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Lying in a shallow continental shelf cyclically affected by oscillating sea levels since the Miocene, the Seychelles islands are particularly interesting for evolutionary studies. Recent molecular studies are generating an emerging picture of the origin of its biota, yet very little is known regarding their phylogeographic structure or on the factors promoting diversification within the archipelago. Here we aimed to obtain a detailed depiction of the genetic structure and evolution of one of the most widespread vertebrate groups in the archipelago: the day-geckos of the genus Phelsuma. In parallel, we aimed to infer divergence times between species and subspecies, testing a long-standing hypothesis that argues for different time since sympatry between species as the cause of their different morphological differentiation across the archipelago.
Molecular data corroborated the existence of two main lineages, corresponding to the two currently recognized species. Divergences between species likely date back to the Mio-Pliocene, while more recent, Pleistocenic, divergences are suggested within each species. Populations from outer islands share mtDNA haplotypes with inner island populations, suggesting very recent dispersals (or introductions). We found no evidence of current gene flow between species, but results pointed to the possibility of gene flow between (now allopatric) subspecies. Time estimates suggest a synchronous divergence within each species (between island groups).
The geographic patterns of genetic variation agree with previous taxonomic subdivisions within each species and the origin of outer islands populations is clearly tracked. The similar intraspecific divergence time estimates obtained suggest that the differential body-size differentiation between species within each group of islands may be driven by factors other than character displacement proportional to time since sympatry, as previously suggested. These factors could include different habitats/resources available within each island group, niche differentiation and/or character displacement. We also bring again into consideration the hypothesis of body size being influenced by the distribution of native vegetation and social systems within this group, although it remains to be tested. Our results highlight not only the necessity of clarifying the role of ecology and interspecific interactions in this group's morphological diversification and community assemblage, but also the importance of co-evolutionary mechanisms and their importance for appropriate conservation of island biodiversity. Further, we provide a detailed description of the phylogeographic structure of these taxa across these islands, which still remain poorly characterized in this respect.
Keywords:Phelsuma; Seychelles; Phylogeography; Species-trees; Diversification; Morphological evolution; Character displacement; Biogeography
The Western Indian Ocean islands of Madagascar, Comoros, the Mascarenes, and the Seychelles are one of the 34 "hotspots" defined by Conservation International [1]. While there is an emerging picture from molecular data about the origin of their biota (see [2-7] for recent reviews), diversification mechanisms within each archipelago and island are less understood. That is particularly true for the Seychelles, where only a handful of studies have explored the genetic structure of different species within the archipelago [8-13]. The separation of the Seychelles and the Indian subcontinent is estimated to have been completed 65 Mya and the Seychelles include about 155 islands that can be divided in 3 groups: granitic, low and raised coralline islands (Figure 1). The granitics are a group of about 40, clustered together on the undersea shelf of granite, that is the Seychelles bank. These comprise the islands of North, Silhouette, Mahé, Fregate, Praslin, La Digue, Curieuse and several smaller ones encircling them. The low coralline islands are very recent, probably less than 6,000 years ago, and formed from marine sediments cemented with guano deposits [14]. The raised coralline islands (Aldabra, Assumption, Astove and Cosmoledo) are also oceanic, formed by reef-building corals acting on submerged volcanic seamounts that may have formed some 20 million years ago (Mya) [15] and were submerged and re-emerged several times since their formation.
Figure 1. Sampling sites for Phelsuma species along the granitic and coralline Seychelles islands. Species and subspecies are coded using different symbols: dark blue triangles - P. astriata astriata; light blue triangles - P. astriata semicarinata; white triangle - P. astriata (Fregate population); dark green circles - P. sundbergi longinsulae; light green squares - P. sundbergi sundbergi; light green circles - P. sundbergi ladiguensis. Within the granitic islands (insets a and b), different shadings show areas that would be emerged at −30 m (dark grey) and −50 m (light grey) below present sea-level stands. Low-stands below 60 m would allow contact between all islands in this granitic group. Current data on Indian Ocean sea level changes support low-stands of up to 145 ± 5 m below present sea level (bpsl) at six episodes in the last 0.5 Myr, some of which persisted for up to 50,000 years at a time see [6] and geological records going back further suggest low-stands of 80–120 m bpsl at 0.64, 0.88, 1.04, 1.26 and 1.54 Mya, and eleven additional low-stand episodes of 50 m or more bpsl during the last 5 Myr [18]. Detailed locations and accession numbers are given in Additional file 1.
Additional file 1. List of all analysed specimens with details on geographic origin and Genbank numbers.
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Sea-level oscillations occurred throughout the Pleistocene and back through the Miocene, with low stands of up to 145 m below present sea-level (bpsl) [16-18]. All the islands in the plateau would have been united with sea-levels of roughly 60 m bpsl, but even less pronounced minima greatly increased the size of each island and their connectivity (Figure 1a, b). It is probable that these cycles of allopatry and contact between islands shaped distribution patterns, organismal divergence and diversification within the archipelago. Nevertheless, intra-archipelago genetic structure of Seychellois taxa is poorly known, as well as the relative influence of isolating vs. connecting periods on species diversification. As in other archipelagos cyclically isolated and connected, cryptic diversity might be higher than currently appreciated [19,20].
Historically, Mahé and surrounding islands, plus Silhouette and North have been considered to form a biogeographic unit separated from the Praslin-La Digue (and surrounding islands) group. Fregate, located roughly in the middle of the two groups of islands, is generally regarded as an isolated or intermediate unit [8,21,22]. Distribution data for insects [21], molluscs [23], and a few studies of intraspecific variation within amphibians [24] and reptiles [8,25] are consistent with this pattern. However, this does not seem to stand for some other groups [9,26]. Most of the few existing studies on intraspecific genetic variation suggest deep and cryptic divergences among the granitic Seychelles [12,13,27,28], highlighting the need for thorough sampling and phylogeographic analyses across these islands. At the same time, it becomes important to understand also the relative influence of historical and ecological factors in the generation and maintenance of such diversity. Day-geckos of the genus Phelsuma are ideal in this respect, as its two Seychellois species are widespread across most of the islands, with both sympatric and allopatric populations. Phelsuma mostly diversified in Madagascar, from where they colonized the surrounding archipelagos and often speciated (reviewed in [28]). Within each island clade, Phelsuma species diverged ecologically and morphologically, evolving a wide variety of colour patterns and sizes and occupying a broad range of arboreal habitats (see [29] and references therein). However the relative contribution of geographic isolation, local adaptation and interspecific interactions, including hybridization, in the generation of these species assemblages is rather unclear.
In the granitic Seychelles, Phelsuma is represented by a small endemic clade [8,30]. The classification of the Seychelles "forms" of Phelsuma has been a persistent problem due to the considerable variation in colour pattern and size across islands. Cheke [31] and Gardner [25,32,33] assessed the systematics of the Seychelles forms and currently two endemic species are recognized with an unclear number of subspecies. Within P. sundbergi, up to six subspecies have been recognized: P. sundbergi longinsulae, inhabiting Mahé (and associated islands) plus Fregate; P. (sundbergi) longinsulae rubra and umbrae, respectively from North and Silhouette sensu [34] reflecting previous consideration of P. longinsulae as a separate species; P. s. sundbergi, from Praslin, Curieuse, Aride and surrounding islands, and P. s. ladiguensis from La Digue, Mariane, Grande and Petite Soeur and surrounding islands (Figure 1). Gardner [25,33] examined variation within these species based on body size, scalation and colour pattern variables and found support for only three of the groups, P. s. longinsulae, P. s. sundbergi and P. s. ladiguensis (the latter two being clearly more similar), but not for the distinction of the North and Silhouette forms, rubra and umbrae. These were synonymised with P. sundbergi longinsulae, as was also P. (sundbergi) longinsulae menaiensis sensu [35], a putative subspecies from the coralline islands of Cosmoledo described in some detail by Cheke [31]. Within P. astriata two allopatric subspecies are recognized; P. a. astriata and P. a. semicarinata, from the southern and northern group of islands respectively (Figure 1), with populations from Fregate described as "intermediate forms" and hypothesized to be hybrids between the two subspecies [31]. Individuals from Astove (raised coralline island) were considered by Cheke [31] to be closely related to the nominate astriata on Mahé.
The degree of morphological differentiation between and within species and subspecies varies geographically. Notably, body size variation shows no clear geographic pattern within P. astriata, while clearly increases from south to north within P. sundbergi; with P. s. sundbergi and P. s. ladiguensis individuals being significantly larger than P. s. longinsulae[8,32]. Radtkey [8] proposed allopatric speciation followed by asynchronous dispersals across island groups plus resource competition generating character displacement as the drivers of speciation and body size evolution within this group. More specifically, he proposed that after the invasion of the Seychelles archipelago by a single species of Phelsuma, sea-level changes led first to allopatric speciation, with the ancestor of P. sundbergi and P. astriata differentiating in the southern and northern groups of islands, respectively, and then to the asynchronous dispersal of these species across island groups. The differences in the relative timing of the dispersal between island groups (with the ancestor of P. s. sundbergi and P. s. ladiguensis colonizing the northern group of islands much earlier than the ancestor of P. astriata astriata colonized the southern group) would have resulted in P. sundbergi evolving an intermediate body size in the group of islands associated with Mahé (southern) and a large body size in the group of islands associated with Praslin and La Digue (northern), due to character displacement, and given its longer period of sympatry with P. astriata in the northern islands. Nevertheless, this is not the only possible explanation for the different degrees of body size disparity. Body size distributions may be governed instead by different ecological variables and interactions happening within each island group [32,36]. For example, competition and selective pressures for body size within each island or island group may be distinct and lead to distinct patterns of morphological differentiation between species within island group, independently of the time since sympatry. In fact, under character displacement, phenotypic divergence accelerates in sympatry [37] but needs not be correlated with time since sympatry [38,39].
Gardner [32] proposed instead that body size in Seychellois Phelsuma has probably been influenced by the distribution of vegetation and social systems. The use of defensible pollen and nectar food supplies, especially the male inflorescences of Lodoicea maldivica, native to (and only found in) the northern islands of Praslin and Curieuse, may have resulted in the evolution of the larger body size of P. sundbergi in the northern islands while hardwood forests, shrub and seabird islands vegetation may have led to smaller sizes in the south. The competitive relationships between the species could then be influenced by their body size differences and help to support the selective pressures within the system. Sympatric species within each island may be partitioning their habitat differently or diversifying along different axis eg. [40-43], resulting in the observed pattern, without the need to invoke asynchronous sympatry as its cause. In the Mascarenes for example, sympatric species of Phelsuma often differ across several resource axes at the same time [44]. If the time since contact between the different species in the Seychelles is similar, ecological factors shall play a much more important role in morphological diversification of Seychellois Phelsuma than currently appreciated. It is also not known if these species hybridize on any island group.
Our objectives here were first to assess the intraspecific genetic variation within species and its geographical distribution, inferring possible colonization routes and the geographic origins of outer island populations. Second, using multilocus data, to explore the possibility of gene flow across species and subspecies and infer the underlying population's history. Ultimately, we aimed to test the asynchronous divergence within each species, simultaneously providing a time scale for the diversification of this clade. Such a phylogeographic approach will help us to better understand the relative influence of oscillating sea-levels in shaping diversification across these islands, and also should provide a valuable framework upon which the ecological determinants of diversification within this group can be further explored.
Mitochondrial DNA variability and genetic structure
The two species, P. sundbergi and P. astriata correspond to two divergent mtDNA clades (Figure 2) (Nei's Da = 11%). Intraspecifically, Phelsuma sundbergi longinsulae clearly differentiates from P. s. sundbergi and P. s. ladiguensis (Da = 2.5%) (Figure 2b). Within P. s. longinsulae, the North and Silhouette populations share or carry haplotypes very closely related to Mahé. Silhouette haplotypes are paraphyletic relative to Mahé, and individuals from North exhibit a single haplotype, shared with individuals from Mahé (Figure 2b). Furthermore, all individuals from Fregate carry a unique haplotype, which is just one nucleotide substitution away from the most abundant haplotype from Mahé. The haplotype of one P. sundbergi individual from Mahé (3MA206) clusters with P. sundbergi haplotypes from Praslin. We provisionally interpreted this as a possibly anthropogenic related pattern (but see Discussion) and excluded this individual for the inference of divergence times and gene flow. Concerning the P. sundbergi populations from the northern group of Praslin, La Digue and associated islands, i.e., P. sundbergi sundbergi and P. s. ladiguensis, they form two slightly divergent mtDNA clades, in agreement with their morphological variation: Praslin and Curieuse populations in one clade and La Digue and Grande Soeur populations in the other clade (Figure 2b). Yet, two individuals from La Digue exhibit haplotypes that cluster within those from Praslin.
Figure 2. a) MtDNA haplotype phylogeny. Midpoint-rooted Bayesian 50% majority-rule consensus tree with posterior probabilities above 0.5 indicated above branches. The scale bar refers to branch lengths (expected changes per site). Median-Joining networks for P. sundbergi (b) and P. astriata (c). Islands are colour-coded and circle size is proportional to the number of individuals. Full grey dots along branches represent missing or unsampled haplotypes. Islands labelling is as follows: Aride (ARD), Curieuse (CUR), Grande Soeur (BS), Praslin (PL), La Digue (LD), Cousine (CNE), Fregate (FG), Cerf (CF), Mahé (MA), Silhouette (SILH) and North (NOR). Highlighted haplotypes (***) correspond to the individuals from Mahé and La Digue found to cluster within P. sundbergi sundbergi (from Curieuse and Praslin) (see text). The outer islands of Cosmoledo, Astove (AT), Poivre and Alphonse (APH) are identically coloured in white (see Results for detailed information).
Within P. astriata the non-overlapping geographic distribution of mitochondrial lineages agrees also with the currently recognized subspecies (Figure 2c). The divergence between these lineages is not large, with just three substitutions separating haplotypes from P. a. astriata and P. a. semicarinata, and with two and three substitutions separating both these subspecies from individuals from Fregate, where the three individuals sampled exhibit the same mtDNA haplotype.
All the outer island populations share mtDNA haplotypes with the granitic islands populations (Figures 2b and 2c, in white colour). The Cosmoledo populations of P. sundbergi longinsulae exhibit a single haplotype, the central one in the phylogenetic network and the most abundant in Mahé. Similarly, P. astriata astriata haplotypes from Alphonse are also shared with individuals from Mahé, while the Astove population of P. astriata astriata shares its (again single) haplotype with Mahé, Cerf and Silhouette individuals. Finally, individuals of P. sundbergi sundbergi from Poivre exhibit a single and same haplotype as most of the individuals from Praslin.
Nuclear DNA genealogies
The nuclear variation is almost completely sorted between species (Figure 3). P. astriata and P. sundbergi share only a single haplotype for one of the genes (PDC), which is central in the phylogenetic network and the most abundant in P. astriata. Overall, divergence between species is not high and the conspecific subspecies in all cases share a high number of haplotypes. The only nuclear gene where intraspecific structure is noticeable is MC1R, with two subgroups within each species, with a strong geographic component.
Figure 3. MJ networks representing nuclear DNA variation. Islands are colour-coded and circle size is proportional to the number of individuals. Labelling is the same as in Figure 2. Full grey dots along branches represent missing or unsampled haplotypes. Clades above and below the dashed horizontal line correspond to P. astriata and P. sundbergi, respectively (except for PDC where haplotype sharing is observed – inset network). The gene to which each network corresponds is identified on its inferior left corner.
Genetic variability and neutrality tests
Considerable variation was observed in the levels of polymorphism across loci (Table 1), with Cytb and RELNint61 being the polymorphic ones. The variability levels within each subspecies were not remarkably different, although they were slightly higher within P. sundbergi longinsulae. Tajima's D and Fu's Fs were usually negative, while R2 values were usually small, suggesting demographic growth across all groups, although only in a few instances (P. sundbergi longinsulae for the mitochondrial and Rag-2 markers and P. astriata semicarinata for Cytb) were values significant for all statistics.
Table 1. Summary diversity statistics, tests of neutrality and indexes of populations growth for all subspecies of P. sundbergi and P. astriata
Time and gene flow estimates under the isolation-with-migration model
Using the IM model we obtained consistent estimates of demographic parameters across all runs (Table 2), with high effective sample size (ESS) values, convergence and a good sampling of the parameter space. Assuming a mean rate substitution of 1% per lineage per million years (Myr) at this marker, the estimated time since divergence between the two species was 6.39 Myr, with a 95% high posterior density (HPD) interval of 4.264 – 8.397. No gene flow was detected between species and the ancestral effective population size estimate was very low (Table 2, bottom row). Estimates of the splitting times between the southern and northern clades of both species were fairly coincident (~500,000 years old) as so were their posterior density intervals (Table 2 and Figure 4a), which were, nevertheless, quite large. The effective population size estimates for P. sundbergi doubled those for P. astriata, irrespectively of the island group. Accordingly, the estimated effective ancestral population sizes were also higher in P. sundbergi. Even having excluded the few individuals presumed to be the result of anthropogenic introductions (as mentioned above), significant migration rates were still recovered within both species, especially for P. sundbergi (Table 2 and Figure 4b). Neither recombination nor selection was detected at any locus.
Figure 4. a) Probability distribution for the splitting time between northern and southern clades of P. astriata (light blue) and P. sundbergi (dark green); b) number of migrant gene copies per generation (2 Nm) between those same clades within P. astriata (blue, upper) and P. sundbergi (green, down).
Table 2. Isolation-with-migration estimates
Seychelles' Phelsuma species tree
*BEAST analyses recovered consistent results across replicates, with high ESS values for all parameters of interest. Estimates without data recover completely different values and distributions, showing that the data, and not the prior, are primarily responsible for the posterior distributions. The maximum clade credibility species tree (Figure 5) was in general agreement with the inferences made under the IM model in respect to the ages of the branching events. The initial split between the two species (P. sundbergi and P. astriata) was dated to circa 3.6 Myr, although the 95% HPD age intervals were very wide (1.33 – 6.86). These estimates are slightly more recent than those obtained under the IM model (Table 2), but the HPD intervals considerably overlap. Estimates of 95% HPD intervals for the north–south split within both species again clearly overlap. The population of P. astriata from Fregate (included here though excluded from the IMa analysis so data could fit a two populations model) was recovered as sister taxa to P. astriata semicarinata, with moderate support.
Figure 5. "Species" tree of the Phelsuma day-geckos endemic from the Seychelles. The tree shown corresponds to the maximum clade credibility tree with mean estimates used for node heights and it is based on the multispecies coalescent analysis of the five molecular markers and assuming a rate of evolution of 0.01 (SD = 0.0027) substitutions per lineage per My for Cytb. Clade posterior probability is indicated above each branch. Node bars correspond to the 95% high posterior density intervals for node height (age), which are given below/in front of each node. Horizontal axis corresponds to time in million years before present (Mya).
Species divergence and intraspecific genetic structure
Here we analyzed in detail for the first time the genetic structure of the Phelsuma day-geckos in the Seychelles based on a comprehensive sampling across the archipelago. Molecular data from widespread populations across these islands corroborate the two recognized species as two single main lineages and place their divergence in the late Miocene or early Pliocene. That is earlier than the Pliocene/Pleistocene divergence estimates within the similarly distributed Seychellois crab genus Seychellum[12] but in the same range as the north–south split within the co-distributed Seychellois gecko species Urocotyledon inexpectata[13].
Very recently, Carranza et al. [45] obtained rate estimates for part of the same Cytb fragment used in this study that are higher that the one we used (approximately the double and up to three times higher, depending on the group of taxa, although with high standard deviations). Pending further confirmation of these rate estimates, we preferred to keep the use here of a more conservative (and widespread) average rate of 1% substitutions per lineage per Myr. Still, we note the need of taking these absolute age estimates with caution. We note also that using a faster rate would lead us to more recent estimates of species and subspecies diversification (although yet early Pliocenic and Pleistocenic, respectively) but that it would not change the relative timing between these events. Importantly, it would not change the inference of a simultaneous split within each species.
We did not find evidences of past or current gene flow between the two species. While the application of the IM model is debatable given the high levels of population structure observed, Strasburg and Rieseberg [46] showed that most parameter estimates in IMa are fairly robust to population structure over a wide range of parameter values, down to Nm = 0.1 among conspecific populations. It is nevertheless possible that estimates of population sizes and divergence times are slightly biased upwards in these cases [46].
Within each species, our data are congruent with previous multivariate morphometric studies from Gardner [25], with Cheke's [31] taxonomic classification and with previous inferences from Radtkey [8]: three lineages within P. sundbergi and two lineages within P. astriata. The limited number of individuals of P. astriata sampled from Fregate, as well as the little intraspecific differentiation found between the northern and southern islands did not allow us, though, to infer whether the individuals from Fregate are in fact the result of hybridization between distinct clades (as previously proposed) or are just a distinct, equidistant, clade.
Due to the reciprocal monophyly of the main clades within each species at the mtDNA level and the low variation and lack of sorting at the nuclear markers, it seems too speculative to depict historical colonization sequences and routes within the granitic Seychelles. Several scenarios could fit the observed patterns. Moreover, extinction and demographic changes may have affected populations differently, further obscuring any historical signal to be inferred. As argued by Gardner [32], current diversity could be explained by the sectioning of pre-existing clinal variation due to rising sea levels. It seems likely therefore that divergence between and within each species in the granitic islands was mostly driven by vicariance through cycles of allopatry between populations, rather than through stepping-stone colonization events. However, if isolation was due solely to oscillating sea-levels we would expect that the main split occurred between Silhouette and North versus remaining islands, such as in [12] (see Figure 1). It seems though, that distance was also an important factor for these species, and they were likely able to perform short overwater dispersals more often than long ones, leading to the main subdivision being between northern (Praslin-group) and southern (Mahé group, including Silhouette and North) islands.
Within P. sundbergi, some observations point to the possibility of (past or ongoing) gene flow between subspecies (island groups): one individual collected in Mahé as P. s. longinsulae exhibited in fact an haplotype clustering within P. s. sundbergi (from Curieuse and Praslin), and the same happened with two individuals collected in La Digue that grouped within P. s. sundbergi. Given that we did not collect morphological measurements or photos that would allow us to reconfirm the identification of these specimens, and given previous records of occasional dispersals that do not seem to form viable populations [31] we provisionally considered these individuals as possible cases of anthropogenic or very recent dispersals, and eliminated them from further analyses when appropriate. Nevertheless, even without considering these individuals, the IMa analyses returned positive estimates of gene flow within P. sundbergi (specifically, into the northern clade of P. s. sundbergi and P. s. ladiguesis), suggesting that the possibility of gene flow should be investigated in more detail. Also between P. astriata there were signs of possible gene flow, albeit weaker. Given the estimated Pleistocenic divergence between these subspecific clades, and low sea levels assumed during this period [6,18], which could have facilitated contact between island groups, it is reasonable to expect that gene flow may have occurred. Pending further studies shedding light on gene flow estimates and possible ecological differences across island groups, we consider the current taxonomy of this group appropriate, with only two species, P. sundbergi and P. astriata, that include three and two subspecies (or lineages; we are not necessarily advocates of the subspecies rank) respectively. Phelsuma astriata population from Fregate remains unassigned as P. astriata ssp.
The phylogeographic pattern of P. sundbergi (and P. astriata to a certain extent) mirrors the patterns found within U. inexpectata, a gecko species co-distributed with Phelsuma across the granitic Seychelles [13] and may thus reflect a more general biogeographic pattern. Many similarities can also be found with a co-distributed endemic freshwater crab species, Seychellum allaudi[12] with the difference that in this case Silhouette populations split first from the remaining populations, which seems logical due to their lower dispersal capacity.
The origins of the outer island populations
Cheke [31] discussed the status of the populations from Cosmoledo and Astove, keeping their designation as separate subspecies (P. longinsulae menaiensis and P. astriata astovei) based on slight colour variations. Gardner [25,33] found no morphological differentiation between P. sundbergi populations from the coralline and granitic islands and argued for their probable recent colonization or introductions by man (in the coralline islands). Accordingly, the fact that all individuals analysed from each of these populations possess the same mtDNA haplotype, shared with populations from the granitic islands, supports their very recent origin, either by natural dispersion or as a result of human-driven introductions. Specifically, based on mtDNA variation, the population from Cosmoledo could be assigned to P. sundbergi longinsulae, Poivre to P. sundbergi sundbergi, and the populations from Astove and Alphonse to P. astriata astriata.
The timing and mechanisms driving diversification of Phelsuma in the Seychelles
Perhaps the most unexpected result of this study was the estimate of simultaneous divergence of the northern and southern lineages within both species. In the biogeographic scenario previously proposed by Radtkey [8], the asynchronous divergence between these clades within each species would be responsible for the different degrees of morphological differentiation observed between the two species within each island group (the degree of character displacement being proportional to time since contact between the different species). Our results contradict this hypothesis, implying that other factors should be responsible for the morphological differentiation between species within each island group. The biogeographic scenario proposed by Radtkey [8] was largely based on his estimate of the mtDNA phylogeny, and on the different pairwise genetic distances between the different mtDNA clades within each species. However, this variance in gene divergence could also be due to stochasticity and different levels of ancestral polymorphism within each ancestral population [47,48]. The degree to which gene divergence predates population divergence can be substantial when the effective population size (Ne) of the common ancestor is large relative to the divergence time [47]. In fact, recent studies emphasize the need for model-based approaches to test the effects of shared historical factors in species/clades divergence [49,50]. Moreover, with just a single non-recombining locus, and especially if reciprocal monophyly has been achieved, it is not possible to differentiate between variance in sequence divergence due to divergence times and that resulting from coalescence. Given the different levels of intraspecific genetic divergence seen at the mtDNA, very different long-term population sizes would have to be assumed for each species to make them compatible with a synchronous north–south split. Multiple unlinked loci are extremely useful in addressing this question [47,51], as the expected variance in pairwise genetic distances from unlinked nuclear loci is directly proportional to the ancestral Ne, thereby providing information on coalescent processes in ancestral populations and reducing confidence intervals surrounding estimated divergence times eg. [49]. Accordingly, we estimated higher current and ancestral population sizes within P. sundbergi than within P. astriata. Interestingly, these estimates oppose current census sizes for both species, especially in the case of P. sundbergi populations of the northern islands [43,52], possibly indicating wide demographic changes across the evolutionary history of these species.
Eco-morphological divergence
Assuming a simultaneous divergence between both the northern and southern clades in each species leads us to hypothesize that the different ecological/habitat conditions in the different island groups had a predominant role on the different degrees of body-size divergence within each island group. As shown with body size measurements from Gardner [32] and Radtkey [8], the mean difference in snout-vent length (SVL) between P. sundbergi and P. astriata within the northern group (Praslin, La Digue, and associated islands) is significantly higher than the corresponding difference in the southern group. Gardner [32] demonstrated the importance of body size in resource use with observations of the differential use of palm tree pollen and nectar. Palms from the genera Lodoicea, Deckenia and Cocos all produce male inflorescences that are actively defended for highly nutritious resources, and in all reported cases the largest individual displaced smaller individuals, regardless of the intruding species [32]. This author further suggested that the large body size of P. sundbergi in the northern group likely evolved through interference competition associated with the differential distribution of palm trees. More specifically, he argued that because Lodoicea maldivica (the larger palm species) occurs only in the northern islands of Praslin and Curieuse, the selection pressure necessary to drive the evolution of larger body size was absent in the southern group. Radtkey [8] discarded this hypothesis arguing that competition in Cocos nucifera plantations was equally intense, and that this palm, along with Deckenia, was distributed throughout all the Seychelles. Nevertheless, L. maldivica (native only to the northern islands of Praslin and Curieuse), was the dominant tree on these islands, occurring across a broad range of habitats from the coast to the uplands, prior to human colonization [11] and recent data highlights the clear association of P. sundbergi with adult L. maldivica males in Praslin [43]. Harmon et al. [44] investigating the competition and community structure within Phelsuma from the Mascarene islands also found that one of their unique characteristics compared to other diurnal arboreal lizards is the fact that they partition the habitat between palms and non-palm trees. They also reported that interspecific interactions exist and that species rapidly react to other species presence or removal shifting their habitat use. As argued by Gardner [32], it is therefore possible that in palm forests selection will favour adults with large body-size as they will be able to defend inflorescences successfully, ensuring a constant food supply. In fact, all large species of this genus in the Mascarenes are also associated with potentially defensible items, in which large size may be advantageous [32]. In other habitats small size is probably more effective.
All these aspects become relevant given our divergence time estimates and warrant further investigation on the ecological determinants of eco-morphological diversification within Phelsuma in the Seychelles. According to our results, the intermediate size of P. sundbergi in the southern group is not the result of its evolution as a solitary species to use a wider resource distribution; rather other variables should be considered. Species are likely partitioning their habitat differently within each island, which leads to different outcomes in terms of selection and body size evolution. The low overlap of structural niche categories shows for example that P. astriata from Praslin and Silhouette use different parts of the tree [32]. For example, on Silhouette P. astriata are more abundant than P. sundbergi in coconut plantations, where they use a similar microhabitat to P. sundbergi on Praslin. In Silhouette habitat segregation is considerable (with P. astriata reaching larger sizes than P. sundbergi and dominating nectiferous resources) while in Mahé species seem to co-exist without obvious resource partitioning, although some segregation is possible (P. astriata seems to be much less abundant at the medium perch height where P. sundbergi is most frequently observed; S. Rocha, pers. obs). Further investigation on habitat use and niche partitioning within both species across island groups will be determinant for the understanding of the role of the habitat and interspecific competition in the microevolutionary patterns within this group.
Geographic patterns of genetic variation within Phelsuma species from the Seychelles support current taxonomy, with the taxonomic status of P. astriata population from Fregate remaining undetermined. The data are also congruent with the very recent origin of all outer islands populations. Despite extensive sympatric distributions, no gene flow seems to occur between the two species, whose divergence most probably pre-dates the Pleistocene. The genetic structure within each species is well correlated with geography, with the main mtDNA clades (subspecies) being distributed exclusively across northern and southern islands. Such differentiation is consistent with isolation caused by oscillating sea levels, which also seems to have allowed post-divergence gene flow within both species. The divergence between the main (northern and southern) clades within each species seems to have been approximately simultaneous, and highlights the importance of studying the potential role of habitat and ecological factors in microevolutionary patterns within this group.
Sampling and molecular data collection
Tissue samples (tail tips) of Phelsuma sundbergi and P. astriata, covering almost their entire distribution were collected and stored in 100% ethanol. Sampling spanned a 4-year period (2005–2008) with sampling locations not repeated, thus the likelihood of having sampled the same individual more than once is minimal. No vouchers were collected. DNA extraction followed standard salt or phenol-chloroform protocols [53,54]. Cytochrome-b (Cyt-b) was amplified in 126 P. sundbergi individuals and 73 P. astriata individuals, comprising all recognized morphological variation within each species (Figure 1). Between one and ten individuals per island per species were genotyped for four additional nuclear markers (see Additional file 1).
Primers for Cyt-b were CBL14753 [55] and CB3H [56] with amplification conditions as given by Rocha et al. [30]. Phosducin (PDC) amplification was made with primers and conditions from Gamble et al. [57], recombination-activating gene 2 (Rag-2) as in Rocha et al. [30], melanocortin-1 receptor (MC1R) and intron 61 of the reelin gene (RELNint61) using primers and conditions from Pinho et al. [58]. No evidences of multiple copies (size variation, modifications of reading frame) were found for any fragment. Sequences were determined by a commercial facility (Macrogen, Seoul, Korea). For most individuals we sequenced both strands of nuclear fragments to assure double peaks were real and not base calling errors.
Sequences were aligned manually using BioEdit [59]. For the nuclear genes, the haplotype phases were resolved using two complementary methods: first, for sequences that were heterozygous for insertions or deletions (some individuals for the RELNint61 fragment), we used the method described by Flot et al. [60], after which we applied the Bayesian algorithm implemented in PHASE software [61] using the known phases of haplotypes determined by the previous method, when available. For the remaining genes PHASE was applied directly to the datasets. We considered all positions for which phases were resolved with a posterior probability higher than 0.8 (though the majority of them were actually resolved with 1 or > 0.9 probability). Remaining positions were coded as missing data (N) or with ambiguity codes for subsequent sequence analysis as appropriate, but complete individuals were never excluded. We performed a minimum of three runs of PHASE for each dataset and checked phase calls for consistency. Sequence accession numbers are KC347737-KC348370. All sequence files used in downstream analyses can also be downloaded from the DRYAD database (doi:http://10.5061/dryad.6q3t1 webcite).
Genetic variability, neutrality tests and gene genealogies estimation
To assess how genetic variability was distributed across and within species we calculated a series of summary statistics for each marker and putative subspecies. As only three individuals of P. astriata from the Fregate population were collected (the "intermediate form" sensu [31]), summary statistics were not computed in this case.
For each locus, we calculated the number of haplotypes, the number of segregating sites, haplotype diversity, nucleotide diversity π, and population mutation parameter θ [62]. We tested for non-neutral evolution by computing Tajima's D [63] and further tested each population for signals of demographic expansions by calculating Fu's Fs [64] and R2[65]. We tested significance of the results of the three tests using 10,000 coalescent simulations. All these analyses were conducted in DnaSP v5 [66].
We used MrBayes v3.1.2 [67] to obtain an estimate of the Cytb phylogeny (BI). The dataset was reduced to single haplotypes using ALTER [68] and the best fitting model estimated using jModeltest [69] and PhyML [70], under the AICc criteria [71,72]. Two runs of 11 million generations with default heating parameters were performed. Convergence and congruence across runs were assessed using AWTY [73]. MEGA v4 [74] was used to estimate the mean distance between clades (Nei's Da). Median-joining networks [75] with MP posterior optimization [76] were constructed using NETWORK v4.510 [77] and used to illustrate the relationships between nuclear and mitochondrial haplotypes (applied separately for each species in the latter).
Testing the asynchronous colonization hypothesis: estimates of divergence times and gene flow under the isolation-with-migration model
Based on the results on population structure, we fitted a subset of each species data to a two-populations model (the northern and southern clades of each species), and used the isolation-with-migration (IM) model as implemented in the program IMa2 [78,79] to estimate the six parameters involved: ancestral and current population sizes (θA, θ1, and θ2), migration in both directions (m1 and m2) and time since the population split (T). One individual of P. sundbergi longinsulae from Mahé island that carried a haplotype that clustered within P. sundbergi sundbergi haplotypes from Praslin was excluded from the analysis, as it could possibly reflect an anthropogenic introduction (see results and discussion). Within P. astriata, the Fregate population (N = 3) was also excluded due to its intermediate position relative to the other two groups of haplotypes.
The HKY [80] model was used for the mtDNA dataset and also for Rag-2, RELNint61 and MC1R nuclear gene fragments, where homoplasic positions were detected, and the IS model [81] for PDC. Prior bounds were first selected based on the summary statistics and optimized and adjusted in a few preliminary runs, to ensure that prior distributions covered the whole range of possible values for that parameter. Multiple subsequent runs of each dataset were then performed and effective sample size of parameters (ESS) and trend plots examined to assure proper mixing and convergence. The significance of the estimated migration rates was measured by the LLR test of Nielsen and Wakeley [82]. A substitution rate of 1% per lineage per Myr (mtDNA, see below) and a generation time of one year, as approximately estimated by Gardner [32] for P. astriata (9 months to attain maturity and approximately 2.5 months egg incubation time) were used to convert parameter estimates into biologically meaningful quantities.
Additionally, to obtain an estimate of the divergence time between P. sundbergi and P. astriata, and to test for the existence of post-divergence gene flow across species, we also fitted the IM model to the whole dataset, treating each species as a separate population.
Because IMa2 assumes no intragenic recombination or selection we tested each locus for recombination using the pairwise homoplasy index [83] implemented in PhiPack [84], and for selection performing multiple loci HKA tests [85] using SITES and HKA [86].
Species-tree inference
To obtain a multilocus perspective of the diversification within the whole clade, we employed the Bayesian MCMC method from Heled and Drummond [87], *BEAST, implemented in BEASTv1.6.1 [88]. This method makes use of the multispecies coalescent model to co-estimate the multiple gene trees and the "species-tree" they are embedded within, together with divergence times and effective population sizes of extant and ancestral species. Especially at this shallow level of divergence, it is likely that multiple processes such as demography and incomplete lineage sorting influence the gene genealogies and the implementation of a method that properly accommodates this variation, rather than a concatenation approach, becomes especially relevant [89,90] and references therein. A minimum of two individuals per "species" (the term species not necessarily referring to the taxonomic rank but to any diverging "population structure" – in our case the five subspecies) per loci is needed, while additional individuals increase accuracy [87]. We therefore constructed a dataset with a subsample of our individuals, using some of those that were genotyped for the five genes: 19 P. sundbergi longinsulae, 8 P. sundbergi sundbergi, 5 P. sundbergi ladiguensis, 20 P. astriata astriata, 22 P. astriata semicarinata and finally 2 individuals from P. astriata from Fregate (Additional file 1).
We estimated the appropriate model of evolution for each fragment as previously and implemented it choosing the most similar within the ones available in BEASTv1.6.1, with all related parameters (gamma; proportion of invariant sites, transition/transversion rates) being co-estimated along the runs. The multispecies coalescent allows for gene-specific mutation rates. Throughout, the substitution rate of the mitochondrial locus was set to a normal distribution prior of mean 0.01 (~1% per lineage per Myr) and standard deviation of 0.0027, as in [91]. Similar rates have been consistently recovered for mtDNA fragments of other reptile and amphibian groups at the intrageneric level, ranging from 0.99% in Hydromantes[92], to 1.35% in lacertids [93] and references therein], estimates available from calibrated trees for gekkonid falling within this range [94]; 1, 15% for Hemidactylus). Rate estimates around 1% per lineage per million year were also obtained by Chapple et al. [95] (mean 1.01%; David Chapple pers. comm) and Cox et al. [96] (S. Carranza pers comm) and the range 1.15-1.35% used also by Miralles and Carranza [97]. In the absence of any available external calibration, we believe that our rate distribution prior, which encompasses these values, is appropriate to obtain an approximate estimate of the time-scale of the diversification of this group (although see Discussion for the possibility of faster substitution rates). Nuclear rates were co-estimated along the run, relative to the mitochondrial one. An uncorrelated relaxed clock model was assumed for the Cytb dataset, whereas a strict clock was assumed for the nuclear gene fragments given their low variability (causing runs under relaxed clock models to fail to converge). We ran the dataset under both a Yule and a birth-death prior for the species-tree and a population size model of continuous growth and constant root (as recommended by the authors; J. Heled, pers. comm.). Default values were used for all other parameters. Multiple runs of 100–500 million generations were performed and sampled at appropriate intervals to obtain 10,000 final samples (trees). To test the influence of the priors on the posterior estimates, an additional run of identical length was made without data, sampling only from the prior. Tracer v1.5 was used to visualize the results of each run, to check the effective sample size of each parameter and chose appropriate burnin values [98]. After discarding the burnin samples, a consensus species tree (Maximum Clade Credibility tree), mean node heights and supports were obtained using TreeAnnotator v1.6.1 [88] and visualized using FigTree v1.3.1 [99].
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
SR and DJH collected the samples in the field. SR performed the laboratorial work, analysed the data and drafted the manuscript. SR, DJH and DP devised the analytical strategy. DJH and DP provided critical corrections and comments on the paper. All authors read and approved the final manuscript.
This research was supported by grants from Fundação para a Ciência e Tecnologia SFRH/BD/1745/2004 (to SR), PTDC/BIA-BDE/65745/2006 and the Gulbenkian Society. We are grateful to SBS of Seychelles for permits for fieldwork and tissue collection and to the Ministry of Environment and Natural Resources for logistic support during fieldwork. We thank NPTS, ICS Seychelles, SIF and several entities and persons that made fieldwork across all islands possible. A special acknowledgement is due to Raquel Vasconcelos, Ana Perera and Andreia Silva for help during fieldwork and also to Justin Gerlach from NPTS for support and information in earlier phases of the study. Thanks to Ana Perera for Figure 1 and to Ricardo Pereira for insightful discussions. We further thank Nancy Bunbury, Anthony Cheke, Miguel Carretero, Luke Harmon and Ricardo Pereira for helpful discussions and comments on previous versions of the manuscript. Cover picture from Christopher K. Bunbury.
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Nielsen R, Wakeley J: Distinguishing migration from isolation: a Markov chain Monte Carlo approach.
Genetics 2001, 158:885-896. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Bruen TC, Philippe H, Bryant D: A simple and robust statistical test for detecting the presence of recombination.
Genetics 2006, 172:2665-2681. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
PhiPack: PHI test and other tests of recombination.
available at http://www.maths.otago.ac.nz/~dbryant/software.html webcite
Hudson RR, Kreitman M, Aguadé M: A test of neutral molecular evolution based on nucleotide data.
Genetics 1987, 116:153-159. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
SITES and HKA.
available at http://genfaculty.rutgers.edu/hey/software webcite
Heled J, Drummond AJ: Bayesian inference of species trees from multilocus data.
Mol Biol Evol 2010, 27:570-580. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Drummond AJ, Rambaut A: BEAST: Bayesian evolutionary analysis by sampling trees.
BMC Evol Biol 2007, 7:214. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text
Edwards SV: Is a new and general theory of molecular systematics emerging?
Evolution 2009, 63:1-19. PubMed Abstract | Publisher Full Text
Leaché AD: Species trees for spiny lizards (genus Sceloporus): identifying points of concordance and conflict between nuclear and mitochondrial data.
Mol Phylogenet Evol 2010, 54:162-171. PubMed Abstract | Publisher Full Text
Paulo OS, Pinheiro J, Miraldo A, Bruford MW, Jordan WC, Nichols RA: The role of vicariance vs. dispersal in shaping genetic patterns in ocellated lizard species in the western Mediterranean.
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Carranza S, Romano A, Arnold EN, Sotgiu G: Biogeography and evolution of European cave salamanders, Hydromantes (Urodela: Plethodontidae), inferred from mtDNA sequences.
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Mol Phylogenet Evol 2009, 52:470-487. PubMed Abstract | Publisher Full Text
Cox SC, Carranza S, Brown RP: Divergence times and colonization of the Canary Islands by Gallotia lizards.
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Miralles A, Carranza S: Systematics and biogeography of the Neotropical genus Mabuya, with special emphasis on the Amazonian skink Mabuya nigropunctata (Reptilia, Scincidae).
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Tracerv1.5. 2007.
Available from http://beast.bio.ed.ac.uk/Trace webcite
FigTreev1.3.1. 2009.
Available from http://tree.bio.ed.ac.uk/software/figtree/ webcite


text:
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Getting there is truly more than half the fun! I am able to enjoy several hours of this every week. On weekends, I am usually able to get in sessions of several hours before the wife gets up or while she is out shopping. A man needs to know how to handle himself !View Thread
I have pics and a bad vid posted but no face.
I can't see how showing my facet would help me since I am married with grown children-more downside than up, I would think..View Thread
I envy your situation. It wouldn't work for me because I really don't think I could enjoy jo with anyone I knew beforehand (so to speak).
I can 'compartmentalize' and would hope my eventual jo bud(s) become friends but only afterward, not before. I would prefer to jo with someone that I'd met only for that purpose. Weird, but that's my fantasy.View Thread
I like everything about a woman's body and enjoy the emotional and intellectual closeness with women. So, women are the whole package, men pretty much just the package.View Thread
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Freedom of Information Act Office
IC Directors' Meeting Highlights
December 22, 2005
|IC Directors
|Director, Executive Secretariat
|IC Directors' Meeting Highlights —July 28, 2005
Discussion Items
I. Registration of NIH-Sponsored Clinical Trials
Dr. Lindberg updated the group on the status of ClinicalTrials.Gov, noting that it has been a big success under the 1997 Food and Drug Administration Modernization Act (FDAMA), which required the NIH to establish a registry of clinical trials for both federally and privately funded trials of experimental treatments for serious or life-threatening diseases. However, he stressed that potential legislation, the Fair Access to Clinical Trials Act (FACT) of 2005, would enforce against any non-compliance with registration.
While NIH Institutes have strongly supported, helped, and advised Clinical Trials.Gov, Dr. Lindberg pointed out areas that must be improved in order to comply fully, the most important being that a number of NIH trials are not registered and the status of the trials are frequently inaccurate. He also pointed out occasions on which ICs issued press releases on clinical resource results where the clinical trial has not been posted on ClinicalTrials.Gov. He urged that all ICs work together to ensure nothing less than perfect and consistent compliance by NIH. Dr. Lindberg also raised other areas requiring discussion, such as the need for trial numbering.
The group then offered suggestions for ways to ensure that the posting of NIH-conducted and -supported research is consistent and complete. Finally, Dr. Zerhouni asked Dr. Lindberg to work with other IC Directors to achieve comprehensive registration of NIH-funded trials in ClinicalTrials.gov
II. NIH Public Access Policy: Update
Dr. Hays presented a time-line summarizing both the pre-release and post-release milestones of the policy, including the March 1, 2005, Report to Congress. He noted that the Public Access Policy Working Group made two key recommendations, among others, at its July 11 meeting:
- Need to evaluate why submissions are low, and
- Need for program administrators to inform grantees about the policy.
He then reviewed a detailed time-line of the ongoing outreach activities to both NIH staff and the public, noting that —
- After todays meeting, an NIH staff package will be released to help engage more intramural and extramural staff.
- On August 2, investigators and institutions can use the manuscript submission process to fulfill the existing requirement to provide publications as part of progress reports. A reference to the PMC submission identification number may be included in progress reports in lieu of submitting a hard copy of the publication. In addition, information about the policy will begin to appear in Notices of Grant Award, Program Announcements, and Requests for Applications.
- In the fall, articles are scheduled to appear in the OER Insider and the NIH Record.
Next steps include continued outreach to stakeholders and investigators; evaluation of the policy in reaching its objectives and of the effectiveness of NIH outreach; batch uploading by IRPs, libraries, repositories, and publishers; and continued communication with NIH staff.
Dr. Zerhouni thanked Dr. Hays and the WG for all of the hard work toward creating this new venue. He asked that they continue to find ways to ensure that NIH intramural scientists clearly understand the policy and that they possibly create a compendium of what professional organizations are saying about the policy to share with the intramural scientists. He also asked that the group be kept posted on progress and concerns.
III. Minority Research Training Programs Update
Dr. Ruffin discussed the recently published National Research Council report, Assessment of NIH Minority Research Training Programs: Phase 3, prepared under the auspices of the Committee for the Assessment of NIH Minority Research Training Programs, co-chaired by Drs. John Bailar III and Willie Pearson Jr. (see http://books.nap.edu/catalog/11329.html). In 2001, NCMHD contracted with NAS to undertake an independent phase three assessment. The study charge asked that the study address the following questions:
- Do the NIH minority research training programs work?
- Which minority programs and which features of minority programs have been most successful?
- Which minority programs have been least successful and why?
- What additional factors contribute to minority trainee success?
- How can a system be set up that would better address assessment questions in the future?
The Committee was also charged with developing policy recommendations for an improved coordinated tracking system.
Dr. Ruffin then introduced Peter Henderson, who served as an Acting Study Director through two different periods during 2004. Mr. Henderson summarized the Committees findings and recommendations for the group — the recommendations are located at http://www.nap.edu/books/0309095751/html/159.html et seq.
The group discussed the issues and recommendations, and Dr. Berg mentioned that a working group of the National Advisory General Medical Council (NAGMC) has been discussing similar issues. Dr. Zerhouni asked that Dr. Ruffin work with Dr. Berg to use this NAGMC working group as a base for addressing the NAS recommendations.
Dale Johnson
cc: OD Senior Staff


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Exactly James. The whole point is to have a limited life on the tyre to make the race interesting.
It is the usual marketing bullshit, competitors taking the piss out of Pirelli for doing what they have been contracted to do, so they make their tyres last longer and the racing becomes dull again.


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David,If you are debating between a long sleeve crew and a long sleeve zip, I would go for the long sleeve zip. You'll appreciate the zipper when it's warm. I've not had wear problems under my pack straps with my Smartwool or other wool shirts.
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Friends pray for recovery of fellow injured biker in Abilene vigil
At the same hour Wednesday that Scott Sanford of Abilene was scheduled for surgery at Hendrick Medical Center, about 20 of his friends from Mercy Church Motorcycle Ministries were meeting at the "Jacob's Dream" garden sculpture to pray for his recovery.
"This is a prayer vigil of Christians who believe in the power of prayer," said Terry Taillon of Hawley, pastor and president of Mercy Church Motorcycle Ministries.
"We're praying for Scott and every doctor that comes in contact with him and his family," Taillon added. "We're praying that God will use His miraculous power."
The prayer vigil began at 6:30 p.m. at the sculpture on the campus of Abilene Christian University in Wednesday's cold temperatures, wind and rain.
Taillon said Sanford, 56, is a maintenance worker at an Abilene apartment complex who was seriously injured March 18 while riding his motorcycle and struck by a pickup truck. Sanford received several internal injuries, according to Taillon, and his left leg was severely injured enough that doctors say it likely will have to be amputated.
Family members from Virginia and New Mexico are in Abilene to join Sanford's brother in caring for the injured biker.
Taillon called Sanford one of the group's "core members" although he was relatively new to Abilene. "We started about two years ago," Taillon said of the church, "with seven members and nowhere to meet."
Mercy Church Motorcycle Ministries meets each Sunday at 11 a.m. at 721 South First St. at the former location of the bar Fast Lane. "We're a biker ministry," Taillon said, "but our church services are open to anyone.
"We urge anyone to come worship with us and pray for Scott," he added. "We're really trying to pick up the people who have fallen through the cracks going through the highways and byways as the Bible commands us to do."
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So sorry, but only 4 items are allowed for comparison. I blame the gnomes and so should you.
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text:
Belching is the act of bringing up air from the stomach.
Alternative Names
Burping; Eructation; Gas - belching
Belching is most often a normal process. The purpose of belching is to release air from the stomach. Every time you swallow, you also swallow air, along with fluid or food.
The buildup of air in the upper stomach causes the stomach to stretch. This triggers the muscle at the lower end of the esophagus (the tube that runs from your mouth to the stomach) to relax. Air is allowed to escape up the esophagus and out the mouth.
Excessive or repeated belching may be caused by swallowing air without realizing it (aerophagia).
Belching may last longer or be more forceful depending on what is causing it. Symptoms such as nausea, dyspepsia, and heartburn may be relieved by belching.
- Pressure caused by the unconscious swallowing of air
- Acid reflux disease (GERD) and heartburn
Home Care
You can get relief by lying on your side or in a knee-to-chest position until the gas passes.
Avoid chewing gum, eating quickly, and eating gas-producing foods and beverages.
When to Contact a Medical Professional
Most of the time belching is a minor problem. Call a health care provider if the belching does not go away, or if you also have other symptoms.
What to Expect at Your Office Visit
Your health care provider will examine you and ask questions about your medical history and symptoms, including:
- Is this the first time this has occurred?
- Is there a pattern to your belching? For example, does it happen when you are nervous or after you have been consuming certain foods or drinks?
- What other symptoms do you have?
You may need more tests based on what the health care provider finds during your exam and your other symptoms.
Functional Gastrointestinal Disorders: Irritable Bowel Syndrome, Dyspepsia, and Functional Chest Pain of Presumed Esophageal Origin. In: Goldman L, Schafer AI, eds. Cecil Medicine. 24th ed. Philadelphia, Pa: Saunders Elsevier; 2011:chap 139.
Last reviewed 10/13/2013 by George F. Longstreth, MD, Department of Gastroenterology, Kaiser Permanente Medical Care Program, San Diego, California. Also reviewed by David Zieve, MD, MHA, Bethanne Black, and the A.D.A.M. Editorial team.
- The information provided herein should not be used during any medical emergency or for the diagnosis or treatment of any medical condition.
- A licensed medical professional should be consulted for diagnosis and treatment of any and all medical conditions.
- Call 911 for all medical emergencies.
- Links to other sites are provided for information only -- they do not constitute endorsements of those other sites.
©1997 - A.D.A.M., Inc.Any duplication or distribution of the information contained herein is strictly prohibited.


text:
Climate sensitivity and aerosol forcings
In a new review paper in Nature this week, Andreae, Jones and Cox expand on the idea that uncertainty in climate sensitivity is directly related to uncertainty in present day aerosol forcing (see also this New Scientist commentary). This was discussed here a couple of months back in the Global Dimming and the climateprediction.net posts, and so it is worth revisiting the question in the light of their analysis.
Over the observational period there is a great deal of uncertainty in the climate forcings – mainly (though not exclusively) associated with various aerosol effects. Given this uncertainty, it has therefore been very difficult to calculate climate sensitivity (how much the globe warms as a result of a change in forcings) using the modern changes in global temperature. Such estimates have tended to have very broad error bars.
The basic idea of the AJC paper is that any uncertainty in aerosol effects (which is large) translates directly into uncertainty in climate sensitivity. This gives a range of climate sensitivity that is much larger than the IPCC range (1.5 to 4.5 deg C for a doubling of CO2) , and which therefore translates to wider bounds on possible climate projections both at the high end and low end.
We contended here before that this is a bit of a leap, since there are other constraints on climate sensitivity (such as the last ice age) and other sources of uncertainty (solar, ozone, land use, etc.). Both of these factors reduce the direct influence that the aerosol forcing alone should have on our ideas of climate sensitivity.
This paper is nonetheless interesting for the link that they make to the carbon cycle and the potential for feedbacks that may amplify the CO2 concentration in the future that will depend on the warming, and hence on climate sensitivity. However, the fundamental point which underlies all their calculations is the link between aerosol forcing and sensitivity, which I would argue is not as strong as they have posited. (James Annan has also made a similar point).
To demonstrate why this is may be an overstatement, one can look at the results from the GISS ModelE simulations for the 20th Century (illustrated here). In these experiments the climate sensitivity was 2.7 deg C for a doubling of CO2, the net aerosol forcing from 1940 to 2000 was around -0.7 W/m2 (55% of the total forcing, -1.27, from 1850 to 2000), and the ocean uptake of heat was well-matched to recent observations. However, this amount of present day aerosol forcing would imply a sensitivity of about 4 deg C according to figure 1 in AJC (right), with 2.7 outside their error bar. What is therefore missing from their calculation? Firstly, there is approximately 0.35 in other forcings over this period (from solar, tropospheric ozone, land use and volcanoes), which if taken into account moves the curve down and to the right. Secondly, there is more uncertainty in the GHG and other forcing terms than they allow for. This actually widens the error bars so much that for large aerosol forcings, you cannot rule out even zero climate sensitivity. These details are not inconsequential because most of the conclusions in their paper stem from the rapid increase in climate sensitivity between 1.0 and 2.0 W/m2 aerosol forcing.
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AOL News has a new home! The Huffington Post.Click here to visit the new home of Politics Daily!
I agree with many of the other comments out here - the question is not whether or not this person was evil or not - the question is: Can we do things to prevent this type of tragedy? The answer is: Yes, we can do things to make this less likely and to make these type of things happen less often - not that things won't happen occassionally in a free society. But a free society can allow people to hunt and bear arms, but not allow people with a history of mental illness easy access to guns. (And, yes - I know some criminals will always get guns, but you don't have to make it easy.) And you don't have to have the type of rhetoric that Palin and O'Reilly - and - ok - even some on the left like Randi Rhodes - seem to have adopted. As a political leader, Palin should be particularly appalled. A simple, sincere apology and explanation that she understands the influence her words can have on some people who are not able to think clearly is what is needed. It certainly makes me wonder if violence and intimidation isn't part of her agenda.January 12 2011 at 2:30 PM Permalink
Once again Sarah Palin has managed to make an issue all about her and how it affects her instead of seeing the bigger picture and speaking as an adult would. Here we go back to junior high again. Can we please get past this aggressive, unpleasant woman whose words nearly always serve to further divide us? I'm not sure how she manages it, but she has done it consistently ever since her first public appearance. Enough already. There are important issues to talk about and work on together and people who just want to attack others are in the way, no matter which side of the political spectrum they belong to.
As far as the Christian aspect of all of this, I have this to say:There are some people in this country who say they are Christians. If these people have actually read the Bible, and all the words of Jesus, they would know that Christians do not threaten much less perpetrate violence in word or in deed. Of course, they make mistakes and fall short, but a true follower of the Messiah gets back up, applies the Word to his life and learns a more excellent way. God's way. The way of peace.
True Christians do not own or use weapons for the killing of people. True Christians do not wish to see harm done to anyone, but pray even for their enemies. True Christians are the medics and the clerics on the battlefield of this earth, trying to preserve and save life, which is God's gift to all of us. True Christians do not wish to live in luxury at the expense of their fellow human beings. True Christians lift people up and encourage them. They do not demonize or ridicule others who hold different political or world views.
In these difficult times, I have struggled mightily with this. I have to fight the urge to want to verbally strike back in anger when I hear the bile that is spewing out from extremist speakers. Some of them say they are Christian, but as Jesus said, we will know them by their fruit. Perhaps I should thank those who are so clearly hate and fear-mongers. Because of them, I am learning to forgive something I find nearly unforgivable. And I'm learning to pray that they will see the damage they cause, repent of it, learn the better way and become a peacemaker and a healer. Amen.
If you are a Christian, pray this for all those who spread division, no matter what "side" they are on.
Blaming Sarha Palin the Tea Party, conservetive rhetoric, Fox News ,Russ Limbaugh,makes me sick. Who did we bame when President Reagan was shot, and his Press Secretary Jim Brady, who was shot in the head just like the Congresswoman.
Who did we blame when President Ford was shot at and again by Sqeeky Frome's attempt. We didn't blame the liberal press or political ideals.
On both sides the politicians are making fools of themselves more protection, gun laws,disallowing what is said on the media and so on.
Does anyone remember the James Brady hand gun legislation?
All of the above shooters including the current killer had some kind of mental illness.(Period) The diffence with this nut he had a semiautomatic instead of a saturday night special. I do blame the liberal media ie: Kieth Olbermann and others trying to turn it into a political game instead of changing the laws for the crimminally insane in this country.I beleive in the Constitution leave it alone. Since 01/08/2011 the day of the tragety, how many people have been murdered in this country by madmen?
My prayers are with Congresswoman Gifford her family and all the victums of this tragety.
The terrible shootings in Tucson were done by a young man who had a history of psychological troubles. To attempt to fix a catalyst to his behavior with unsubstantiated finger pointing is un-American. Please, let us stick to the facts of the case and not blame innocent people, especially to try to advance one's political idealogies. Jared was the shooter, he was old enough to know right from wrong, he choose to do wrong, he is solely responsible for his actions. Jared is to blame for the horiffic shootings.January 12 2011 at 2:26 PM Permalink
From what I have heard and seen on various interviews about the person doing the shooting I can draw only one conclusion and that is no one really knows what was going throught this young mans mind. I believe all the speculation made by the various media and political people can be destructive. Talking about what they feel could have caused the shooting has no value and add's nothing to the discussion. As far as I'm concerned news people polititions and talk show people should all keep quiet and let the police and the eventual trial tell the story. Spend time gathering the evidence and wait to talk about it at proper time. You all seem to be trying to create a reality that you know nothing about.Stop PleaseJanuary 12 2011 at 2:26 PM Permalink
The words that come out of your mouth refelect what the intent of the heart really is. When children play violent video games we say they may or can be influenced by the violent games. Then we look at the game makers and wonder why they don't take more responsibility for what they endorse. Therefore I understand why people are looking at Sarah Palan and placing her words and her map in the hot seat. Words are powerful influences in this society, rather meant to to be postive or negitive.January 12 2011 at 2:25 PM Permalink
WHAT IN THE HELL IS WRONG WITH YOU PEOPLE? Do you honestly think that Sara Palin or any politican in America would want a child 9 years of age DEAD, to prove a point? If you truly beleive that you must be a card carring member of the Westboro Babtist Church and really need help. IT IS JUST WHAT IT WAS, A NUT ON A SELF MINDED RAMPAGE, It is a wonder to me after reading most of these left wing rants and hate of MS palin that this has not happened before and more often. People please knock it off.January 12 2011 at 2:24 PM Permalink
Sarah Palin is right. In the end political rhetoric or not we all make our own decisions good or bad and we all should be held accountable alone for our decisions good or bad. Nobody should get away with blaming anyone else for their bad actions. We'd have a lot less crime if we stopped giving people excuses to misbehave and started holding people beginning with children on up to the eldest of adults responsible for their actions with appropriate consequenses. Anyone who commits murder or engages in violent crime is somehow mentally unstable and mentally ill. Since they did not seek appropriate help for themselves and it is abundently available they too should be held accountable for their crimes alone with nobody else to blame but them. There are many many mentally unstable and mentally ill people who do what they need to do in order to not commit such violent acts.January 12 2011 at 2:24 PM Permalink
Can we get any more divided than we are now? I hope not! It is time for our President to step up to the plate and try to stop all this blame throwing.Please MR. President,try to bring us back togeather.January 12 2011 at 2:23 PM Permalink
Sarah Palin did not cause the shooting, but her words and actions when processed by a mentally ill person, may have contributed to this horrible event! The misguided right wing, which Palin speaks for, are entitled to their opinions but their public statements and the tone in their speeches should be tempered. We need political discussion, not the ferment of violent revolution.January 12 2011 at 2:22 PM Permalink
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