Anadenobolus excisus is a large species of millipede endemic to the Caribbean Island of Jamaica. Initial detailed morphological studies showed little or no discrete variation across this species' distribution in somatic or, in particular. genitalic morphology. However, a molecular survey based on approximately 1000 base pairs of the mitochondrial (mtDNA) 16S rRNA gene that examines 242 individuals sampled from 54 localities reveals three highly divergent mtDNA lineages. A lack of discrete morphological differentiation suggests that genetic and morphological divergence may be decoupled, a pattern inconsistent with a number of evolutionary models. In contrast to minimal morphological divergence, size variation among mtDNA lineages suggests that character displacement has occurred and that these lineages are cohesive in sympatry. We conclude that A. excisus is actually a complex of three cryptic species and that morphological approaches to delineating millipede species may sometimes underestimate evolutionary diversity.     

Regional specialization of Sarcodes sanguinea (Ericaceae) on a single fungal symbiont from the Rhizopogon ellenae (Rhizopogonaceae) species complex

We have sampled the mycorrhizal roots of 76 snow plants (Sarcodes sanguinea, Monotropoideae, Ericaceae) in two areas of the Sierra Nevada of California that are ～180 km apart. To identify the fungal symbionts associated with these plants, we first analyzed restriction fragment length polymorphisms (RFLPs) of the internal transcribed spacer region (ITS) of the fungal nuclear ribosomal repeat. Fungal ITS-RFLPs were successfully produced from 57 of the 76 plants sampled, and all symbionts shared the same DNA fragment pattern. The morphology of S. sanguinea mycorrhizae was consistent with that expected from a Rhizopogon species in section Amylopogon. To confirm and refine this identification, a total of six fungal ITS sequences were determined from S. sanguinea mycorrhizae. These sequences were analyzed together with eight existing and eight newly determined ITS sequences from Rhizopogon section Amylopogon. The newly determined sequences include an ITS sequence from the fungal symbiont of pine drops (Pterospora andromedea, Monotropoideae, Ericaceae), a plant that was previously reported to be exclusively associated with the Rhizopogon subcaerulescens group. When these sequences were analyzed together, the Sarcodes symbionts grouped tightly with several collections of R. ellenae including the holotype, one collection of R. idahoensis, and one collection of R. semireticulatus. A different lineage comprised collections of R. subgelatinosus, R. subcaerulescens, another collection of R. semireticulatus, and the Pterospora symbiont. We conclude that S. sanguinea associates exclusively with a single species in the R. ellenae species complex throughout our sampling range. These results indicate a much higher level of specificity in S. sanguinea than was previously reported and confirm the emerging pattern that nonphotosynthetic, monotropoid plants generally associate very specifically with a narrow range of ectomycorrhizal fungi.     

Exploring species boundaries with multiple genetic loci using empirical data from non‐biting midges

Over the past decade, molecular approaches to species delimitation have seen rapid development. However, species delimitation based on a single locus, for example, DNA barcodes, can lead to inaccurate results in cases of recent speciation and incomplete lineage sorting. Here, we compare the performance of Automatic Barcode Gap Discovery (ABGD), Bayesian Poisson tree processes (PTP), networks, generalized mixed Yule coalescent (GMYC) and Bayesian phylogenetics and phylogeography (BPP) models to delineate cryptic species previously detected by DNA barcodes within Tanytarsus (Diptera: Chironomidae) non‐biting midges. We compare the results from analyses of one mitochondrial (cytochrome c oxidase subunit I [COI]) and three nuclear (alanyl‐tRNA synthetase 1 [AATS1], carbamoyl phosphate synthetase 1 [CAD1] and 6‐phosphogluconate dehydrogenase [PGD]) protein‐coding genes. Our results show that species delimitation based on multiple nuclear DNA markers is largely concordant with morphological variation and delimitations using a single locus, for example, the COI barcode. However, ABGD, GMYC, PTP and network models led to conflicting results based on a single locus and delineate species differently than morphology. Results from BPP analyses on multiple loci correspond best with current morphological species concept. In total, 10 lineages of the Tanytarsus curticornis species complex were uncovered. Excluding a Norwegian population of Tanytarsus brundini which might have undergone recent hybridization, this suggests six hitherto unrecognized species new to science. Five distinct species are well supported in the Tanytarsus heusdensis species complex, including two species new to science.     

Eunicid polychaetes (Annelida) from Scandinavian and adjacent waters. Family Eunicidae

Four species of Eunice [E. pennata (0. F. Müller, 1776), E. dubitata Fauchald, 1974, E. norvegica (Linné, 1767), and E. harassii (Audouin & Milne-Edwards, 1833)] and one species of Marphysa [M . sp., close to M. sanguinea (Montagu, 1815)] are described and a branchial formula is proposed. The genus Marphysa is reported from Scandinavian waters for the first time. The specimen of Marphysa studied has tridentate composite falcigers as well as composite spinigers in the anterior setigers, whereas M. sanguinea has been reported to have composite spinigers only. The geographical distribution of all five species is given and intraspecific variation is discussed. Reports of E. oerstedii (Stimpson, 1853) from Europe are discussed and specimens of this species are compared with E. dubitata . The behaviour of E. pennata and E. dubitata has been observed in aquaria.     

Exceptional cryptic diversity and multiple origins of parthenogenesis in a freshwater ostracod

The persistence of asexual reproduction in many taxa depends on a balance between the origin of new asexual lineages and the extinction of old ones. This turnover determines the diversity of extant asexual populations and so influences the interaction between sexual and asexual modes of reproduction. Species with mixed reproduction, like the freshwater ostracod (Crustacea) morphospecies Eucypris virens, are a good model to examine these dynamics. This species is also a geographic parthenogen, in which sexual females and males co-exist with asexual females in the circum-Mediterranean area only, whereas asexual females occur all over Europe. A molecular phylogeny of E. virens based on the mitochondrial COI and 16S fragments is presented. It is characterised by many distinct clusters of haplotypes which are either exclusively sexual or asexual, with only one exception, and are often separated by deep branches. Analysis of the phylogeny reveals an astonishing cryptic diversity, which indicates the existence of a species complex with more than 40 cryptic taxa. We therefore suggest a revision of the single species status of E. virens. The phylogeny indicates multiple transitions from diverse sexual ancestor populations to asexuality. Although many transitions appear to be ancient, we argue that this may be an artefact of the existence of unsampled or extinct sexual lineages.     

Disentangling taxonomy within the Rhabditis (Pellioditis) marina (Nematoda, Rhabditidae) species complex using molecular and morhological tools

Correct taxonomy is a prerequisite for biological research, but currently it is undergoing a serious crisis, resulting in the neglect of many highly diverse groups of organisms. In nematodes, species delimitation remains problematic due to their high morphological plasticity. Evolutionary approaches using DNA sequences can potentially overcome the problems caused by morphology, but they are also affected by theoretical flaws. A holistic approach with a combination of morphological and molecular methods can therefore produce a straightforward delimitation of species. The present study investigates the taxonomic status of some highly divergent mitochondrial haplotypes in the Rhabditis ( Pellioditis ) marina species complex by using a combination of molecular and morphological tools. We used three molecular markers (COI, ITS, D2D3) and performed phylogenetic analyses. Subsequently, morphometric data from nearly all lineages were analysed with multivariate techniques. We included R. ( P. ) mediterranea and R. (R.) nidrosiensis to infer species status of the observed lineages. The results showed that highly divergent genotypic clusters were accompanied by morphological differences, and we created a graphical polytomous key for future identifications. This study indisputably demonstrates that R. ( P. ) marina and R. ( P. ) mediterranea belong to a huge species complex and that biodiversity in free-living marine nematodes may be seriously underestimated.  © 2008 The Linnean Society of London, Zoological Journal of the Linnean Society , 2008, 152 , 1–15.