Markedly discordant mitochondrial DNA and allozyme phylogenies of tube-nosed fruit bats, Nyctimene, at the Australian–Oriental biogeographical interface

Australo-Papuan tube-nosed fruit bats of the genus Nyctimene reach their most westerly distribution on the island archipelagos of eastern Indonesia. A recent morphological examination indicates three species occur on Moluccan islands. Both allozyme electrophoresis and mitochondrial DNA (mtDNA) sequencing reveal there are only two species present, Nyctimene cephalotes and Nyctimene albiventer , but there is considerable disparity between the results obtained from the two genetic data sets. Allozyme data indicate N. albiventer occurs on Wokam, which sits on the Australian continental shelf and was joined to Australo-Papua during the last glacial maximum, and N. cephalotes on the other Moluccan islands, all of which are off the continental shelf. Divergence of these two species is dated at approximately 1.5 Mya. By contrast, the mtDNA gene tree shows two deep clades, one containing all specimens from Wokam and Yamdena, and the second all the specimens from the other islands. This especially marked incongruence between the two genetic data sets is ascribed to either a single introgression event of N. albiventer mtDNA into N. cephalotes on Yamdena at around 100 000 ya or lineage sorting of very old mtDNA lineages that coalesce a rather long time before the speciation event. These results highlight that caution should be exercised when relying on mtDNA as barcodes in species taxonomy. The continental-associated N. albiventer on Wokam has higher levels of allozyme heterozygosity and mtDNA nucleotide diversity than the N. cephalotes populations occurring on the more remote islands, indicating that this colonizing species has experienced bottlenecks and/or a low effective population size since speciation.  © 2008 The Linnean Society of London, Biological Journal of the Linnean Society , 2008, 93 , 589–602.     

Phylogenetic analyses of mtDNA sequences reveal three cryptic lineages in the widespread neotropical frog Leptodactylus fuscus (Schneider, 1799) (Anura, Leptodactylidae)

Leptodactylus fuscus is a neotropical frog ranging from Panamá to Argentina, to the east of the Andes mountains, and also inhabiting Margarita, Trinidad, and the Tobago islands. We performed phylogenetic analyses of 12S rRNA, 16S rRNA, tRNA-Leu, and ND1 mitochondrial (mt) DNA sequences from specimens collected across the geographic distribution of L. fuscus to examine two alternative hypotheses: (i) L. fuscus is a single, widely distributed species, or (ii) L. fuscus is a species complex. We tested statistically for geographic association and partitioning of genetic variation among mtDNA clades. The mtDNA data supported the hypothesis of several cryptic species within L. fuscus. Unlinked mtDNA and nuclear markers supported independently the distinctness of a 'northern' phylogenetic unit. In addition, the mtDNA data divided the southern populations into two clades that showed no sister relationship to each other, consistent with high differentiation and lack of gene flow among southern populations as suggested by allozyme data. Concordance between mtDNA and allozyme patterns suggests that cryptic speciation has occurred in L. fuscus without morphological or call differentiation. This study illustrates a case in which lineage splitting during the speciation process took place without divergence in reproductive isolation mechanisms (e.g. advertisement call in frogs), contrary to expectations predicted using a biological species framework.  © 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 87 , 325–341. No claim to original US government works.     

Intraspecific vicariant history and the evolution of adaptive morphological diversity in a fish species (Osmerus mordax)

Vicariant geographic isolation and resource partitioning have long been independently identified as processes contributing to the morphological divergence of closely-related species. However, little is known about the extent to which vicariant history influences the adaptive ecological divergence associated with resource partitioning and trophic specialization within species. The present study thus quantified the contribution of vicariant historical genetic divergence to the adaptive contemporary morphological divergence of intraspecific feeding specialists in the Rainbow smelt (Pisces: Osmerus mordax ). This species is characterized by the polyphyletic origin of two lacustrine feeding specialists originating in two intraspecific lineages associated with independent glacial refuges. The historical genetic segregation was initiated approximately 350 000 years ago, whereas the lacustrine trophic segregation arose within the past 10 000 years. Wild caught lacustrine smelt populations were grouped a priori based on known historical genetic identities (Acadian and Atlantic mitochondrial DNA clades) and contemporary feeding specializations (microphageous and macrophageous morphotypes). The present study demonstrated that independent suites of correlated morphological traits are associated with either vicariant history or contemporary feeding specializations. Second, functionally-similar feeding specialists exhibit distinct morphologies resulting largely from vicariant historical processes. Although, the evolutionary processes producing historical phenotypes remains unknown, the results obtained demonstrate how adaptive radiation associated with ecological resource partitioning and feeding specializations can be strongly influenced by intraspecific phenotypic diversification resulting from relatively recent vicariant histories.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 97 , 140–151.     

THE UPPER CAMBRIAN TRILOBITE OLENUS AT ANDRARUM,SWEDEN: A CASE OF ITERATIVE EVOLUTION?

Abstract:  The morphological changes in four species-level lineages of the Late Cambrian trilobite Olenus from the Alum Shale at Andrarum, south-east Scania, Sweden, have been analysed with the aim of re-examining the classical study of iterative evolution by Kaufmann (1933 a ). New material comprising 647 pygidia was collected for a restudy of Olenus , and for comparison the skeletal development of 918 cephala and 568 pygidia from the associated Homagnostus obesus were included. The re-examination shows that (1) only pygidia of the O. transversus – O. truncatus – O. wahlenbergi lineage display a statistically significant directional change towards a narrower shape; (2) all other lineages, including the agnostids, show net morphological stasis; (3) hence iterative directional evolution cannot be confirmed. Based on morphological investigations it is tempting to conclude that the lineage O. transversus – O. truncatus – O. wahlenbergi represents just one species displaying gradual morphological changes. Whether this is an evolutionary or an ecophenotypic phenomenon remains uncertain. The vertical distributions of olenids and agnostids are compared by using vanadium/(vanadium + nickel) ratios from the Olenus Zone of the nearby Gislövshammar-2 drill-core. It appears that incursions of olenids were linked to increases in bottom-water oxygen levels and that Homagnostus obesus was tolerant of lower dysoxia than olenids.     

Evolutionary and taxonomic relationships among Far-Eastern salmonid fishes inferred from mitochondrial DNA divergence

Mitochondrial DNA (mtDNA) restriction analysis was used to examine the evolutionary and taxonomic relationships among 11 taxa of the subfamily Salmoninae. The genera Brachymystax and Hucho were closely related, diverging by sequence divergence estimates of 3.1%. Because the mtDNA sequence divergence between blunt- and sharp-snouted forms of Brachymystax (2.24%) was similar to divergence level of Brachymystax and Hucho , then taking into account the distinct morphological, ecological and allozyme differences between them, it is possible to recognize these forms as two separate species. The subgenus Parahucho formed a very distinct group differing by 6.35–7.08% (sequence divergence estimate) from both Brachymystax and Hucho and must be considered as a valid genus. The UPGMA and neighbour-joined phenograms showed that the five genera studied are divided into two main groupings: (1) Hucho, Brachymystax and Salvelinus ; and (2) Oncorhynchus and Parahucho species. The mtDNA sequence divergence estimates between these groupings were about 8.1%. However, the subsequent bootstrap analysis of mtDNA RFLP data did not support the monophyly of the latter grouping. The concordance of morphological and mtDNA phylogenetic patterns is discussed.     

Reticulate evolution: ancient introgression of the Adriatic brown trout mtDNA in softmouth trout Salmo obtusirostris (Teleostei: Salmonidae)

Two populations of softmouth trout ( Salmo obtusirostris ) from the rivers Neretva (Bosnia and Herzegovina) and Jadro (Croatia), along with two neighbouring populations of brown trout ( Salmo trutta ) were analysed with a suite of genetic markers (two mtDNA genes, two nuclear genes, and nine microsatellites) as well as morphological characters. The Jadro softmouth trout were fixed for a brown trout mtDNA haplotype of the Adriatic lineage, which is 1.7% divergent from a previously described haplotype characteristic for the Neretva softmouth trout. All other genetic markers, as well as morphological analysis, supported the clear distinction of softmouth trout from the rivers Neretva and Jadro from brown trout in neighbouring populations, and thus a mtDNA capture event is assumed. Population specific microsatellite allele profiles, as well as a high number of private alleles for both populations of softmouth trout, support the hybridization between brown trout and the Jadro softmouth trout most likely being of ancient origin, thus leading to a reticulate evolutionary pattern of mtDNA in this taxon.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 90 , 139–152.     

Delimiting species in the hoplonemertean genus Tetrastemma (phylum Nemertea): morphology is not concordant with phylogeny as evidenced from mtDNA sequences

The marine genus Tetrastemma contains monostiliferous hoplonemertean (phylum Nemertea) species which mostly undergo direct development with no free-swimming stages of larvae. The lack of a pelagic phase, and the fact that many benthic species lay eggs attached to the bottom substrate, are obvious restrictions on dispersal and gene flow. Nevertheless, some of the species, for example T. candidum and T. melanocephalum , are described as ubiquitous and are reported from waters all over the world. We studied genetic variation and evolutionary relationships in order to assess whether they are concordant with external characters in samples of nine morphologically distinct forms formally named as different Tetrastemma species, from different geographical localities. We estimated the phylogeny and species network based on 539 base pairs of the mitochondrial protein-coding gene cytochrome oxidase-1 (CO1) for 30 ingroup specimens. From this, we assessed the evolutionary history and phylogenetic relationships between these forms. We conclude that in most cases there was no correspondence between evolutionary lineage and morphotype. Our results thus indicate that morphological species delimitation in nemerteans may be questionable, and that this in turn may have a profound effect upon estimates of species diversity within the phylum.  © 2005 The Linnean Society of London, Biological Journal of the Linnean Society , 2005, 86 , 201–212.     

Cryptic speciation in the field vole: a multilocus approach confirms three highly divergent lineages in Eurasia

Species are generally described from morphological features, but there is growing recognition of sister forms that show substantial genetic differentiation without obvious morphological variation and may therefore be considered ‘cryptic species’. Here, we investigate the field vole (Microtus agrestis), a Eurasian mammal with little apparent morphological differentiation but which, on the basis of previous sex‐linked nuclear and mitochondrial DNA (mtDNA) analyses, is subdivided into a Northern and a Southern lineage, sufficiently divergent that they may represent two cryptic species. These earlier studies also provided limited evidence for two major mtDNA lineages within Iberia. In our present study, we extend these findings through a multilocus approach. We sampled 163 individuals from 46 localities, mainly in Iberia, and sequenced seven loci, maternally, paternally and biparentally inherited. Our results show that the mtDNA lineage identified in Portugal is indeed a distinct third lineage on the basis of other markers as well. In fact, multilocus coalescent‐based methods clearly support three separate evolutionary units that may represent cryptic species: Northern, Southern and Portuguese. Divergence among these units was inferred to have occurred during the last glacial period; the Portuguese lineage split occurred first (estimated at c. 70 000 bp ), and the Northern and Southern lineages separated at around the last glacial maximum (estimated at c. 18 500 bp ). Such recent formation of evolutionary units that might be considered species has repercussions in terms of understanding evolutionary processes and the diversity of small mammals in a European context.     

Taxonomic status of two South American sympatric squat lobsters, Munida gregaria and Munida subrugosa (Crustacea: Decapoda: Galatheidae), challenged by DNA sequence information

We investigated the taxonomic status of two sympatric morphospecies of squat lobsters from southern South America (Beagle Channel, Strait of Magellan, and Burdwood Bank), Munida gregaria and Munida subrugosa , by DNA sequence analysis of three mitochondrial (mt)DNA gene fragments [416 bp of 16S rDNA(165), 566 bp of cytochrome c oxidase subunit I(COI) and 418 bp of NADH dehydrogenase subunit 1 (ND1)]; and the nuclear rDNA internal transcribed spacer (ITS) 1 (883–952 bp). We obtained a total of 79 sequences from 32 individuals. The 16S sequences of all M. gregaria and M. subrugosa were invariant and identical, whereas COI and ND1 showed 12 and 15 variable sites, respectively. These polymorphisms were shared between morphospecies. Interspecific Tamura–Nei distances for COI and ND1 sequences were 0.0024 and 0.0032, respectively, and were not significantly different from intraspecific distances (Kruskal–Wallis tests: P  = 0.58 and P  = 0.69, for COI and ND1, respectively). Similar to the results obtained from the mtDNA sequences, no relationship was found between the ITS1 maximum parsimony tree topology and the morphologic classification of specimens in M. gregaria and M. subrugosa . We conclude that M. gregaria and M. subrugosa from southern South America may either represent a case of a dimorphic species, or a case of incomplete lineage sorting. The fact that these two morphospecies did not show fixed differences over a total of 1947 bp analysed reinforces the hypothesis of a single dimorphic species.  © 2008 The Linnean Society of London, Biological Journal of the Linnean Society , 2008, 94 , 421–434.     

On the use of DNA sequences for determining the species limits of a polymorphic new species in the stink bug genus Halys (Heteroptera: Pentatomidae) from Pakistan

Abstract.  We describe a new species of Halys Fabricius (Pentatomidae: Pentatominae: Halyini) based on morphological and DNA sequence data, and demonstrate the value of DNA sequences for taxonomic problems that are difficult to resolve on the basis of morphology alone. Halys sindillus Memon, Meier & Manan, sp.n. varies with regard to characters that are usually constant within the genus (spermathecal bulb of females; blade of male clasper; ratio between the second and third antennomeres; length of labium). The surprising levels of variation raised the question as to how many species were represented in three series of specimens from Pakistan. Because the morphological variability was largely continuous, we hypothesized the presence of one new species, and confirm this result here using sequence data from two mitochondrial markers. The data reveal very little molecular variation within the newly described species (COI: 730 bp: 0–0.16%; COI/tRNA_Leu/COII: 563 bp: 0–0.36%), that is, morphology and DNA sequences show very different patterns of variability. The new species is compared with the closely related Halys sulcatus (Thunberg) whose sequences are distinctly different and whose spermathecal bulbs are largely invariable (I: 2.87–3.28%; II: 2.13–2.49%). We discuss the shortcomings of mitochondrial data in taxonomy and compare the genetic distances in Halys with frequency distributions of intra- and interspecific distances obtained for all 878 Hemiptera COI sequences in GenBank. We conclude that the observed distances for Halys are consistent with our taxonomic conclusions, thus demonstrating the usefulness of DNA sequences for Halys taxonomy. However, the observed overlap between intra- and interspecific sequence variability in Hemiptera is so wide that it questions the feasibility of approaches to taxonomy based predominantly on DNA sequences (e.g. DNA taxonomy, DNA barcoding).     

Close relatedness between mitochondrial DNA from seven Anser goose species

The phylogenetic relationships of seven goose species and two of the subspecies representing the genus Anser were studied by approximately 1180 bp of mitochondrial DNA tRNAglu, control region and tRNAphe sequences. Despite obvious morphological and behavioural affinities among the species, their evolutionary relationships have not been studied previously. The small amount of genetic differentiation observed in the mitochondrial DNA indicates an extremely close evolutionary relationship between the Anser species. The sequence divergences between the species (0.9–5.5%) are among the lowest reported for avian species with speciation events of Anser geese dating to late Pliocene and Pleistocene. The species grouped into four mtDNA lineages: (1) snow and Ross’ goose, (2) greylag goose, (3) white‐fronted goose, and (4) bean, pink‐footed and lesser white‐fronted goose. The phylogenetic relationships of the most closely related species, bean, pink‐footed and lesser white‐fronted goose, indicate a period of rapid cladogenesis. The poor agreement between morphological relationships and the phylogenetic relationships indicated by mtDNA sequences implies that either ancestral polymorphism and lineage sorting, hybridization and introgression or convergent evolution has been involved.     

Genome sizes of cyclopoid copepods (Crustacea): evidence of evolutionary constraint

Genome sizes for 36 species of cyclopoid copepods were determined by DNA-Feulgen cytophotometry of nuclei from adults collected from diverse habitats and locales in North America, South America, Europe, and Asia. Genome sizes are small, show a 20-fold range ( C  = 0.10–2.02 pg DNA), and vary in a discontinuous fashion. The genomes of cyclopoid copepods are remarkably small and constant within each species, unlike the large and variable genomes of marine calanoid species. These differences may reflect the evolutionary antiquity of marine copepods in relation to marine, brackish, and freshwater copepods, as well as differences in mechanisms used to modulate genome size. The small genome sizes of contemporary cyclopoids provide substantive evidence of evolutionary constraint, possibly favouring small genomes, rapid replication rates and accelerated development as adaptive strategies for survival in often fragmented, stressful, and changing habitats. © 2006 The Linnean Society of London, Biological Journal of the Linnean Society , 2006, 87 , 625–635.     

Nomenclature in the dock. Overclassification leads to instability: a case study in the horticulturally important genus Cyclamen (Myrsinaceae)

Nine different classifications have been produced in the last 70 years for the horticulturally valuable genus Cyclamen , a small genus with fewer than 30 species. These classifications, generated by intuitive methods and cladistic analyses, incorporated a total of four infrageneric ranks above that of species and were based on data from morphology, cytology and DNA sequencing. Our results, based on cladistic analyses of three independent data sources − nrDNA ITS, cpDNA trn L intron and morphological data − reveal good resolution only in nrDNA sequence data. However, when these three data sources are combined they provide stronger resolution and support for three major clades, only one of which, subgenus Psilanthum , has been consistently supported in previous classifications. The differing infrageneric classifications produced in Cyclamen result from varying taxon sampling, differing interpretation of morphological data, changes in the sources and analysis of data, and inconsistent application of names. Extensive subdivision of small genera in the absence of adequate data that could provide evidence for consistent patterns of relationship is premature and leads to a proliferation of names.  © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society , 2004, 146 , 339–349.     

Three extant genera of freshwater thalassiosiroid diatoms from Middle Eocene sediments in northern Canada

The evolutionary history of diatoms is only constrained partially by the fossil record. The timing of several key events, such as initial colonization of freshwater habitats by marine taxa, remains poorly resolved. Numerous specimens of the genera Cyclotella, Discostella, and Puncticulata (Ochrophyta: Thalassiosirales) have been recovered in Middle Eocene lacustrine sediments from the Giraffe Pipe locality in the Northwest Territories, Canada. These diatoms extend the fossil record of the family Stephanodiscaceae to at least 40 million years before present (Ma) and thus provide a new evolutionary milepost for the thalassiosiroid diatoms, an important clade of centric diatoms with global representation in both marine and freshwater environments. The quality of the fossil material enables detailed investigations of areolae, fultoportulae, and rimoportulae, revealing direct morphological affinities with a number of extant taxa. These observations extend the antiquity of several characters of phylogenetic importance within the thalassiosiroid diatoms, including the fultoportula, and imply that the entire lineage is considerably older than prior constraints from the fossil record, as suggested independently by several recent molecular phylogenies.     

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.     

Their Day in the Sun: molecular phylogenetics and origin of photosymbiosis in the 'other' group of photosymbiotic marine bivalves (Cardiidae: Fraginae)

The subfamily Fraginae (Cardiidae) is a morphologically diverse group of small-bodied marine clams inhabiting shallow seas worldwide. Like the exclusively photosymbiotic giant clams (Cardiidae: Tridacninae), some fragines are known to host zooxanthellae photosymbionts. However, surveys to widely determine photosymbiotic status and the lack of a comprehensive phylogeny have hindered attempts to track the evolution of photosymbiosis in the group. Worldwide sampling of all fragine genera and subgenera with phylogenetic reconstructions based on four gene regions [nuclear (28S) and mtDNA (16S, cytochrome oxidase I, cytochrome b )] does not support a monophyletic Fraginae. Sampled taxa form four restructured clades: (1) the ' Fragum ' group, (2) the ' Trigoniocardia ' and ' Ctenocardia ' groups, (3) the ' Parvicardium ' group and (4) the ' Papillicardium ' group. Maximum likelihood analyses strongly support a clade of European cardiids uniting species from three subfamilies. Live examination of > 50% of species reveals that less than half of derived genera and subgenera host photosymbionts, supporting a single and relatively late origin of photosymbiosis in the Fraginae. The evolutionary implications for a small and little modified earliest diverging photosymbiotic lineage are discussed.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 97 , 448–465.     

Mitochondrial DNA, ecology and morphology: interpreting the phylogeography of the Nesotes (Coleoptera: Tenebrionidae) of Gran Canaria (Canary Islands)

The genus Nesotes (Coleoptera: Tenebrionidae) is represented in the Canary Islands by 19 endemic species, the majority of which are single island endemics. Nesotes conformis and N. fusculus are described on four and three islands, respectively, but each forms a paraphyletic assemblage between Gran Canaria and the other islands. The other described species for Gran Canaria are N. quadratus, N. lindbergi and N. piliger. Thirty-six individuals representing the five species on Gran Canaria have been sequenced for 675 bp of the mitochondrial DNA (mtDNA) cytochrome oxidase II gene. Neighbour-joining analysis of maximum likelihood distances resulted in five distinct mtDNA lineages for N. quadratus, two of which also include mitotypes of N. conformis. Each of the other three species is found on only one mtDNA lineage. We propose from the molecular data that differentiation in a widespread N. quadratus-type ancestor was followed by morphological adaptation to coastal, pine and laurel forest habitats.     

Speciation mirrors geomorphology and palaeoclimatic history in African laminate-toothed rats (Muridae: Otomyini) of the Otomys denti and Otomys lacustris species-complexes in the 'Montane Circle' of East Africa

We adopted an integrated systematic approach to delimit evolutionary species and describe phylogeographic, morphometric and ecological relationships in Otomys denti (from the Albertine Rift, Southern Rift in Malawi and the northern Eastern Arc Mountains) and Otomys lacustris (from the Southern Rift in Tanzania and Zambia, and the southern Eastern Arc Mountains). Molecular [cytochrome (cyt) b sequences, 1143 bp, N  = 18], craniometric (classical, N  = 100 and geometric, N  = 60) and ecological (Partial Least Squares regression of shape and ecogeographic variables) approaches show a profound, parallel disjunction between two groups: (1) Eastern Arc and Southern Rift (including the Malawi Rift) ( O. lacustris and Otomys denti sungae ) and (2) Albertine Rift ( Otomys denti denti and Otomys denti kempi ) taxa. Within both groups, cyt b sequences or craniometric analysis provided evidence for the differentiation of both southern and northern Eastern Arc from Southern Rift lineages (across the so-called Makambako Gap). Within the Albertine Rift ( denti – kempi ) lineage, populations from individual mountain ranges differed significantly in skull shape (but not size), but were similar genetically. Over-reliance in the past on very few morphological characters (e.g. number of molar laminae) and a polytypic species concept has obscured phylogenetic relationships and species discrimination in this group. We recognize at least three species in this group, and distinct lineages within two of these species. Each species or lineage was endemic to one of three regions: the Albertine Rift, the Malawi Rift or the Eastern Arc. Our result echo conclusions of recent studies of other mammalian and bird taxa and reflect the geomorphology and palaeoclimatic history of the region.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 96 , 913–941.     

Rubyspira, new genus and two new species of bone-eating deep-sea snails with ancient habits

Rubyspira, a new genus of deep-sea snails (Gastropoda: Abyssochrysoidea) with two living species, derives its nutrition from decomposing whalebones. Molecular phylogenetic and morphological evidence places the new genus in an exclusively deep-sea assemblage that includes several close relatives previously known as fossils associated with Cretaceous cold seeps, plesiosaur bones, and Eocene whalebones. The ability to exploit a variety of marine reducing environments may have contributed to the evolutionary longevity of this gastropod lineage.