Cryptic diversity in a Eurasian water snake (Natrix tessellata, Serpentes: Colubridae): Evidence from mitochondrial sequence data and nuclear ISSR-PCR fingerprinting

The dice snake, Natrix tessellata (Laurenti, 1768), is a suitable study organism to address questions of Eurasian phylogeography due to its wide Palearctic distribution. We analysed complete mitochondrial cytochrome b sequences and nuclear ISSR-PCR fingerprints of more than 300 specimens representing nearly the entire geographic range. Nine major mitochondrial lineages were discovered based on mtDNA sequences. The three most basal lineages comprised populations from Iran, Jordan–Egypt, and Greece, respectively. Other lineages were associated with samples from the Turkish peninsula, the Caucasus, the Aral Sea, and eastern Kazakhstan. A sister-group relationship was found between two lineages from Crete and the European mainland. Assuming an evolutionary rate of 1.35% sequence divergence per million years, among-lineage p-distances of 1.7–8.4% suggest that intraspecific differentiation might date back as far as the Miocene/Pliocene transition 5–6 million years ago. The pattern of genetic differentiation in mitochondrial phylogeny with regard to Asia Minor and the region of the Aral Sea was not congruent with the results of the nuclear ISSR-PCR analyses, and suggests admixing within some mtDNA clades at contact zones. The taxonomic implications of the high intraspecific variation in the dice snake are discussed.     

Evidence for cryptic speciation in Carchesium polypinum Linnaeus, 1758 (Ciliophora: Peritrichia) inferred from mitochondrial, nuclear, and morphological markers

Protist diversity is currently a much debated issue in eukaryotic microbiology. Recent evidence suggests that morphological and genetic diversity might be decoupled in some groups of protists, including ciliates, and that these organisms might be much more diverse than their morphology implies. We sought to assess the genetic and morphological diversity of Carchesium polypinum, a widely distributed peritrich ciliate. The mitochondrial marker cytochrome c oxidase subunit I and the nuclear small subunit ribosomal RNA were used to examine genetic diversity. For the morphological assessment, live microscopy and Protargol staining were used. The mitochondrial marker revealed six robust, deeply diverging, and strongly supported clades, while the nuclear gene was congruent for three of these clades. There were no major differences among individuals from the different clades in any of the morphological features examined. Thus, the underlying genetic diversity in C. polypinum is greater than what its morphology suggests, indicating that morphology and genetics are not congruent in this organism. Furthermore, because the clades identified by the mitochondrial marker are so genetically diverse and are confirmed by a conserved nuclear marker in at least three cases, we propose that C. polypinum be designated as a "cryptic species complex." Our results provide another example where species diversity can be underestimated in microbial eukaryotes when using only morphological criteria to estimate species richness.     

Phylogeny of the Colubroidea (Serpentes): new evidence from mitochondrial and nuclear genes

The Colubroidea contains over 85% of all the extant species of snakes and is recognized as monophyletic based on morphological and molecular data. Using DNA sequences (cyt b, c-mos) from 100 species we inferred the phylogeny of colubroids with special reference to the largest family, the Colubridae. Tree inference was obtained using Bayesian, likelihood, and parsimony methods. All analyses produced five major groups, the Pareatidae, Viperidae, Homalopsidae, the Elapidae, and the Colubridae. The specific content of the latter two groups has been altered to accommodate evolutionary history and to yield a more stable taxonomy. We propose an updated classification based on the reallocation of species as indicated by our inferred phylogeny.     

A phylogenetic analysis of Pseudonaja (Hydrophiinae, Elapidae, Serpentes) based on mitochondrial DNA sequences

A phylogenetic analysis of mitochondrial ND4 and adjacent tRNA sequences for a geographically extensive series of specimens reveals nine major clades within Pseudonaja, of which six are largely coincident with nominal taxa (P. affinis, P. guttata, P. inframacula, P. ingrami, P. modesta, and P. textilis). The remaining three clades are composed of specimens presently referred to P. nuchalis. Two of these clades correspond with the "Darwin" and "Southern" morphs of previous authors, while the third clade incorporates the "Orange with black head" and "Pale head, grey nape" morphs. We are unable to confirm the presence of consistent karyotypic differences between "Orange with black head" and "Pale head, grey nape" specimens, however, P. inframacula, P. textilis, and P. nuchalis "Darwin" are found to exhibit distinctive karyotypes, as previously reported. These results, in conjunction with additional observations of karyotpic and morphological variation, are consistent with nine historically-independent lineages (i.e., species) within Pseudonaja. There is strong support for a clade composed of P. affinis, P. inframacula, P. ingrami, P. textilis, and the three P. nuchalis lineages, and for the relationships (P. inframacula, P. nuchalis "Southern") and (P. nuchalis "Darwin", P. nuchalis "Orange with black head"--"Pale head, grey nape" ).     

Post-ovipositional development of the monocled cobra, Naja kaouthia (Serpentes: Elapidae)

External morphological development between oviposition and hatching of the monocled cobra, Naja kaouthia, is described. Ten developmental stages are diagnosed according to nine features. These include fusion of the body wall musculature along the ventral midline, appearance of the endolymphatic ducts, formation of the eyelid, and the appearance of scales on the head and body. Additional observations of the developing skull are made at four of these ten stages, based on cleared and stained heads.     

Species boundaries in Malagasy snakes of the genus Madagascarophis (Serpentes: Colubridae sensu lato) assessed by nuclear and mitochondrial markers

We studied mitochondrial divergence in 27 individuals of colubrid snakes of the genus Madagascarophis Mertens from most of its distribution area in Madagascar. Combined analyses of 16S rRNA and cytochrome b sequences identified six major clades which only partly agreed with previously proposed classifications. Analysis of nuclear DNA sequences of the c-mos gene as well as of ISSR fingerprints revealed consistent differences only among three clades which we consider as distinct species: a widespread Madagascarophis colubrinus (Schlegel), with M. citrinus (Boettger) as a junior synonym, a southern M. meridionalis Domergue, and a presumably undescribed species from the extreme north of Madagascar. The species M. ocellatus Domergue was not available for our study. Within M. colubrinus there are two populations from the north-west, each showing two divergent haplotypes with pairwise divergences of up to 5.2% in the cytochrome b gene. Maximum divergence in this gene within M. colubrinus was 7.1%. These high values emphasise that caution needs to be applied before genetic distance values are used for species delimitation. Phylogeographically, most of the genetic variation in M. colubrinus is found in northern Madagascar, indicating that the species might have originated in this region. Later one haplotype clade colonised western and eastern Madagascar, with a putative secondary introgression into north-western populations.     

Population systematics of the snake genus Naja (Reptilia: Serpentes: Elapidae) in Indochina: Multivariate morphometrics and comparative mitochondrial DNA sequencing (cytochrome oxidase I)

We analyze the population systematics of Asiatic cobras in Indochina, China and the Andaman Islands by means of comparative sequencing of the cytochrome oxidase subunit I gene of the mitochondrial DNA molecule and multivariate analysis of morphological characters. Canonical variate analysis and mtDNA sequence information reveal that the cobras of this region comprise four distinct species: Naja atra from China and northern Vietnam, Naja kaouthia from Burma, central Thailand, Cambodia and southern Vietnam, Naja siamensis from Thailand, Cambodia and southern Vietnam, and Naja sagittifera from the Andaman Islands. The subspecies N. kaouthia suphanensis Nutaphand 1986 shows no mtDNA sequence difference from typical N. kaouthia from central Thailand, and multivariate analysis does not reveal differences in general phenotypic profile; the subspecies is therefore synonymised with Naja kaouthia. The cytochrome oxidase subunit I gene, little used in molecular taxonomy, is shown to be well suited for studies at the species level, as it shows taxonomically useful levels of interspecific divergence but low levels of intraspecific variation; this is particularly relevant for studies of rare species, where sample size is a problem. The combination of multivariate morphometrics and molecular systematics can be particularly powerful in resolving systematic problems in such cases.     

A fish prey found in the coral snake Micrurus alleni (Serpentes: Elapidae) in Costa Rica

A fish prey found in the coral snake Micrurus alleni (Serpentes: Elapidae) in Costa Rica. The presence of a small specimen of the swamp eel Synbranchus marmoratus (84 mm total length) in the stomach contents of an adult coral snake Micrurus alleni with 692 mm total length from the Caribbean versant of Costa Rica is reported. This eel was swallowed headfirst.     

The phylogeny of cobras inferred from mitochondrial DNA sequences: evolution of venom spitting and the phylogeography of the African spitting cobras (Serpentes: Elapidae: Naja nigricollis complex)

We use phylogenetic analysis of 1333 bp of mitochondrial DNA sequence to investigate the phylogeny and historical biogeography of the cobra-like elapid snakes, with special reference to the evolution of spitting and the phylogeography of the African spitting cobras, a radiation widespread in open vegetational formations throughout sub-Saharan Africa. Our results suggest that spitting adaptations appear to have evolved three times in cobras, but alternative scenarios cannot be rejected. The Asiatic Naja are monophyletic and originate from a single colonization of Asia from Africa. The radiation of the African spitting Naja appears to date back to the early Miocene and many speciation events in the group predate the Pliocene expansion of grasslands and the radiation of large grazing mammals in Africa. The cladogenic events in this complex appear to have been triggered by both ecological changes and tectonic events associated with the formation and expansion of the African Rift Valley. Taxonomically, our data confirm the inclusion of Boulengerina and Paranaja within Naja, and reveal a clade of African rainforest cobras including N. melanoleuca, Paranaja multifasciata and Boulengerina that constitutes the sister clade of the African open-formation non-spitting cobras. Naja nigricollis is polyphyletic, and we therefore recognize N. nigricincta as a separate species, more closely related to N. ashei and N. mossambica than to N. nigricollis.     

Monophyly of elapid snakes (Serpentes: Elapidae). An assessment of the evidence

The defining morphological characters of the family Elapidae are analysed in an attempt to evaluate whether the front-fanged, proteroglyphous, snakes constitute a natural (monophyletic) group or whether proteroglyphy is more likely to be a condition achieved independently by a number of higher snake lineages. The evidence relating to presumed elapids whose affinities have been questioned, namely a South African genus Homoroselaps and New World proteroglyphs (Micrurus and Micruroides) , is examined. It concluded that Homoroselaps is a genuinely equivocal case, the evidence for its inclusion in the Elapidae is balanced by features which suggest that it is more closely related to the Aparallactinae. However, Micrurus and Micruroides seem clearly to be more closely related to undisputed elapids than to any other caenophidians. It is suggested that, at least for the present, the family Elapidae be retained in its broad sense to include all proteroglyphous snakes.     

A new species of slender coralsnake from Colombia, and its clinal an ontogenetic variation (Serpentes, Elapidae: Leptomicrurus)

Leptomicrurus renjifoi is described from tropical semi-deciduous forest of the eastern Colombian Ilanos. It is one of the smallest species in the genus, is most similar to L. scutiventris, and it may be distinguished from known congeners by a combination of color, pattern, and scale characters. Evidence for the recognition of Leptomicrurus is convincing, although its members were recently thought to comprise a closely related assemblage within Micrurus. A supposedly aberrant specimen of L. scutiventris may indicate clinal or ontogenetic variation in pattern.     

Integrating phylogeny,environment and space to explore variation in macroecological traits of Viperidae and Elapidae (Squamata: Serpentes)

We used eigenvector mapping in space and phylogeny to investigate the relationships among space, phylogeny and environment on body size and range size variation across two groups of venomous snakes – Viperidae and Elapidae – from the New World. Data on species geographic range sizes, maximum body sizes and phylogenetic relationships were compiled from the available literature. The distributional data were also used to calculate the latitudinal and longitudinal midpoint and the environmental centroids for each species. The eigenvectors extracted from the pair wise spatial and phylogenetic distance matrices were integrated with environmental variables into a method of variation partitioning where the variation in each trait was quantitatively attributed to ‘pure’ and/or shared effects of phylogeny, environment and space. Our results showed that variation in body size was predominantly determined by phylogeny in both groups of snakes. For Viperidae, we found that pure ‘effects’ of phylogeny were the strongest, indicating that most of the body size evolution that was phylogenetically determined in this group occurred independently of environment and geographical proximity. Regarding range sizes, pure phylogenetic influences were very low in both groups, whereas the largest single fraction of explained variation corresponded to overlapped influences of the three sets of predictors, especially for Elapidae. Along with this, we found evidence that niche conservatism is an important processes underlying variation in body size and range size in both groups of snakes.     

Uncovering cryptic species diversity of a termite community in a West African savanna

To uncover the termite species diversity of a natural African savanna ecosystem, we combined morphological analyses and sequencing of three gene fragments (cytochrome oxidase I, cytochrome oxidase II and 28SrDNA, total length about 2450 bp) to infer putative species from phylogenetic trees. We identified 18 putative species clusters with high support values and which we retrieved consistently. Samples from two genera (Ancistrotermes and Microcerotermes) were excluded from the mitochondrial phylogenetic analyses as they might represent nuclear mitochondrial sequences (NUMTs). In total, our data suggest a species richness of at least 20 species, all but one belonging to the Termitidae (higher termites), and among them the fungus-growing Macrotermitinae were most prevalent with at least nine putative species. Within the fungus-growers the most species-rich genus was Microtermes and its four putative species were all cryptic species. Their abundance in the samples suggests that they play an important ecological role which is completely unstudied also due to the lack of reliable identification means. Our study shows that morphological traits are unreliable means of species identification for several termite taxa. Yet reliable and consistent identification is necessary for studying the functional role of termites in ecosystem and global processes.     

The Muscoidea (Diptera: Calyptratae) are paraphyletic: Evidence from four mitochondrial and four nuclear genes

Approximately 5% of the known species-level diversity of Diptera belongs to the Muscoidea with its approximately 7000 described species. Despite including some of the most abundant and well known flies, the phylogenetic relationships within this superfamily are poorly understood. Previous attempts at reconstructing the relationships based on morphology and relatively small molecular data sets were only moderately successful. Here, we use molecular data for 127 exemplar species of the Muscoidea, two species from the Hippoboscoidea, ten species representing the Oestroidea and seven outgroup species from four acalyptrate superfamilies. Four mitochondrial genes 12S, 16S, COI, and Cytb, and four nuclear genes 18S, 28S, Ef1a, and CAD are used to reconstruct the relationships within the Muscoidea. The length-variable genes were aligned using a guide tree that was based on the protein-encoding genes and the indel-free sections of the ribosomal genes. We found that, based on topological considerations, this guide tree was a significant improvement over the default guide trees generated by ClustalX. The data matrix was analyzed using maximum parsimony (MP) and maximum likelihood (ML) and yielded very similar tree topologies. The Calyptratae are monophyletic and the Hippoboscoidea are the sister group to the remaining calyptrates (MP). The Muscoidea are paraphyletic with a monophyletic Oestroidea nested within the Muscoidea as sister group to Anthomyiidae+Scathophagidae. The monophyly of three of the four recognized families in the Muscoidea is confirmed: the Fanniidae, Muscidae, and Scathophagidae. However, the Anthomyiidae are possibly paraphyletic. Within the Oestroidea, the Sarcophagidae and Tachinidae are sister groups and the Calliphoridae are paraphyletic.     

Phylogeny of Cidaroida (Echinodermata: Echinoidea) based on mitochondrial and nuclear markers

We present the first molecular phylogeny of Cidaroida, one of the most problematic groups within the echinoids. Two genes??the nuclear ribosomal gene 28?S rRNA and the mitochondrial protein-encoding gene COI??were obtained from 21 specimens representing 17 genera and 20 species, among which 13 species belong to Cidaroida. Phylogenetic analyses of the combined molecular data using parsimony and maximum likelihood optimality criteria resulted in a well-resolved phylogeny. Our results are broadly compatible (with the notable exception of Cidaris cidaris) with previous results obtained from morphological data. We find that Cidaroida represent a monophyletic group sister to the non-cidaroid Echinoidea. The family Cidaridae sensu Mortensen (1928) and Fell (1966) is paraphyletic because of the placement of Psychocidaris ohshimai as sister-group to Histocidaris elegans. Inside the Stylocidarina, we show that the two Atlantic species Stylocidaris affinis and Stylocidaris lineata constitute a well-supported clade. However, these two taxa could also represent two morphotypes within a single species showing high morphological variation.     

Feeding behavior and venom toxicity of coral snake Micrurus nigrocinctus (Serpentes: Elapidae) on its natural prey in captivity

The feeding behavior and venom toxicity of the coral snake Micrurus nigrocinctus (Serpentes: Elapidae) on its natural prey in captivity were investigated. Coral snakes searched for their prey (the colubrid snake Geophis godmani) in the cages. Once their preys were located, coral snakes stroke them with a rapid forward movement, biting predominantly in the anterior region of the body. In order to assess the role of venom in prey restraint and ingestion, a group of coral snakes was 'milked' in order to drastically reduce the venom content in their glands. Significant differences were observed between snakes with venom, i.e., 'nonmilked' snakes, and 'milked' snakes regarding their behavior after the bite. The former remained hold to the prey until paralysis was achieved, whereas the latter, in the absence of paralysis, moved their head towards the head of the prey and bit the skull to achieve prey immobilization by mechanical means. There were no significant differences in the time of ingestion between these two groups of coral snakes. Susceptibility to the lethal effect of coral snake venom greatly differed in four colubrid species; G. godmani showed the highest susceptibility, followed by Geophis brachycephalus, whereas Ninia psephota and Ninia maculata were highly resistant to this venom. In addition, the blood serum of N. maculata, but not that of G. brachycephalus, prolonged the time of death of mice injected with 2 LD(50)s of M. nigrocinctus venom, when venom and blood serum were incubated before testing. Subcutaneous injection of coral snake venom in G. godmani induced neurotoxicity and myotoxicity, without causing hemorrhage and without affecting heart and lungs. It is concluded that (a) M. nigrocinctus venom plays a role in prey immobilization, (b) venom induces neurotoxic and myotoxic effects in colubrid snakes which comprise part of their natural prey, and (c) some colubrid snakes of the genus Ninia present a conspicuous resistance to the toxic action of M. nigrocinctus venom.