Molecular Barcoding of Aquatic Oligochaetes: Implications for Biomonitoring

Aquatic oligochaetes are well recognized bioindicators of quality of sediments and water in watercourses and lakes. However, the difficult taxonomic determination based on morphological features compromises their more common use in eco-diagnostic analyses. To overcome this limitation, we investigated molecular barcodes as identification tool for broad range of taxa of aquatic oligochaetes. We report 185 COI and 52 ITS2 rDNA sequences for specimens collected in Switzerland and belonging to the families Naididae, Lumbriculidae, Enchytraeidae and Lumbricidae. Phylogenetic analyses allowed distinguishing 41 lineages separated by more than 10 % divergence in COI sequences. The lineage distinction was confirmed by Automatic Barcode Gap Discovery (ABGD) method and by ITS2 data. Our results showed that morphological identification underestimates the oligochaete diversity. Only 26 of the lineages could be assigned to morphospecies, of which seven were sequenced for the first time. Several cryptic species were detected within common morphospecies. Many juvenile specimens that could not be assigned morphologically have found their home after genetic analysis. Our study showed that COI barcodes performed very well as species identifiers in aquatic oligochaetes. Their easy amplification and good taxonomic resolution might help promoting aquatic oligochaetes as bioindicators for next generation environmental DNA biomonitoring of aquatic ecosystems.     

Molecular phylogeny and phylogeography of flightless beetles Parechthistatus gibber and Hayashiechthistatus inexpectus (Coleoptera: Cerambycidae) inferred from mitochondrial COI gene sequences

To elucidate the speciation patterns of two endemic flightless cerambycid beetles, Parechthistatus gibber and Hayashiechthistatus inexpectus, molecular phylogenetic analysis was carried out. A 1144 bp region of the cytochrome oxidase subunit I gene was sequenced for individuals from 51 local populations of these species. There were nine haplotype lineages of P. gibber, and H. inexpectus was included within a P. gibber lineage. These lineages were highly divergent and occurred in different regions. Based on previously published molecular change rates for the COI gene (1.5–2.3% per million years), the time of divergence of P. gibber COI haplotypes was inferred to be 3.0–4.6 million years ago, in the Pliocene.     

Decoupling of molecular and morphological evolution in deep lineages of a meiobenthic harpacticoid copepod

Molecular and biochemical genetic analyses have revealed that many marine invertebrate taxa, including some well-studied and presumably cosmopolitan species, are actually complexes of sibling species. When morphological differences are slight and estimated divergence times are old, data suggest either unusually high rates of sequence evolution or long-term morphological stasis. Here, five gene regions (mitochondrial cytochrome oxidase subunit I and large-subunit ribosomal 16S rDNA and nuclear ITS1, 5.8S rDNA, and ITS2) were analyzed in four geographic samples of the meiobenthic harpacticoid copepod Cletocamptus deitersi. Molecular sequences revealed four extremely differentiated molecular lineages with unalignable nuclear intergenic spacers and mitochondrial uncorrected divergences reaching 25% (cytochrome oxidase) and 36% (16S rDNA). These levels of divergence are greater than those reported previously for congeneric species in diverse invertebrate taxa, including crustaceans. The nominally intraspecific divergence matches or exceeds the corresponding divergence from a known congener (Cletocamptus helobius). A molecular clock applied to the cytochrome oxidase subunit I data suggests that these lineages split in the Miocene, consistent with the fossil record of a North American Cletocamptus from the same period. Morphological differences among the major lineages are subtle but congruent with the patterns of genetic differentiation. Our conclusion, based on concordant patterns of variation in two mitochondrial and three nuclear gene regions, as well as morphological observations, is that C. deitersi in North America is composed of at least four separate species by the genealogical concordance, phylogenetic, and morphological-species criteria. Alternative explanations for the deep phylogenetic nodes and apparent morphological stasis, including high rates of sequence evolution, balancing selection, and genetic signatures of historical events, are considered unlikely.     

Large amounts of genetic divergence among Italian species of the genus Orchesella (Insecta, collembola) and the relationships of two new species

The phylogenetic position of two putative new species of the collembolan genus Orchesella was investigated by comparison with four other Italian species of the genus using a fragment of the mitochondrial gene encoding for subunit I of cytochrome c oxidase (COI). The gene showed the well-known A + T bias, typical of insect mitochondrial DNA, although A + T content was not as high as that observed in species belonging to more derived insect orders. The large number of variable sites in 3rd codon positions (85.2% variable) suggested that these sites contain significant homoplasy due to multiple hits. Despite the lack of morphological differentiation, the COI portion examined shows remarkable levels of genetic divergence between the putative species and their closest relatives. Phylogenetic analysis suggests that one of the putative new species is related to O. villosa, whereas the other is included in a clade with O. cincta and O. ranzii. The species O. chiantica appears to be related to O. villosa, agreeing with previous allozyme data.     

Molecular evidence suggesting species in the zoanthid genera Palythoa and Protopalythoa (Anthozoa: Hexacorallia) are congeneric

Taxonomic status of the zoanthid genera Palythoa and Protopalythoa has been in question for almost a century. Separation of the two genera has been based on traditional morphological methods (colony and polyp form, nematocyst size and form, and number of septa), with Palythoa polyps embedded in a well developed coenenchyme and Protopalythoa polyps standing free and clear of the coenenchyme. Here we sequenced two mitochondrial regions, the cytochrome oxidase I (COI) gene and 16S ribosomal DNA (16S rDNA) genes, from Palythoa and Protopalythoa samples from various parts of the world and performed phylogenetic analyses of the sequence data. The phylogenetic trees for both COI and 16S rDNA from Palythoa and Protopalythoa show four monophyletic groups (designated Palythoa tuberculosa, Palythoa heliodiscus, Palythoa mutuki 1, and Palythoa mutuki 2), with levels of sequence divergence (COI and 16S rDNA divergence approximately 0.0 approximately 1.1%) similar to or lower than that previously found among congeneric species within the closely related genus Zoanthus. Surprisingly, sequence differences among Palythoa tuberculosa, Palythoa mutuki 1, and Palythoa mutuki 2 were negligible (0.0 approximately 0.2% for both COI and 16S rDNA), potentially indicating relationships below the species level. Our sequences align well with the few Palythoa and Protopalythoa sequences reported to date. These findings strongly indicate that our samples represent a minimum of two and possibly up to four species (the Palythoa tuberculosa - P. mutuki 1 - P. mutuki 2 group, and P. heliodiscus) within the genus Palythoa, and that the genus Protopalythoa is erroneous nomenclature.     

Cryptic divergence and strong population structure in the colonial invertebrate Pycnoclavella communis (Ascidiacea) inferred from molecular data

We studied sequence variation in the mitochondrial gene cytochrome c oxidase subunit I (COI) for 135 individuals from eight Mediterranean populations of the colonial ascidian Pycnoclavella communis across most of its presently known range of distribution in the Mediterranean. Three haplotypes from Atlantic locations were also included in the study. Phylogenetic, phylogeographic and population genetic analyses were used to unravel the genetic variability within and between populations. The study revealed 32 haplotypes for COI, 29 of them grouped within two Mediterranean lineages of P. communis (mean nucleotide divergence between lineages was 8.55%). Phylogenetic and network analyses suggest the possible existence of cryptic species corresponding to these two lineages. Population genetic analyses were restricted to the five populations belonging to the main genetic lineage, and for these localities we compared the information gleaned from COI sequence data and from eight microsatellite loci. A high genetic divergence between populations was substantiated using both kinds of markers (COI, global Fst=0.343; microsatellite loci, global Fst=0.362). There were high numbers of private haplotypes (COI) and alleles (microsatellites) in the populations studied. Restricted gene flow and inbreeding occur in the present range of distribution of the species. Microsatellite loci showed a strong incidence of failed amplifications, which we attribute to the marked intraspecies variability that hampered the application of these highly specific markers. Our results show important genetic variability at all levels studied, from within populations to between basins, possibly coupled to speciation processes. This variability is attributable to restricted gene flow among populations due to short-distance dispersal of the larvae.     

Phylogeography and phylogeny of the genus Acanthonyx (Decapoda,Epialtidae) in the north‐east Atlantic and Mediterranean

The genus Acanthonyx Latreille, 1828 (Majoidea, Epialtidae) contains 17 or 18 known species, depending on competing taxonomic views, that are widely distributed across the world. Morphologically, most species look superficially alike, and therefore, similar taxonomic concepts have been described under different names. Consequently, there is today a considerable list of synonyms, which further complicates the taxonomy of the genus Acanthonyx. In this study, we conducted a phylogeographical and phylogenetic analysis of populations of the genus Acanthonyx in the NE Atlantic, Mediterranean and Macaronesia using the mitochondrial cytochrome oxidase subunit I (COI) and the nuclear 28S rRNA loci. Our phylogenetic and phylogeographical results revealed that Acanthonyx lunulatus sensu lato is a complex of three distinct lineages: one corresponding to the previously described Acanthonyx brevifrons, another to A. lunulatus sensu stricto and a third to a yet undescribed group. Whereas our results confirms that A. brevifrons deserves the status of a species, as it can be easily distinguishable from A. lunulatus by a few morphological traits, we could not find any such traits suitable for the discrimination between A. lunulatus sensu stricto and the third lineage. Furthermore, the degree of COI divergence between this lineage and A. lunulatus is below average levels for Decapoda species. Yet, no shared haplotypes have been detected between them. The differences found in the nuclear gene (indels), together with the sympatric occurrence of the two forms, prompt for a more detailed analysis of this group. Overall, the results show that significant genetic differentiation between specimens with similar morphology occurs in the Epialtidae, thus reinforcing the importance of combining morphological and genetic tools to fully resolve the taxonomy of these decapods.     

Integrative taxonomy in two free-living nematode species complexes

Integrative taxonomy considers species boundaries from multiple, complementary perspectives, with the main objective being to compare the observed data against the predictions of the methodologies used. In the present study we used three methods for delineating species boundaries within the cosmopolitan nematode species Rhabditis ( Pellioditis ) marina and Halomonhystera disjuncta . First, phylogenetic relationships among molecular sequences from the mitochondrial cytochrome oxidase c subunit 1 gene (COI), and from two nuclear regions, internal transcribed spacer (ITS) and D2D3, were analysed. Subsequently, multivariate morphometric analysis was used to investigate whether concordant molecular lineages were also morphologically distinct. When morphological differences were found, typological taxonomy was performed to identify fixed or non-overlapping characters between lineages. Interbreeding experiments were conducted between the two closest related lineages of R . ( P. ) marina to investigate potential reproductive isolation. This integrative approach confirmed the presence of several species within each nominal species: molecular lineages were concordant across two independent loci (COI and ITS), and were characterized by significant morphological divergence. Most lineages were also detectable in the D2D3 region, but were less resolved. The two lineages investigated in our study did not produce offspring. Our results highlight that classical taxonomy grossly underestimates species diversity within the phylum Nematoda.  © 2008 The Linnean Society of London, Biological Journal of the Linnean Society , 2008, 94 , 737–753.     

Two mitochondrial lineages in Korean freshwater Corbicula (Corbiculidae: bivalvia)

The Korean freshwater Corbicula was surveyed genetically by sequencing 614-bp homologous fragment of mitochondrial cytochrome oxidase I subunit. Among a total of 127 individuals collected from 12 Korean freshwater localities we found only two COI haplotypes and these differed by a total of 9 base substitutions. Although the sequence divergence between the two haplotypes is moderate (p = 1.47%), placing the two mitotypic sequences in the context of Asian mtDNA phylogeny reveals that Korean freshwater Corbicula is comprised of two independent freshwater mitochondrial lineages. These results are in serious disagreement with the long-standing conclusions of earlier conchology-based taxonomic work on Korean Corbicula in which a number of species names (a minimum of 10 nominal species) have been used. This indicates that morphological characteristics alone are poor criteria for species-level identification in this group. In addition, our COI dataset shows that there is an extremely low level of genetic variation in Korean freshwater populations, suggesting that these populations have passed through a severe population bottleneck that greatly reduced their genetic variability. Our data also provide new information on the biogeographic distribution of Korean freshwater Corbicula. When haplotypic frequencies were compared, it was evident that the two Korean freshwater mitochondrial lineages have achieved very different distribution ranges: the predominant lineage (FWKR1) is widely distributed in Korean freshwater systems, whereas the minor lineage (FWKR2) is restricted to a relatively narrow range.     

Population Structure and Historical Demography of the Oriental River Prawn (Macrobrachium nipponense) in Taiwan

The oriental river prawn (Macrobrachium nipponense) is a non-obligatory amphidromous prawn, and it has a wide distribution covering almost the entire Taiwan. Mitochondrial DNA fragment sequences of the cytochrome oxidase subunit I (COI) and 16S rRNA were combined and used to elucidate the population structure and historical demography of oriental river prawn in Taiwan. A total of 202 individuals from six reservoirs and three estuaries were separately collected. Nucleotide diversity (π) of all populations was 0.01217, with values ranging from 0.00188 (Shihmen Reservoir, SMR, northern Taiwan) to 0.01425 (Mingte Reservoir, MTR, west-central Taiwan). All 76 haplotypes were divided into 2 lineages: lineage A included individuals from all sampling areas except SMR, and lineage B included specimens from all sampling locations except Chengching Lake Reservoir (CLR) and Liyu Lake Reservoir (LLR). All F _ST values among nine populations were significantly different except the one between Jhonggang River Estuary (JGE, west-central Taiwan) and Kaoping River Estuary (KPE, southern Taiwan). UPGMA tree of nine populations showed two main groups: the first group included the SMR and Tamsui River Estuary (TSE) (both located northern Taiwan), and the second one included the other seven populations (west-central, southern and eastern Taiwan). Demographic analyses implied a population expansion occurred during the recent history of the species. The dispersal route of this species might be from China to west-central and west-southern Taiwan, and then the part individuals belonging to lineage A and B dispersed southerly and northerly, respectively. And then part individuals in west-central Taiwan fell back to and stay at estuaries as the sea level rose about 18,000 years ago.     

Heterogeneous colonization pattern of European Cyprinids, as highlighted by the dace complex (Teleostei: Cyprinidae)

The dace (Leuciscus leuciscus), with a very large geographic distribution all over Europe, represents an interesting species model for studies of the global mechanisms underlying aquatic system biodiversity. To assess the congruence with the past colonization process hypothesis of the freshwater fauna in Western Europe, we investigated the evolutionary history of this species, by integrating morphological variation (eight meristic characters), mitochondrial (cytochrome b, 16S rDNA and control region, over a total of 2169 bp) and nuclear (12 allozymes loci) phylogenetic relationships, and investigating population dynamics via expansion, migration, bottleneck, and divergence time analyses. We carried out nested clade phylogeographic analysis for a total of 663 specimens from 31 populations taken from all over the distribution area. Unlike previous studies, we found that L. leuciscus is currently constituted by five lineages belonging to two clades (yielding 6.3% of pairwise divergence). The relationships between these lineages were accounted for by complex biogeographical patterns due to Pliocene and Pleistocene paleoclimatic events, validating the identification of new glacial refuges for freshwater fish in Western Europe. Finally, we demonstrated hybridization between L. leuciscus and Leuciscus idus.     

Vicariance of Pyrocoelia fireflies (Coleoptera: Lampyridae) in the Ryukyu islands,Japan

We performed molecular phylogenetic analyses based on the mitochondrial COI gene (687 bp) and the nuclear 28S rRNA gene (715 bp) and reconstructed phylogenetic trees of the Pyrocoelia fireflies in the Ryukyu Islands and eastern Asia. Age calibration was done using a robust geological constraint: the Okinawa trough and associated straits began to rift at 1.55 Ma, isolating the Ryukyu Islands from the Chinese continent, Japanese islands, Taiwan island and some of the islands from each other. We suggest that the physical isolation of these islands began to generate the allopatric speciation within these islands, so the timing of this isolation was assigned to an appropriate node. The topology is completely concordant among phylogenetic trees reconstructed using MEGA (maximum‐likelihood), raxmlGUI (maximum‐likelihood) and BEAST (Bayesian inference; including combined analysis of COI and 28S rRNA genes). Two lineages are recognized, related to their emergence time; spring to summer, and autumn. In each lineage, vicariance is inferred to have begun at 1.55 Ma from our phylogenetic and geological analyses. In lineage 1, P. oshimana (Amami), P. matsumurai (Okinawa), P. discicollis (W. Japan), P. fumosa (E. Japan) and P. abdominalis (Yaeyama) were differentiated. In lineage 2, P. rufa (Tsushima and Korea), P. miyako (Miyako‐jima), P. atripennis (Ishigaki‐jima) and P. praetexta (Taiwan and HongKong) were differentiated. Pyrocoelia analis (Taiwan and China) emerges throughout the year except for winter, and constitutes another lineage. We suggest that Pyrocoelia fireflies differentiated at 2 Ma to generate these three lineages. The base substitution rate for the COI gene is estimated as 4.48% Myr^–1 and that for the 28S rRNA gene is 0.394%  Myr^–1, and these rates were used in a combined BEAty analysis in BEAST.     

Phylogeny of Glaucosomatidae inferred from molecular evidence

Most species of glaucosomatids (Teleostei: Glaucosomatidae) are endemic to Australia, except Glaucosoma buergeri that is widely distributed from Australia to Japan. This study elucidated phylogenetic relationships among glaucosomatids based on the morphological characters of the saccular‐otolith sagitta, in addition to molecular evidence of mitochondrial 16S rDNA, cytochrome oxidase I (COI) and cytochrome b (cyt b) sequences, and nuclear rhodopsin sequences. The topologies of individuals' phylogenetic trees, based on 16S rDNA, COI and cyt b sequences, were statistically indistinguishable from one another, and were only slightly different from a tree based on rhodopsin sequences. These molecular tree topologies, however, differed from species relationships in morphology‐based phylogenetic hypothesis proposed in previous studies. Specimens of G. buergeri from Australia and Taiwan showed differences in the sagitta and molecular differentiation at the four genes, suggesting a possible speciation event. Both molecular and morphological evidences indicate that Glaucosoma magnificum is the plesiomorphic sister species of other glaucosomatid species. Glaucosoma hebraicum is the sister species of a clade composed of G. buergeri and Glaucosoma scapulare. Molecular and morphological evidences also support the species status of G. hebraicum.     

The genetic differentiation of a cricket (Velarifictorus micado) with two modes of life cycle in East Asia after the middle Pleistocene and the invasion origin of the United States of America

The cricket Velarifictorus micado is widely distributed in East Asia and colonized the United States of America (the USA) in 1959. It has two life cycles: egg and nymph diapause. We aimed to investigate the biogeographic boundary between them and determine when and why V. micado diverged. Mitochondrial fragments including COI and CytB were used for haplotype network, demographic analysis, and divergence time estimation in individuals of East Asia. We selected several samples from the USA to find out the colonization origin. The haplotype network indicated there were three lineages based on COI, NE lineage (the egg diapause and mainly distributed in the northern regions), SE lineage (the egg diapause and mainly distributed in the southern regions), and SN lineage (the nymph diapause and mainly distributed in the southern regions). The molecular chronograms indicated that the first divergence of V. micado into two main lineages, NE and southern lineages (SE and SN), was essentially bounded by the Yangtze River. It occurred around ~0.79 Ma (95% HPD: 1.13–0.46 Ma) in the Middle Pleistocene Transition. This was followed by the divergence of the southern lineage into two sublineages, SE and SN lineage, occurred around ~0.50 Ma (95% HPD: 0.71–0.25 Ma), corresponding to the time of development of glaciers in various parts of the Qinghai–Tibet Plateau (QTP) (0.73–0.46 Ma). SE lineage might originate from southwestern China based on the comparison between the haplotype network based on COI and CytB. Our study suggested that divergences of lineages have twice co‐occurred with tendency of cooling climatic in Asia after the Mid‐Pleistocene, and the life‐history strategy may play an important role in lineage diversification. Additionally, our results indicated that the USA populations were revealed at least twice separate Asian invasions. These both belonged to the egg diapause, which might provide a new perspective for invasion control.     

Investigating niche and lineage diversification in widely distributed taxa: phylogeography and ecological niche modeling of the Peromyscus maniculatus species group

The relationship between lineage formation and variation in the ecological niche is a fundamental evolutionary question. Two prevailing hypotheses reflect this relationship: niche conservatism and niche divergence. Niche conservatism predicts a pattern where sister taxa will occupy similar niche spaces; whereas niche divergence predicts that sister taxa will occupy different niche spaces. Widely distributed species often show distinct phylogeographic structure, but little research has been conducted on how the environment may be related to these phylogenetic patterns. We investigated the relationship between lineage divergence and environmental space for the closely related species Peromyscus maniculatus and P. polionotus utilizing phylogenetic techniques and ecological niche modeling (ENM). We estimated the phylogenetic relationship among individuals based on complete cytochrome b sequences that represent individuals from a majority of the species ranges. Niche spaces that lineages occupy were estimated by using 12 environmental layers. Differences in niche space were tested using multivariate statistics based on location data, and ENMs were employed using maximum entropy algorithms. Two similarity indices estimated significant divergence in environmental space based on the ENM. Six geographically structured lineages were identified within P. maniculatus. Nested within P. maniculatus we found that P. polionotus recently diverged from a clade occupying central and western United States. We estimated that the majority of the genetic lineages occupy distinct environmental niches, which supports a pattern of niche divergence. Two sister taxa showed niche divergence and represent different ecomorphs, suggesting morphological, genetic and ecological divergence between the two lineages. Two other sister taxa were observed in the same environmental space based on multivariate statistics, suggesting niche conservatism. Overall our results indicate that a widely distributed species may exhibit both niche conservatism and niche divergence, and that most lineages seem to occupy distinct environmental niches.     

A molecular revision of the taxonomic status of mermithid parasites of black flies from Quebec (Canada)

The four currently recognized mermithid (Nematoda) species parasitizing black flies (Diptera: Simuliidae) from Northeast America were distinguished using discriminatory PCR primers aimed at COI and 18S rDNA. Isomermis wisconsinensis, Gastromermis viridis and Mesomermis camdenensis were easily differentiated using either genomic target, even for juvenile mermithids damaged beyond morphological recognition. However, specimens from Mesomermis flumenalis being identical in external morphology and producing a unique-sized PCR product were classified by sequence data into four clearly distinguished molecular variants. This quartet was made of two winter and two summer ‘physiological variants’, including one which also belonged to, but diverged early from the rest of the Mesomermis genus. Combining the multiplex PCR and sequencing approaches allowed for the characterization of a multiple parasitism which simultaneously implicated I. wisconsinensis and two M. flumenalis variants. With another instance where parasites were identified by morphology only, this is the first report of black fly parasitism by multiple mermithid species. A phylogenetic tree built by combining our sequences to previous GenBank entries likely indicates a monophyletic origin for the mermithid family, but also suggests that differentiation between parasite genera sometimes occurred before the evolutionary emergence of the actual host group.     

Colour variation is incongruent with mitochondrial lineages: cryptic speciation and subsequent diversification in a Gulf of California reef fish (Teleostei: Blennioidei)

The Gulf of California endemic reef fish, Acanthemblemaria crockeri (Blennioidei, Chaenopsidae), reportedly has two colour morphs, one with melanic lateral spots ('Gulf' morph) and one with orange spots ('Cape' morph). In this study, we recorded colour morph in both males and females and collected mitochondrial DNA sequence data for cytochrome c oxidase I (COI) and tRNA-Pro/D-loop of specimens from throughout the Gulf to explore the genetic basis of the colour morphs. Two highly divergent (HKY + I distance = 11.9% for COI), reciprocally monophyletic lineages were identified, consistent with the presence of two parapatric species. A 30-km gap between the distributions of mitochondrial lineages roughly corresponds to a hypothesized former seaway across the Baja California peninsula north of La Paz, although the estimated divergence time (1.84 million years ago) is more recent than the hypothetical seaway (3–4 million years ago). Surprisingly, the distribution of mitochondrial species is not congruent with the distribution of either male or female colour morphs. Our analysis also revealed significant population differentiation within both species and no shared haplotypes among populations. The northern Gulf species includes four populations (NB, CB, NM and CM) corresponding to northern and central Baja and northern and central mainland sites, while the Cape species includes two populations (SB and SM) corresponding to the Baja and mainland sides of the southern Gulf. The NB/CB division corresponds to a hypothesized Plio–Pleistocene mid-peninsular seaway. The level of genetic divergence documented in this lineage is extraordinary for a marine fish with a pelagic larval stage within a semi-enclosed basin.     

Morphological and genetic discordance in two species of Bornean Leptobrachium (Amphibia, Anura, Megophryidae)

Recent phylogenetic studies of Southeast Asian megophryid Leptobrachium, while clarifying (1) distinct specific status of three Philippine populations and (2) high genetic diversities within Bornean Leptobrachium montanum, posed two questions, (1) relationships and divergence histories of two Philippine species and Bornean Leptobrachium gunungense, and (2) possible discordance between phylogenetically and morphologically defined lineages. In order to solve these questions, and especially reviewing current taxonomy of Bornean species, we estimated the phylogenetic relationships of endemic Bornean species together with their putative relatives from Philippines and Sumatra, using 2451 bp sequences of the 12S rRNA, tRNA(val), and 16S rRNA of mitochondrial DNA genes. With respect to Leptobrachium hasseltii and Leptobrachium chapaense, lineages from Borneo, Philippines, and Sumatra formed a monophyletic group with Leptobrachium lumadorum from Mindanao as the basal clade, while two other Philippine species from Palawan and Mindoro formed a clade and nested in Bornean lineages. Sister species relationship of the two Philippine species and L. gunungense is not supported, rejecting the hypothesis of Philippine origin of L. gunungense. Phylogeny does not conform to morphological variation, and the topotypic L. montanum and one lineage of Leptobrachium abbotti are genetically very close despite their clear difference in ventral color pattern. Furthermore, each of these species forms a paraphyletic group and contains several lineages, each of which is a candidate of good species. These results clearly indicate that current taxonomy of Bornean species based on several morphological characteristics requires complete revision. Detailed studies on adult and larval morphology, as well as acoustic characteristics, are necessary to evaluate the taxonomic status of all lineages recovered.     

Opening a can of worms: unprecedented sympatric cryptic diversity within British lumbricid earthworms

Earthworms play a major role in many aspects of soil fertility, food web ecology and ecosystem functioning, and hence are frequently the subjects of, for example, ecological and toxicological research. Our aim was to examine the genetic structure of common earthworm species, to identify cryptic lineages or species that may be distinct ecotypes or biotypes (and hence confound current research based upon morphotypes) and to try to explain the massive cryptic diversity that eventually emerged. We demonstrated that species such as Allolobophora chlorotica, Aporrectodea longa, Aporrectodea rosea and Lumbricus rubellus all comprise highly divergent lineages with species-level divergence at the mitochondrial cytochrome oxidase I (COI) gene. In Allo. chlorotica alone, we found 55 haplotypes for COI, with 35 of these being found in pink and 20 in green morph worms. There were no cases of the two colour morphs sharing COI haplotypes. Phylogenetic analyses of mitochondrial COI and 16S genes showed the presence of five highly divergent lineages, suggesting the presence of multiple cryptic species within Allo. chlorotica. There was no clear geographical pattern to lineage distribution and many populations were polymorphic for both mitochondrial DNA lineage and colour morph. Amplified fragment length polymorphism results, based on two primer combinations, were broadly congruent with mitochondrial DNA results with one significant exception. Despite showing over 14% divergence at COI, amplified fragment length polymorphism markers showed that the two green morph lineages may be interbreeding and therefore represent a single taxon. The cryptic diversity revealed by these results has profound consequences for all areas of earthworm research.     

A molecular and karyological approach to the taxonomy of Nautilus

Nautiloids, the externally shelled cephalopods of Cambrian origin, are the most ancient lineage among extant cephalopods. Their ancestral characters are explored based on morphological and molecular data (18S rDNA sequence) to investigate the evolution of present cephalopod lineages. Among molluscs, nautilus 18S rDNA gene is the longest reported so far, due to large nucleotidic insertions. By comparison with other 18S sequences, the complete gene of N. macromphalus helps to clarify the taxonomic status of the three universally recognised Nautilus species. The range of interspecific molecular differences supports separation of the present species into two surviving ectocochleate genera, Nautilus and Allonautilus. Nautiloid 18S is considered as corresponding to the ancestral form of 18S as is the number of chromosomes in Nautilus (52), the lowest among cephalopods. Comparison of karyological characteristics amongst cephalopods in a phylogenetic context suggests a possible correlation between duplication events and lineage divergence.