Adaptation of the Plasmodium falciparum FCB strain for in vitro and in vivo analysis in squirrel monkeys (Saimiri sciureus)

The asexual blood stages of the Plasmodium falciparum parasite are responsible for inducing the clinical symptoms and the most severe presentations of malaria infection that causes frequent mortality and morbidity in tropical and subtropical areas of the world, making the blood stages of infection a key target of new malaria treatment and prevention strategies. Progress towards the development of more effective treatment and prevention strategies has been hindered by the limited availability of infection models that permit the sequential analysis of blood stage parasites in vitro followed by in vivo analysis to confirm therapeutic benefits. To advance a model for in vitro and in vivo analysis of blood stage parasites, we examined nine laboratory strains of P. falciparum to determine which strains could adapt to growth in vivo in splenectomized squirrel monkeys (Saimiri sciureus). Only one of the nine laboratory strains tested, the FCB strain, adapted to in vivo growth. Morphological analysis show that the adapted ring-stage parasites have a different morphology from original parasites cultured in vitro, and more often they were found to localize at the edge of the infected red blood cell. No remarkable differences were observed for both trophozoites and schizonts. The adapted strain can be cultured back in vitro similar to the original parasite, indicating that the adapted parasite can develop both in vitro and in vivo. This squirrel monkey-adapted P. falciparum parasite is expected to be suitable and is advantageous for the research and development of vaccines and antimalarial drugs.     

Higher-level salamander relationships and divergence dates inferred from complete mitochondrial genomes

Phylogenetic relationships among the salamander families have been difficult to resolve, largely because the window of time in which major lineages diverged was very short relative to the subsequently long evolutionary history of each family. We present seven new complete mitochondrial genomes representing five salamander families that have no or few mitogenome records in GenBank in order to assess the phylogenetic relationships of all salamander families from a mitogenomic perspective. Phylogenetic analyses of two data sets—one combining the entire mitogenome sequence except for the D-loop, and the other combining the deduced amino acid sequences of all 13 mitochondrial protein-coding genes—produce nearly identical well-resolved topologies. The monophyly of each family is supported, including the controversial Proteidae. The internally fertilizing salamanders are demonstrated to be a clade, concordant with recent results using nuclear genes. The internally fertilizing salamanders include two well-supported clades: one is composed of Ambystomatidae, Dicamptodontidae, and Salamandridae, the other Proteidae, Rhyacotritonidae, Amphiumidae, and Plethodontidae. In contrast to results from nuclear loci, our results support the conventional morphological hypothesis that Sirenidae is the sister-group to all other salamanders and they statistically reject the hypothesis from nuclear genes that the suborder Cryptobranchoidea (Cryptobranchidae + Hynobiidae) branched earlier than the Sirenidae. Using recently recommended fossil calibration points and a “soft bound” calibration strategy, we recalculated evolutionary timescales for tetrapods with an emphasis on living salamanders, under a Bayesian framework with and without a rate-autocorrelation assumption. Our dating results indicate: (i) the widely used rate-autocorrelation assumption in relaxed clock analyses is problematic and the accuracy of molecular dating for early lissamphibian evolution is questionable; (ii) the initial diversification of living amphibians occurred later than recent estimates would suggest, from the Late Carboniferous to the Early Permian (294 MYA); (iii) living salamanders originated during the Early Jurassic (183 MYA), and (iv) most salamander families had diverged from each other by Late Cretaceous. A likelihood-based ancestral area reconstruction analysis favors a distribution throughout Laurasia in the Early Jurassic for the common ancestor of all living salamanders.     

Molecular characterization of major histocompatibility complex class 1 (MHC-I) from squirrel monkeys (<Emphasis Type="Italic">Saimiri sciureus</Emphasis>)

Little is known about the major histocompatibility complex (MHC) class 1 in squirrel monkeys (Saimiri sciureus). We cloned, sequenced and characterized two alleles and the cDNA of the coding region of MHC class 1 in these New World monkeys. Phylogenetic analyses showed that these sequences are related to HLA class 1 genes (HLA-A and HLA-G). The structure and organization of one of the two identified clones was similar to that of a class 1 MHC gene (HLA-A2). All the exon/intron splice acceptor/donor sites are conserved and their locations correspond to the HLA-A2 gene. The sequences of the newly described cDNAs reveal that they code for the characteristic class 1 MHC proteins, with all the features thought necessary for cell surface expression. Typical sequences for the leader peptide, ₁, ₂, ₃, transmembrane and cytoplasmic domains were found.The nucleotide sequence data reported in this paper have been submitted to the GenBank database and have been assigned the accession numbers AJ438576 (Sasc-G*31), AJ438577 (Sasc-G*25), AY282760 (Sasc-G*03), AY282761 (Sasc-G*04) and AY282762 (Sasc-G*05). Sequences were named as recommended by Klein and co-workers (1990)     

A study of correlates of hand preferences in squirrel monkeys (Saimiri sciureus)

The purpose of this study was to test the influence of sex, age, social rank, matriline membership, posture, and visual and tactual motor control on manual preferences inSaimiri sciureus. A well-established social group of 12 squirrel monkeys, aged 2 to 14 yrs and consisting of two matrilines with social rank known for each animal, was presented with four different food-reaching tasks and assessed for hand preferences with a minimum of 100 reaches per animal. Frequency of occurrence of hand preferences at the group level and degree of hand preferences at the individual level depended on posture and on whether the reaching act took place under visual or tactual guidance. Sex, age, social rank, and matriline membership were not found to determine frequency of occurrence, direction or degree of hand preferences with the exception of one task in which a significant negative correlation between the degree of hand preference and age was found. Nine out of 12 monkeys showed task-dependent changes in the hand they used preferentially while only three animals preferred the same hand in all four tasks. Significant preferences for the use of right or left hand on a given task were distributed almost equally between individuals. Thus, the results of this study suggest task-specific demands like posture and/or whether reaching was visually or tactually guided to be the major correlates of hand preferences in food-reaching tasks in squirrel monkeys.     

Discriminating parts from the whole: determinants of odor mixture perception in squirrel monkeys,Saimiri sciureus

In a task designed to simulate olfactory-guided foraging, the ability of squirrel monkeys to discriminate an artificial 12-component odorant from 3-, 6-, 9- or 11- component submixtures was investigated. A combination of factors was found to contribute to the animals' performance:

Morphometric analysis of the zona pellucida and ooplasm of squirrel monkey (Saimiri sciureus) eggs

The objective of this study was to correlate the thickness of the zona pellucida (zona) with egg (oocyte) maturity determined through a widely-used method of assessing oocyte maturation namely, the evidence for cumulus and corona cells expansion. Measurements of the zona of cultured oocytes were recorded at 0, 3, 6, 24, and 48 hr after laparoscopic oocyte retrieval from hormonallystimulated squirrel monkeys. The results indicated that at the time of oocyte retrieval, oocytes that were classified as mature had thicker zonae compared with immature oocytes. The zona layer of the mature oocyte expanded to a maximum after 6 hr of incubation while the zona layer of the immature oocyte became compressed. The diameter of the mature oocyte (minus the zona and perivitelline space) became smaller with time while the immature oocyte diameter remained relatively unchanged. The correlation between the maturational state of the oocyte and the thickness of the zona layer suggest the possible application of zona morphometric evaluations as an indicator of oocyte maturation.     

Molecular phylogenetic relationships among Sichuan snub-nosed monkeys (Rhinopithecus roxellanae) inferred from mitochondrial cytochrome-b gene sequences

The Sichuan snub-nosed monkey (Rhinopithecus roxellanae) is a species endemic to China, and its distribution is the widest among all snub-nosed monkeys in China. To clarify whether there is subspecific differentiation within this species, we determined partial sequences of the cytochrome-b gene from four populations ofR. roxellanae. First, 402bps of the partial sequences fromR. roxellanae were compared with those fromR. bieti andR. avunculus, and the phylogenetic tree was constructed by the neighbor-joining method. The genetic distance was only 0–0.002 among the four populations, and their sequences constituted a monophyletic group. Further, comparison of longer sequences (735bp) among the four populations revealed that there were only four substitutions and the genetic distance was only 0.001–0.005 among them. Thus, we suggest that, at least on mtDNA phylogeny, the difference among the four populations does not reach the subspecies level, and that this species should be recognized as a monotypic species.     

Molecular systematics of theDrosophila hydei subgroup as inferred from mitochondrial DNA sequences

The phylogeny of theDrosophila hydei subgroup, which is a member of theD. repleta species group, was inferred from 1,515 base pairs of mitochondrial DNA sequence of the cytochrome oxidase subunits I, II, and III. Four of the seven species in the subgroup were examined, which are placed into two taxonomic complexes: theD. bifurca complex (D. bifurca) andD. nigrohydei) and theD. hydei complex (D. hydei and (D. eohydei). Both complexes appear to be monophyletic, although theD. bifurca complex is only weakly supported. The evolution of chromosomal change, interspecific crossability, sperm gigantism, and divergence times of the subgroup is discussed in a phylogenetic context. Correspondence to: G. Spicer     

Conditions of failure and recovery of maternal behavior in captive squirrel monkeys (Saimiri)

This paper deals with the functioning and malfunctioning of maternal behavior in relation to external and social conditions. During developmental studies in captive squirrel monkey groups, 49 mother-infant pairs were observed. There were several cases of maternal inadequacy subsequent to (1) postnatal transportation, (2) exaggerated social investigation by immatures,and (3) premature allomothering. Brief mother-infant separations by humans did not yield such failures. One female consistently prevented nursing with at least three of her five viable offspring. Complete or partial failure of maternal behavior did not preclude subsequent recovery and/or adequate allomothering. Dedicated to Prof. Dr. Med. Detlev Ploog on the occasion of his 60th birthday.     

Molecular phylogeny of gibbons inferred from mitochondrial DNA sequences: Preliminary report

We analyzed the 896 base-pair (bp) mitochondrial DNA (mtDNA) sequences for seven gibbons, representative of three out of four subgenera. The result from our molecular analysis is consistent with previous studies as to the monophyly of subgenus Hylobates species, yet the relationship among subgenera remains slightly ambiguous. A striking result of the analysis is the phylogenetic location of Kloss's gibbon (H. klossii). Kloss's gibbon has been considered to be an initial off-shoot of the subgenus Hylobates because of its morphological primitiveness. However, our molecular data strongly suggest that Kloss's gibbon speciated most recently within the subgenus Hylobates. Correspondence to: S. Horai     

The effect of relative humidity on osmoregulation in the squirrel monkey (Saimiri sciureus)

Osmoregulatory balance was studied in four young, tamed squirrel monkeys (Saimiri sciureus, Columbia) after acclimatization to relative humidities (rh) commonly used in laboratories (30% and 50%) and to higher humidities representative of the dry and wet seasons in their natural environment (75% and 95%). The temperature was constant at 25°C and the light-dark cycle was 12:12 hours. The animals were maintained in large metabolism cages and were free moving. Water consumption and urine flow rates increased at each higher humidity from 30% rh to 75% rh and then decreased at 95% rh (p<0.05). Fecal water loss was greater with higher humidity (p<0.05). Evaporative water loss remained relatively constant until a break between 75% and 95% rh, at which point it decreased dramatically (p<0.05). Expressed relative to total water intake, evaporative water loss demonstrated a progressive decrease with increasing humidity: 65%, 56%, 51%, and 42%, at 30%, 50%, 75%, and 95% rh. This indicates that as the humidity approaches maximum saturation, not only is the evaporative water loss component necessarily diminished, but also the apparent precipitation of alternate strategies of thermoregulation leads to a reduction in the overall water requirements of the animals. Potassium/sodium excretion ratios were relatively constant at 30%, 50%, and 95% rh but decreased significantly at 75% rh (p<0.05). As a reflection of adrenocorticosteroid activity, this suggests that at 25°C, 75% rh is an optimal humidity in the maintenance of squirrel monkeys. The significant osmoregulatory alteration occurring between 75% and 95% rh provides further evidence that relative humidity may be an important factor in the seasonal physiological cycles of the squirrel monkey. In conducting the research described in this report the investigators adhered to the “Guide for Laboratory Animal Facilities and Care,” as promulgated by the Committee on the Guide for Laboratory Animal National Academy of Sciences, National Research Council.     

Complete mitochondrial DNA sequence of Aulopus japonicus (Teleostei: Aulopiformes), a basal Eurypterygii: longer DNA sequences and higher-level relationships

For the first step toward resolution of the higher-level relationships of the order Aulopiformes (Teleostei: Eurypterygii) using longer DNA sequences, we determined the complete mitochondrial DNA sequence for Aulopus japonicus (Aulopodidae). The entire genome was purified by gene amplification using a long PCR technique, and the products were subsequently used as templates for PCR with 63 fish-versatile and 3 species-specific primers that amplify contiguous, overlapping segments of the entire genome. Direct sequencing of the PCR products demonstrated that the genome (16 653 base pairs [bp]) contained the same 37 mitochondrial genes (2 ribosomal RNA, 22 transfer RNA, and 13 protein-coding genes) as found in other vertebrates, with the gene order identical to that in typical vertebrates. Maximum-parsimony analysis using nucleotide sequences from the concatenated 12 protein-coding genes (no third codon positions and excluding the ND6 gene) plus 22 tRNA genes (stem regions only) from eight teleosts placed A. japonicus in a reasonable phylogenetic position; those from individual protein-coding genes and the concatenated 22 tRNA genes alone, however, did not reproduce the expected phylogeny with few exceptions, probably owing to insufficient phylogenetic information in these smaller data sets. This result suggests that further taxonomic sampling and sequencing efforts may clarify limits and intra- and interrelationships of this morphologically and ecologically diverse group of fishes using mitochondrial genomic (mitogenomic) data. Received: August 31, 2000 / Revised: December 20, 2000 / Accepted: January 23, 2001     

Making the most of mitochondrial genomes--markers for phylogeny, molecular ecology and barcodes in Schistosoma (Platyhelminthes: Digenea)

An increasing number of complete sequences of mitochondrial (mt) genomes provides the opportunity to optimise the choice of molecular markers for phylogenetic and ecological studies. This is particularly the case where mt genomes from closely related taxa have been sequenced; e.g., within Schistosoma. These blood flukes include species that are the causative agents of schistosomiasis, where there has been a need to optimise markers for species and strain recognition. For many phylogenetic and population genetic studies, the choice of nucleotide sequences depends primarily on suitable PCR primers. Complete mt genomes allow individual gene or other mt markers to be assessed relative to one another for potential information content, prior to broad-scale sampling. We assess the phylogenetic utility of individual genes and identify regions that contain the greatest interspecific variation for molecular ecological and diagnostic markers. We show that variable characters are not randomly distributed along the genome and there is a positive correlation between polymorphism and divergence. The mt genomes of African and Asian schistosomes were compared with the available intraspecific dataset of Schistosoma mansoni through sliding window analyses, in order to assess whether the observed polymorphism was at a level predicted from interspecific comparisons. We found a positive correlation except for the two genes (cox1 and nad1) adjoining the putative control region in S. mansoni. The genes nad1, nad4, nad5, cox1 and cox3 resolved phylogenies that were consistent with a benchmark phylogeny and in general, longer genes performed better in phylogenetic reconstruction. Considering the information content of entire mt genome sequences, partial cox1 would not be the ideal marker for either species identification (barcoding) or population studies with Schistosoma species. Instead, we suggest the use of cox3 and nad5 for both phylogenetic and population studies. Five primer pairs designed against Schistosoma mekongi and Schistosoma malayensis were tested successfully against Schistosoma japonicum. In combination, these fragments encompass 20-27% of the variation amongst the genomes (average total length approximately 14,000bp), thus providing an efficient means of encapsulating the greatest amount of variation within the shortest sequence. Comparative mitogenomics provides the basis of a rational approach to molecular marker selection and optimisation.     

Global phylogeography of the ridley sea turtles (Lepidochelys spp.) as inferred from mitochondrial DNA sequences

The Kemp's ridley sea turtle (Lepidochelys kempi) is restricted to the warm temperate zone of the North Atlantic Ocean, whereas the olive ridley turtle (L. olivacea) is globally distributed in warm-temperate and tropical seas, including nesting colonies in the North Atlantic that nearly overlap the range of L. kempi. To explain this lopsided distribution, Pritchard (1969) proposed a scenario in which an ancestral taxon was divided into Atlantic and Pacific forms (L. kempi and L. olivacea, respectively) by the Central American land bridge. According to this model, the olive ridley subsequently occupied the Pacific and Indian Oceans and recently colonized the Atlantic Ocean via southern Africa. To assess this biogeographic model, a 470 bp sequence of the mtDNA control region was compared among 89 ridley turtles, including the sole L. kempi nesting population and 7 nesting locations across the range of L. olivacea. These data confirm a fundamental partition between L. olivacea and L. kempi (p=0.052-0.069), shallow separations within L. olivacea (p=0.002-0.031), and strong geographic partitioning of mtDNA lineages. The most divergent L. olivacea haplotype is observed in the Indo-West Pacific region, as are the central haplotypes in a parsimony network, implicating this region as the source of the most recent radiation of olive ridley lineages. The most common olive ridley haplotype in Atlantic samples is distinguished from an Indo-West Pacific haplotype by a single nucleotide substitution, and East Pacific samples are distingushed from the same haplotype by two nucleotide substitutions. These shallow separations are consistent with the recent invasion of the Atlantic postulated by Pritchard (1969), and indicate that the East Pacific nesting colonies were also recently colonized from the Indo-West Pacific region. Molecular clock estimates place these invasions within the last 300,000 years. This revised version was published online in July 2006 with corrections to the Cover Date.     

Molecular phylogeny and biogeography of woolly flying squirrel (Rodentia: Sciuridae), inferred from mitochondrial cytochrome b gene sequences

To investigate the genetic diversity between the populations of woolly flying squirrels (Eupetaurus) from the eastern and western extremes of the Himalayas, partial mitochondrial cytochrome b gene sequences (390-810 bp) that were determined from the museum specimens were analyzed using maximum parsimony (MP) and maximum likelihood (ML) methods. The molecular data reveal that the two specimens that were collected in northwestern Yunnan (China) are members of the genus Eupetaurus. Reconstructed phylogenetic relationships show that the populations of Eupetaurus in the eastern and western extremes of the Himalayas are two distinct species with significant genetic differences (12%) and diverged about 10.8 million years ago. Eupetaurus is significantly different from Petaurista and Pteromys. The level of estimated pairwise-sequence divergence observed between Eupetaurus and Petaurista or Pteromys is greater than that observed between Eupetaurus and Trogopterus, Belomys, Glaucomys, or Hylopetes. Considering the divergence time of the two Eupetaurus groups, the glaciations and the uplift of the Himalayas and Qinghai-Tibet plateau during the Pliocene-Pleistocene period might be the major factors affecting the present distribution of Eupetaurus along the Himalayas.     

Increased production of 11beta-hydroxysteroid dehydrogenase type 2 in the kidney microsomes of squirrel monkeys (Saimiri spp.)

In squirrel monkeys (Saimiri spp.), cortisol circulates at levels much higher than those seen in man and other Old World primates, but squirrel monkeys exhibit no physiologic signs of the mineralocorticoid effects of cortisol. These observations suggest that squirrel monkeys have mechanisms for protection of the mineralocorticoid receptor (MR) from these high levels of cortisol. We previously showed that the serum cortisol to cortisone ratio in these animals is low relative to that in human serum, suggesting that production of the MR protective enzyme, 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), is increased in squirrel monkeys. Here, we directly evaluate whether increased production of 11beta-HSD2, which inactivates cortisol to cortisone, is a mechanism for protection of MR. In vitro assays showed that 11beta-HSD2 activity in squirrel monkey kidney microsomes was 3 to 7 times higher than that seen in kidney microsomes from pig or rabbit. 11beta-HSD2 protein detected by Western blot analysis was 4 to 9 times greater in squirrel monkey microsomes than in pig or rabbit microsomes. Comparison of the effect of expression of either human or squirrel monkey 11beta-HSD2 on MR transactivation activity showed similar inhibition of MR response to cortisol by both enzymes, indicating that the intrinsic activities of the human and squirrel monkey enzymes are similar. These findings suggest that one mechanism by which squirrel monkeys protect the MR from activation by high cortisol levels in the kidney is by upregulation of 11beta-HSD2 activity through increased production of the enzyme.     

从细胞色素b基因序列探讨笛鲷属的分子系统发生关系

测定了9种中国南海的笛鲷属鱼类的细胞色素b基因的部分序列,结合来自GenBank中1种分布于菲律宾和9种分布于美国大西洋的笛鲷属鱼类的相应同源序列,用邻接法和最大简约法构建分子系统树。结果显示:红鳍笛鲷(Lutjanuserythropterus)与红笛鲷(L.sanguineus)之间的同源序列碱基差异百分率只有0.32%,支持二者是同种异名的观点;中国南海的笛鲷属鱼类间的平均碱基差异要高于美国大西洋笛鲷属鱼类。在MP和NJ树中,美国大西洋笛鲷表现为亲缘关系较近,来源于中国南海的笛鲷鱼类相对集中在树的基部,分歧较大。这与所研究的笛鲷地理分布和地理隔离基本相一致,同时也说明中国南海笛鲷分化较早并且分歧较大。     

Seasonal changes of serum concentrations of estradiol and progesterone in Bolivian squirrel monkeys (Saimiri sciureus)

Serial measurements of estradiol (E2) and progesterone (P) were used to describe the ovarian cycle of the Bolivian squirrel monkey. Group-caged, sexually mature female squirrel monkeys, housed with males, were sampled daily between 0900 and 1100 hr. Sampling was carried out during the breeding and nonbreeding seasons, for periods of 19–20 days from September 1981 to May 1982. Seasonal differences in serum concentrations of E2 and P were found with low levels of E2 and P and an absence of preovulatory surges of E2 during the nonbreeding season. This pattern was also observed in some animals during the breeding season. An abrupt increase in serum P concentrations in December appeared to signal the onset of cycling. Cycling animals had well-defined peaks of E2 (450–9,500 pg/ml) followed by increasing levels of P, which were >200 ng/ml in some animals. After the breeding season, E2 and P levels returned to their initially low levels. Levels of both steroids in cycling animals were higher than those reported for other primates and for previous measurements made in squirrel monkeys. Cycle length based on time interval between consecutive E2 peaks varied from 6–12 days.     

The origin of land plants: Phylogenetic relationships among charophytes,bryophytes, and vascular plants inferred from complete small-subunit ribosomal RNA gene sequences

Complete nuclear-encoded small-subunit 18S rRNA (=SSU rRNA) gene sequences were determined for the prasinophyte green alga Mantoniella squamata; the charophycean green algae Chara foetida, Coleochaete scutata, Klebsormidium flaccidum, and Mougeotia scalaris; the bryophytes Marchantia polymorpha, Fossombronia pusilla, and Funaria hygrometrica; and the lycopod Selaginella galleottii to get a better insight into the sequential evolution from green algae to land plants. The sequences were aligned with several previously published SSU rRNA sequences from chlorophytic and charophytic algae as well as from land plants to infer the evolutionary relationships for major evolutionary lineages within the Chlorobionta by distance matrix, maximum parsimony, and maximum likelihood analyses. Phylogenetic trees created by the different methods consistently placed the Charophyceae on the branch leading to the land plants. The Charophyceae were shown to be polyphyletic with the Charales (charalean algae) diverging earlier than the Coleochaetales, Klebsormidiales, Chlorokybales, and Zygnematales (charophycean algae) which branch from a point closer to the land plants in most analyses. Maximum parsimony and maximum likelihood analyses imply a successive evolution from charophycean algae, particularly Coleochaetales, to bryophytes, lycopods, and seed plants. In contrast, distance matrix methods group the bryophytes together with the charophycean algae, suggesting a separate evolution of these organisms compared with the club moss and the seed plants. Correspondence to.: V.A.R. Huss     

A phylogeny of Chinese species in the genus Phrynocephalus (Agamidae) inferred from mitochondrial DNA sequences

We investigated the phylogenetic relationships among most Chinese species of lizards in the genus Phrynocephalus (118 individuals collected from 56 populations of 14 well-defined species and several unidentified specimens) using four mitochondrial gene fragments (12S rRNA, 16S rRNA, cytochrome b, and ND4-tRNA(LEU)). The partition-homogeneity tests indicated that the combined dataset was homogeneous, and maximum-parsimony (MP), neighbor-joining (NJ), maximum-likelihood (ML) and Bayesian (BI) analyses were performed on this combined dataset (49 haplotypes including outgroups for 2058bp in total). The maximum-parsimony analysis resulted in 24 equally parsimonious trees, and their strict consensus tree shows that there are two major clades representing the Chinese Phrynocephalus species: the viviparous group (Clade A) and the oviparous group (Clade B). The trees derived from Bayesian, ML, and NJ analyses were topologically identical to the MP analysis except for the position of P. mystaceus. All analyses left the nodes for the oviparous group, the most basal clade within the oviparous group, and P. mystaceus unresolved. The phylogenies further suggest that the monophyly of the viviparous species may have resulted from vicariance, while recent dispersal may have been important in generating the pattern of variation among the oviparous species.