Molecular evidence for species level divergence in African Nile Crocodiles Crocodylus niloticus (Laurenti, 1786)

Relationships of the newly discovered dwarf crocodiles from Mauritania were inferred from mitochondrial 12S sequences. Specimens from 13 different Crocodylus niloticus populations (from East Africa, West Africa and Madagascar) were compared. Additional representatives of the genus Crocodylus (one from Africa and one from Australia), the African genus Osteolaemus and the South American alligatorid Paleosuchus palpebrosus (as outgroup) were included in the analysis. Maximum-likelihood and Bayesian analyses yielded relationships that were strikingly different from currently prevailing phylogenetic hypotheses. Both analyses consistently revealed two groups, one consisting of the monophyletic West- and Central African populations and the other of a paraphyletic group containing the East African and Madagascan populations. High genetic divergence between those groups indicates separation on the species level. Furthermore ‘C’ cataphractus is clearly shown not to be a member of the genus Crocodylus. The resulting nomenclatural changes are discussed. To cite this article: A. Schmitz et al., C. R. Palevol 2 (2003).     

From Scilla to Charybdis – is our voyage safer now?

The genus Charybdis Speta (previously Urginea maritima agg.) was investigated karyologically and genetically throughout its geographic range in the Mediterranean. The different ploidy levels show a strong geographic pattern. Diploid populations are mainly found along the northern coast of Africa with C. pancration extending northwards from Tunisia to southern Italy. Tetraploid populations are most densely distributed in the eastern Mediterranean but are also found in North Africa, on the Balearic and Canary Islands. Hexaploid populations are restricted to the Iberian Peninsula and adjacent Morocco and Algeria. Chloroplast microsatellite data suggest that determination of ploidy levels alone is insufficient to adequately describe the existing populations. Especially the tetraploid and hexaploid populations exhibit additional genetic differentiation and geographic structuring. AFLP data indicate that tetraploid populations from southern Italy are of hybrid origin. Phylogenetic analysis further revealed that the genera Urginea Steinh. and Charybdis are not directly related to each other but have strong ties to genera from southern Africa. A possible colonization scenario of the Mediterranean via NW Africa and Iberia is discussed.     

Genetic characterisation of Cylindrospermopsis raciborskii (Nostocales, Cyanobacteria) isolates from Africa and Europe

The invasive cyanobacterium Cylindrospermopsis raciborskii is increasingly spreading in temperate freshwater habitats worldwide and is of major concern due to its ability to produce potent toxins. It is, therefore, important to understand the mechanisms behind the dispersal of this species. Different hypotheses have been proposed to explain the phylogeography and mechanisms underlying the recent expansion of C. raciborskii into temperate latitudes, but there is still no conclusive evidence whether the obvious ecological success of C. raciborskii is due to selection mechanisms, physiological tolerance, climatic change or radiation after the last ice age. In the present study, new isolates of C. raciborskii from Europe and Africa were genetically characterised by sequencing the ITS1, PC-IGS, nifH and rpoC1 genes and compared to corresponding sequences of C. raciborskii available in GenBank in order to test different phylogeographical hypotheses. The strains were also morphologically examined and screened for production of the hepatotoxic cylindrospermopsin (CYN). We clearly demonstrate a variation among the populations of C. raciborskii from different geographical regions. The phylogenetic analyses revealed a clustering of the strains due to geographic origin. The ITS1 and nifH genes separated into American, European and Australian–African groups, whereas the PC-IGS and rpoC1 separated into American and European/Australian/African groups. An analysis of concatenated data supported the division into American, European and African/Australian groups, and even indicated a subdivision into an African and an Australian group. Our findings do not strongly support any of the existing hypotheses on the phylogeography of C. raciborskii, and most likely a combination of these hypotheses is the best approach to understand the evolution and dispersal of this species.     

Genetic differentiation,gene flow and the origin of infestations of the medfly,Ceratitis capitata

The genetic structure of natural populations of the economically important dipteran species Ceratitis capitatawas analysed using both biochemical and molecular markers. This revealed considerable genetic variation in populations from different geographic regions. The nature of this variation suggests that the evolutionary history of the species involved the spread of individuals from the ancestral African populations through Europe and, more recently, to Latin America, Hawaii and Australia. The observed variation can be explained by various evolutionary forces acting differentially in the different geographic areas, including genetic drift, bottleneck effects, selection and gene flow. The analysis of the intrinsic variability of the medfly's genome and the genetic relationships among populations of this pest is a prerequisite for any control programme.     

Comparative analysis of development and survival of two Natal fruit fly Ceratitis rosa Karsch (Diptera,Tephritidae) populations from Kenya and South Africa

Comparative analysis of development and survivorship of two geographically divergent populations of the Natal fruit fly Ceratitis rosa Karsch designated as Ceratitis rosa R1 and Ceratitis rosa R2 from Kenya and South Africa were studied at seven constant temperatures (10, 15, 20, 25, 30, 33, 35 °C). Temperature range for development and survival of both populations was 15–35 °C. The developmental duration was found to significantly decrease with increasing temperature for Ceratitis rosa R1 and Ceratitis rosa R2 from both countries. Survivorship of all the immature stages of Ceratitis rosa R1 and Ceratitis rosa R2 from Kenya was highest over the range of 20–30 °C (87–95%) and lowest at 15 and 35 °C (61–76%). Survivorship of larvae of Ceratitis rosa R1 and Ceratitis rosa R2 from South Africa was lowest at 35 °C (22%) and 33 °C (0.33%), respectively. Results from temperature summation models showed that Ceratitis rosa R2 (egg, larva and pupa) from both countries were better adapted to low temperatures than R1, based on lower developmental threshold. Minimum larval temperature threshold for Kenyan populations were 11.27 °C and 6.34 °C (R1 and R2, respectively) compared to 8.99 °C and 7.74 °C (R1 and R2, respectively) for the South African populations. Total degree-day (DD) accumulation for the Kenyan populations were estimated at 302.75 (Ceratitis rosa R1) and 413.53 (Ceratitis rosa R2) compared to 287.35 (Ceratitis rosa R1) and 344.3 (Ceratitis rosa R2) for the South African populations. These results demonstrate that Ceratitis rosa R1 and Ceratitis rosa R2 from both countries were physiologically distinct in their response to different temperature regimes and support the existence of two genetically distinct populations of Ceratitis rosa. It also suggests the need for taxonomic revision of Ceratitis rosa, however, additional information on morphological characterization of Ceratitis rosa R1 and Ceratitis rosa R2 is needed.     

Meiotic analyses ofCucumis hybrids and an evolutionary evaluation of the genusCucumis (Cucurbitaceae)

Meiosis in seven interspecificCucumis hybrids has been analysed i.a. inC. metuliferus ×C. zeyheri, where the parents belong to different sections. In the triploid hybrids a remarkably high number of trivalents has been found. Additional data from literature on geographical distribution, cucurbitacins, flavonoid patterns, isozymes, C-banding, genome size, DNA amount and chloroplast DNA are used to discuss species relationships and evolution. The African cross-compatible group is divided into theMyriocarpus subgroup with the diploid speciesC. africanus, C. myriocarpus subsp.leptodermis and subsp.myriocarpus, and theAnguria subgroup withC. anguria, C. dipsaceus, C. ficifolius, C. prophetarum, C. zeyheri and all polyploids (exceptC. heptadactylus). It is argued that the Asian subg.Melo with x = 7 is derived from the African subg.Cucumis with x = 12; the latter contains all the polyploid species and has the most common basic chromosome number of theCucurbitaceae. This phylogenetic advance is interpreted with concepts of the quantum model of evolution.     

Genetic and eco-physiological differences of South American Cylindrospermopsis raciborskii isolates support the hypothesis of multiple ecotypes

The global distribution of the toxic cyanobacterium Cylindrospermopsis raciborskii has recently increased, and it has now been identified in tropical, subtropical and temperate freshwater bodies. The mechanisms underlying its success and expansion are still unknown. Several hypotheses have been proposed, including climate change, natural selection and physiological tolerance to different environmental conditions. In this study, we determined the phenotypic and genotypic characteristics of two recently isolated South American strains of C. raciborskii obtained from Uruguay. We analyzed the morphology, growth preferences, tolerance to low temperature (14 °C) and toxin production of the strains and performed phylogenetic analyses based on the ITS and nifH gene sequences. Both isolates showed significantly different morphology and growth behavior under different light intensities and phosphate supply. When genetic differences were assessed by BOX PCR, cluster analyses revealed that they could also be distinguished genotypically and were clearly distinct from C. raciborskii isolated from other continents. Phylogenetic analysis showed that the Uruguayan strains were closely affiliated to other C. raciborskii isolated from the Americas, especially to those from Brazil. Similar to previous studies, we found three solid clusters (Africa-Australia, Europe and America) according to the geographical origin of the isolates. Interestingly, based on nifH sequences, subclusters were identified in American populations indicating an early spread of the species within the continent. We propose that phenotypic and genetic variability of C. raciborskii populations is linked to the existence of different ecotypes whose success is subject to the local environmental conditions.     

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

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

A molecular phylogeny of the Sylvia cantillans complex: cryptic species within the Mediterranean basin

The subalpine warbler Sylvia cantillans is formally considered a polytypic species, with four subspecies, European S. c. cantillans, albistriata, moltonii (recently resumed name: subalpina) and North African S. c. inornata. They are very similar in external morphology but clearly differ in their vocalizations. We evaluated their uncertain taxonomic status reconstructing the phylogenetic and phylogeographic relationships among populations sampled across major biogeographical areas in the European species’ range, using nucleotide sequences of the mitochondrial cytochrome b gene (mtDNA cyt b). A variety of phylogenetic analyses concordantly led to identify four major groups, only partially corresponding to the three European nominal subspecies. Phylogenetic trees showed a monophyletic group including all moltonii individuals, well diverged from all other taxa. Populations taxonomically assigned to cantillans were polyphyletic being split into two distinct clades (western and southern cantillans), with monophyletic albistriata closely related to southern cantillans. Individuals of moltonii and southern cantillans sampled in sites of sympatry in central Italy were assigned to their respective groups, with perfect concordance between phenotypic and genetic identifications. All findings indicate that moltonii should be ranked as a distinct species. Former subspecies cantillans is polyphyletic, but additional data are needed to define the taxonomic status of its two clades. Albistriata is phylogenetically related to southern cantillans and should be provisionally kept as a subspecies of S. cantillans. The cantillans complex thus provides an interesting case-study illustrating geographical structuring across small geographical ranges, and it exemplifies speciation through differentiation in allopatry leading to reproductive isolation after a secondary contact.     

Molecular Phylogeny of Tuco-Tucos, Genus Ctenomys (Rodentia: Octodontidae): Evaluation of the mendocinus Species Group and the Evolution of Asymmetric Sperm

The phylogenetic relationships among 23 individuals representing 14 species of underground hystricognath rodents of the genus Ctenomys were studied by analyzing variation of complete cytochrome b gene sequences. Maximum parsimony, neighbor joining, and maximum likelihood analyses were performed, using the octodontine genera Octodon and Tympanoctomys as outgroups. Our analyses support previous studies based on chromosomes and skull morphology that suggested a clade comprised of Argentinean and Uruguayan populations of C. rionegrensis. This clade is closely related to one comprised of C. flamarioni and the C. mendocinus species complex. Our analyses provide evidence that the symmetric sperm morph, which is common to other South American hystricognath rodents, is the plesiomorphic character state in Ctenomys and in Hystricognathi. Our analyses do not support the hypothesis that the sperm morphs define two major lineages of tuco-tuco species, because species with asymmetric sperm are diphyletic on the basis of cytochrome b sequences, and this morphology appears to have evolved twice in Ctenomys.     

Electrophoretic study and genetic affinity in theCampanula elatines andC. fragilis (Campanulaceae) rock-plants group from Italy and W. Jugoslavia

Variability and genetic divergence of 11 field populations of seven species of theCampanula elatines andC. fragilis rock-plants group from the Mediterranean and pre-Alpine areas have been studied by starch-gel electrophoretic techniques.Campanula isophylla, C. elatines, C. elatinoides, C. fragilis subsp.fragilis and subs.cavolinii, C. garganica were collected in Italy, whilstC. fenestrellata subsp.istriaca andC. portenschlagiana came from W. Jugoslavia. Twelve enzymatic loci for each population were genetically analysed: PGI-1 and 2, PGM-2, IDH-1 and -2, SKDH, ME, ADH, GOT-2, MPI-1 and -2, SOD-1. The genetic distances among the above mentioned entities have been calculated by Nei's index and depicted in a dendrogram.     

Molecular phylogeny of the blind cavefish <Emphasis Type="Italic">Phreatichthys andruzzii</Emphasis> and <Emphasis Type="Italic">Garra barreimiae</Emphasis> within the family Cyprinidae

The phylogenetic relationships of two cavefish, Phreatichthys andruzzii and Garra barreimiae, belonging to the family Cyprinidae, were investigated by sequencing the mitochondrial cytochrome b gene. These cavefish species are native to Somalia (eastern Africa) and Oman (southeastern Arabian peninsula), respectively, and so far no molecular support to their taxonomy and phylogenetic position was ever provided. The analysis of cytochrome b sequences showed that the species are monophyletic taxa, closely related to each other and to other species of the genus Garra. Molecular clock calculations allowed to date the origin of these hypogaean species back to the Plio-Pleistocene and support the hypothesis that African cyprinids originated from Miocenic immigrations of Asian ancestors.     

Cyclamen (Primulaceae) in tropical Africa

Cyclamen somalense Thulin & Warfa, spec. nova, the first member of the genus known from tropical Africa, is described from the Al Miskat Mts in NE. Somalia. The new species is closely related to the E. MediterraneanC. persicum Mill. The disjunct Mediterranean element in the mountain flora of northern Somalia, to whichC. somalense belongs, is believed to be largely a relict of Tertiary origin.     

Variation in wild populations of annual beet (Beta,Chenopodiaceae)

Among the morphological variation in wild annual populations of sect.Beta only tepal characters show a geographic pattern, and hence can be used to distinguish different taxa. Two morphological types correspond with taxa already described:B. macrocarpa (incl.B. bourgaei) has a Macaronesian and W. to E. Mediterranean andB. adanensis an E. Mediterranean distribution area. A third type in the Aegean region is not well known yet and possibly has to be included inB. macrocarpa. Both diploid and tetraploid (x = 9) cytotypes are found withinB. macrocarpa, the latter exclusively on the Canary Islands.     

Isozyme and seed protein phylogeny of the genusCitrullus (Cucurbitaceae)

Electrophoretic analysis of 26 enzyme coding genes was conducted on accessions of threeCitrullus species and the relatedPraecitrullus fistulosus andAcanthosicyos naudinianus. The isozyme phylogeny of the genusCitrullus and the related species was constructed based on pairwise measurements of the respective genetic distances between the species and races.P. fistulosus andA. naudinianus form two distinct outgroups toCitrullus which is characterized by two main clusters: The first includes twoC. colocynthis races and the second,C. lanatus andC. lanatus var.citroides, which are more closely related to each other than they are toC. ecirrhosus. The isozyme phylogeny is consistent with the variability in six seed protein bands and with the crossability relations among the examined species.     

Genetically diverse but with surprisingly little geographical structure: the complex history of the widespread herb Carex nigra (Cyperaceae)

Aim Based on extensive range‐wide sampling, we address the phylogeographical history of one of the most widespread and taxonomically complex sedges in Europe, Carex nigra s. lat. We compare the genetic structure of the recently colonized northern areas (front edge) and the long‐standing southern areas (rear edge), and assess the potential genetic basis of suggested taxonomic divisions at the rank of species and below. Location Amphi‐Atlantic, central and northern Europe, circum‐Mediterranean mountain ranges, central Siberia, Himalayas. Methods A total of 469 individuals sampled from 83 populations, covering most of the species’ range, were analysed with amplified fragment length polymorphism (AFLP) and chloroplast DNA (cpDNA) markers. Bayesian clustering, principal coordinates analysis, and estimates of diversity and differentiation were used for the analysis of AFLP data. CpDNA data were analysed with statistical parsimony networks and maximum parsimony and Bayesian inference of phylogenetic trees. Results Overall genetic diversity was high, but differentiation among populations was limited. Major glacial refugia were inferred in the Mediterranean Basin and in western Russia; in addition, there may have been minor refugia in the North Atlantic region. In the southern part of the range, we found high levels, but geographically quite poorly structured genetic diversity, whereas the levels of genetic diversity varied among different areas in the north. North American populations were genetically very similar to the European populations. Main conclusions The data are consistent with extensive gene flow, which has obscured the recent history of the taxon. The limited differentiation in the south probably results from the mixing of lineages expanding from several local refugia. Northward post‐glacial colonization resulted in a leading‐edge pattern of low diversity in the Netherlands, Belgium, Scotland and Iceland, whereas the observed high diversity levels in Fennoscandia suggest broad‐fronted colonization from the south as well as from the east. The patterns found in the American populations are consistent with post‐glacial colonization, possibly even with anthropogenic introduction from Europe. Our data also suggest that the tussock‐forming populations of C. nigra, often referred to as a distinct species (Carex juncella), represent an ecotype that has originated repeatedly from different populations with creeping rhizomes.     

Molecular tools separate harmful algal bloom species, Karenia mikimotoi, from different geographical regions into distinct sub-groups

Karenia mikimotoi is a toxic dinoflagellate that is known to form extensive populations in the Eastern North Atlantic and Pacific Oceans that that impacts significantly on recreation and fisheries industries in these areas. Attempts to resolve the complex taxonomy within this “species” have been hampered by the requirement for fine-scale morphological analyses and by the lack of suitable genetic markers. Here we report the use of a novel combination of primer sets designed to facilitate amplification of the rDNA LSU and ITS, and the rbcL genes that can be used to discriminate between K. mikimotoi isolates originating from different geographical regions. We show that isolates from Europe and New Zealand are more closely related to each other than either is to isolates from Japan. Specific PCR-based primers were designed to amplify a region of the rbcL gene for subsequent high resolution analysis of the PCR amplicon melting temperatures. This innovative technique allows us to rapidly discriminate K. mikimotoi from distinct geographic localities and we propose the separation of K. mikimotoi into two distinct sub-species.     

Patterns of genetic differentiation in two annual bromegrasses,Bromus lanceolatus andB. hordeaceus (Poaceae)

Infraspecific genetic differentiation was analysed in two tetraploid annual bromegrasses,Bromus lanceolatus (from N Africa) andB. hordeaceus (from N Africa and France). Genetic analysis of populations was based on allozyme polymorphisms at 17 loci. Different fixed heterozygous phenotypes were scored in both species, according to their allopolyploid origin. In N Africa, more variation occurred among populations ofB. lanceolatus than ofB. hordeaceus. The variation was not randomly distributed among populations of both species. InB. lanceolatus, differentiation was correlated with climatic variables rather than with geographic distance between populations. Higher correlation of genetic differentiation with geographic distance occurred inB. hordeaceus, particularly at large geographic scale, between French and N African populations. Within each region, the populations appeared weakly genetically differentiated, even when belonging to different subspecies.     

Molecular Basis of the Size Polymorphism of the First Intron of the<Emphasis Type="Italic">Adh-1</Emphasis> Gene of the Mediterranean Fruit Fly, <Emphasis Type="Italic">Ceratitis capitata</Emphasis>

The first intron of the gene encoding one of the alcohol dehydrogenase isoenzymes (ADH-1) in Ceratitis capitata is highly polymorphic in size. Five size variants of this intron were isolated from different strains and populations and characterized. Restriction map and sequence analysis showed that the intron size polymorphism is due to the presence or absence of (a) a copy of a defective mariner-like element, postdoc; (b) an 550-bp 3 indel which exhibits no similarity to any known sequence; and (c) a central duplication of 704 bp consisting of part of the 3 end of the postdoc element, the region between postdoc and the 3 indel, and the first 20 bp of the 3 indel. The homologous Adh-1 intron was amplified from the congeneric species, Ceratitis rosa, in order to obtain an outgroup for comparative and phylogenetic analyses. The C. rosa introns were polymorphic in size, ranging from about 1100 to 2000 bp, the major difference between them being the presence or absence of a mariner-like element Crmar2, unrelated to the postdoc element. Phylogenetic analysis suggests that the shorter intron variants in C. capitata may represent the ancestral form of the intron, the longest variants apparently being the most recent.     

Ribosomal DNA non-transcribed spacer polymorphism in subterranean mole rats: genetic differentiation,environmental correlates and phylogenetic relationships

Summary An analysis is presented of genetic differentiation in the non-transcribed spacers of ribosomal DNA (NTS rDNA). Diversity, environmental correlates and the phylogenetic relationships are examined within and between species of the actively speciating subterranean mole rat, superspeciesSpalax ehrenbergi (2n=52, 54, 58, 60) in Israel. This analysis is based on a previous study of the geographic distribution of restriction fragment length polymorphisms of NTS rDNA. Here we present results indicating that NTS rDNA diversity exists mostly (66%) within populations, while 20% is between populations within species, and 14% between species. Multivariate discriminant analysis succeeded in separating 10 of the 13 populations (77%) into their correct chromosomal species, on the basis of the combination of three NTS rDNA repetypes. The phylogenetic relationships suggest that the complex involves two pairs of closely related species (2n=52–54 and 2n=58–60). NTS rDNA diversity, as well as the decrease southward in frequency of repetype C, are correlated with climatic factors of humidity and temperature. These data are discussed in terms of the evolutionary forces of migration and selection which may cause NTS rDNA differentiation. Climatic selection appears to be the major differentiating factor of NTS rDNA.