Barbus neumayeri from two river drainages in Uganda: the Rwembaita Swamp-Njuguta River System and the Dura River. We then examined two indices of population structure, GST and Jost's D, to detect links between oxygen availability and genetic variation and to determine if population genetic structure was associated with (i) dissolved oxygen regime (hypoxia or normoxia), (ii) geographical distance, or (iii) a combination of dissolved oxygen regime and geographical distance. Our results indicate that over a large scale (between drainages), geographical distance significantly affects the genetic structure of populations. However, within a single drainage, dissolved oxygen regime plays a key role in determining the genetic structure of populations. Within the Rwembaita-Njuguta system, gene flow was high between locations of similar oxygen regimes, but low between areas characterized by divergent oxygen regimes. Interestingly, GST analyses appear to yield less realistic measures of population structure than Jost's D, suggesting that caution must be taken when interpreting and comparing the results from different studies. These results support the idea that aquatic dissolved oxygen can act as a selective force limiting gene flow among populations of aquatic species and therefore should be considered when implementing conservation plans and assessing environmental impact of human activities.Population level response to hypoxia has become an issue of global significance because of increased frequency and intensity of hypoxic events worldwide, and the potential for global warming to exacerbate hypoxic stress. In this study, we sequenced two nuclear intronic regions and a single mitochondrial region across seven populations of the African cyprinid,  Nephelopsis obscura) residing in lakes with different dissolved oxygen (DO) regimes, were more consistent with oxygen acting as a selective agent than geographical distance. Similarly, at least two populations of the common skate (Dipturus batis) have been found to be reproductively isolated around the British Isles  dissolved oxygen regime (hypoxia or normoxia), (ii) physical barriers such geographical distance, and/or (iii) both oxygen regime and geography. Nuclear introns accumulate change at a relatively fast rate  and can often be easily amplified from a species of interest using primers designed from the relatively conserved flanking coding regions.Several recent studies have demonstrated strong patterns of interdemic variation in fish populations between low- and high-oxygen environments, particularly in traits related to oxygen uptake such as gill size  and geographical distance. The two main rivers Dura River and the Mpanga River, both eventually feed into the Lake George system . Basic pFish were sampled during the rainy season in January 2010 using baited minnow traps, and body mass and total length were measured in a small plastic bag with water. Pectoral fin clips were taken and stored in 95% ethanol for DNA analyses. Once the fin clips were sampled, fish were allowed to recover in a bucket of well-oxygenated water from their habitat, and they were released at the location of capture. Genomic DNA was extracted using the DNeasy Tissue Kit , and DNA concentration was estimated using an ND-8000 NanoDrop spectrophotometer .We used Polymerase chain reaction (PCR) to amplify two nuclear and one mitochondrial gene regions. The nuclear and mitochondrial loci have demonstrated utility in resolving question of population structure in other studies  nuclear locus was amplified using an oligonucleotide primer set previously optimized for various fish species, including cyprinids (Quattro and Jones The fourth intron of the Triosephosphate Isomeras A (TPI-A) nuclear locus was amplified using locus specific primers designed in a similar fashion to the CK primers (J. M. Quattro pers. comm.). Amplification of sufficient product to allow direct sequencing required semi-nested primary and secondary PCR reactions. The primer sequences are as follows: TPIDF1: 5′-YTG ATY GGS CAG AAG GTG GC-3′; 302 – TPIB4 R1: 5′-AGA ACC ACY TTR CTC CAG TC-3′; TPI4F2 – P301: 5′-GCA TYG GGG AGA AGC TRG AT-3′. The primary amplification was from TPIDF1 to 302 – TPIB4 R1. From this reaction, 2 μL of product was diluted in 100 μL water and 2 μL of this solution was used as template in a second PCR reaction from TPI4F2 – P301 to 302 – TPIB4 R1. In both primary and secondary reactions, the PCR parameters were as follows: an initial denaturation of 2 min at 90°C followed by 44 cycles of 45 sec at 90°C, 60 sec at 48°C, 60 sec at 72°C and a final extension time of 7 min at 72°C.A fragment of the 16S mitochondrial locus was amplified using “universal” primers: 16S AR: 5′-CGC CTG TTT AAC AAA AAC AT-3′; 16S BR: 5′-CCG GTC TGA ACT CAG ATC ACG T-3′ , nucleotide diversity (π), GST and FST-values were all calculated using DnaSP version 4.10  with a final visual optimization of the alignment. The number of haplotypes (within and between location), haplotype diversity , while the Dura River had the lowest diversity (Hd = 0.189). The maximum number of mutational steps between any of the haplotypes was four, although this maximum was only observed between a single individual from Mikana Stream and the haplotype B. The most common haplotype for this intron was haplotype A, found in 42 of 135 individuals . This pattern of FST showing similar, but not significant, patterns of differentiation to those in the GST analysis is consistent across all three loci and we have therefore presented only the GST values. Values for Jost's D dissimilarity ranged from −0.112 to 1.000, indicating variability between the Dura River population and all other populations . Due to the large distance between the Dura River population and all the other populations, additional analyses were conducted without the Dura River population. In the absence of the Dura River population, both GST and Jost's D were associated with the geographical distance in pairwise comparisons . By contrast, we did not find a significant relationship between either genetic metric and dissolved oxygen at this locus, even when the Dura River was not included in the analysis.Overall, we found a significant positive relationship between geographical distance and both Hd = 0.763), while the Dura River was completely fixed at this locus with a haplotype diversity of 0.000. The maximum number of mutational steps between any of the nine haplotypes obtained was two. The most common haplotype for the TPI intron was haplotype B found in 66 of 134 individuals .Similar to CK, ulations . Values pulation . AdditioR2 = 0.38, P < 0.0001 and R2 = 0.46, P < 0.0001 for GST and Jost's D, respectively). Nevertheless, when the Dura River was removed from the analysis, these relationships disappeared . By contrast, the relationships between the genetic metrics and dissolved oxygen between locations were significant when the Dura river was included , but stronger when this population was excluded from the analysis . The Mikana Stream, Njuguta River, and the Rwembaita Swamp all had only a single 16S haplotype and an Hd of 0.000. The most common 16S haplotype was haplotype A found in 112 of 134 individuals (83.6%). Haplotype B was observed in 17 of 134 individuals (12.7%) and only in individuals from the Dura River. The remaining haplotypes were found only in single individuals.We sequenced a 555 bp fragment of the 16S locus and identified seven haplotypes  with an GST and Jost's D analyses suggest significant genetic differences at this locus between some of the populations . However, no correlation was observed between geographical distance and either GST (P = 0.363) or Jost's D (P = 0.834) when the Dura river was excluded from the analysis. Additionally, there was no correlation between the genetic metrics and dissolved oxygen even when the Dura River was removed from the analysis.Similar to the results from the nuclear loci, there was a positive and strong correlation between geographical and genetic distance in the 16S data set . This isolation suggests that habitat-specific selection pressures may contribute to reduced gene flow , as has been suggested in other studies . Such discrepancies may cause misleading conclusions with respect to population structure leading to inappropriate management implications and we therefore advocate for a better investigation of the properties associated with different metrics used to estimate genetic differentiation. Furthermore, it is clear that GST may yield less realistic measure of population differentiation than Jost's D and caution must be taken when interpreting and comparing the results from different studies. Recently, Raeymaekers et al. (D and GST in distinguishing population genetic structure and conclude that no single measure can quantify all aspects of genetic structure. The differences in resolution observed here between D and GST are consistent with this conclusion. Interestingly, both their empirical and theoretical work suggest that D is more useful in distinguishing longer term processes, and GST in recent demographic events, while our empirical results appear to point in the opposite direction. This difference in conclusion suggests that either the separation of the B. neumayeri populations that we have studied is more complicated than we appreciate, or, that more work is necessary to completely understand the difference in performance of these two metrics.This study also allowed us to compare and contrast the results from distinct metrics of genetic structure. Some limitations that have been pointed out for s et al.  empiricaB. neumayeri across dissolved oxygen regimes. We also observe strong genetic differentiation of the Dura River location from all other sites, likely a function of the large swimming distance and the presence of geographical barriers between the Dura River location and all of the other samples sites. The remaining six sampling locations, although geographically close to each other, present levels of genetic divergence associated with dissolved oxygen regime. Earlier studies on the biochemistry of this system found significant differences between different oxygen regimes (Martínez et al. Our study detected significant population differentiation in the African cyprinid


The objective of present study was identification of genetic similarity between wild Iran and captive Azerbaijan Pheasant using PCR-RAPD markers. For this purpose, in overall, 28 birds were taken for DNA extraction and subsequently 15 arbitrary primers were applied for PCR-RAPD technique. After electrophoresis, five primers exhibited sufficient variability which yielded overall 65 distinct bands, 59 polymorphic bands, for detalis, range of number of bands per primer was 10 to 14, and produced size varied between 200 to 1500 bp. Highest and lowest polymorphic primers were OPC5, OPC16 (100%) and OPC15 (81%), respectively. Result of genetic variation between two groups was accounted as nonsignificant (8.12%) of the overall variation. According to our expectation the wild Iranian birds showed higher genetic diversity value than the Azerbaijan captive birds. As general conclusion, two pheasant populations have almost same genetic origin and probably are subpopulations of one population. The data reported herein could open the opportunity to search for suitable conservation strategy to improve richness of Iran biodiversity and present study here was the first report that might have significant impact on the breeding and conservation program of Iranian pheasant gene pool. Analyses using more regions, more birds, and more DNA markers will be useful to confirm or to reject these findings. Pheasants are one of the most endangered species of birds in the world  which apIranian pheasant is one of the unique and unmixed species of world which is on the edge of extinction now. These species are on the list of the world's threatened species. Therefore, conservation program aims to preserve the genetic distinctiveness of the species in the north of Iran . The belAzerbaijan country as neighbor country of Iran has many captive pheasant farms and could be as good potential for importing eggs or live birds for pheasant propagation programs. There is several seasonal immigrations between borders of these counties reported by environmental protection organization of Iran. There is limited scientific information about genetic structure of pheasant in Iran. Identification of genetic similarity between two countries could provide scientific evidence of genetic relatedness between two counties pheasant population.Conservation attempts for wild birds have been limited; management practices are based almost solely on controlled burning, leaving areas of varying postfire age to maintain optimal habitat availability. The analysis of genetic variability is an essential ingredient for conservation programs, and the approach must be based on a combination of phenotypic and genetic data .These indigenous birds such as pheasant are now subjected to fast genetic degradation and dilution because of unplanned conservation and introduction of exotic germplasm, so evaluation of DNA marker is prerequired before designing of any strategy for conservation. The use of molecular markers can aid in the choice of breeds and populations to be conserved, when there is a shortage of resources, as well as the estimation of genetic variability of species breeds and populations .Random amplified polymorphic DNA (RAPD) is a useful approach to assessing genetic variation for conservation of wild populations; it is based on PCR amplification of genomic DNA with arbitrary nucleotide sequence primers. The RAPD marker can detect high levels of DNA polymorphism and can produce fine genetic markers , 9. ThisThe objective of present study was identification of genetic similarity between Iran and Azerbaijan Pheasant birds using PCR-RAPD Markers.n = 16 (coded: 1–16) captives Azerbaijan Pheasant n = 12 (coded: 17–28)) were used for present study.Birds were taken from Arasbaran region  one of the most important border areas of Iran with a varying altitude from 256 m in the vicinity of Aras River to 2896 m and covering an area of 78560 hectares and also commercial farm of Bardeh city near Baku in Azerbaijan country. In overall, 28 birds , 50 ng of genomic DNA, and 1 U Taq DNA polymerase (Promega). The PCR-RAPD protocol included initial denaturation for 3 min at 94°C, followed by 34 cycles of denaturation for 45 s at 94°C, annealing for 45 s at 37°C, extension for 1 min at 72°C, and a final extension at 72°C for 10 min. PCR products were electrophoresed at 85 V for 45 min in 2.5% agarose gels and viewed under UV light. The sizes of alleles were determined in relation to a 100 bp DNA size standard (Fermentas) using a computer software BIO 1D++. PCR was carried out in 25 Produced loci (band), number of polymorphic band, gene diversity Shannon's information, and polymorphic percentage (%) were calculated with POPGENE 3.1 . The anaFive primers among total 15 random primers exhibited sufficient variability for studied populations. To score the band pattern, we assumed that one band corresponded to one locus. After the duplication tests, it was concluded that the RAPD bands acquired in this study are reproducible. The number of polymorphic bands varied from 10 to 14, with a range of 200–1500 bp .The five primers yielded 65 distinct bands, 59 of which were polymorphic Figures , 4 and 5Estimation of polymorphic loci, intrapopulation similarity indices suggested 96.5 percent similarity between two populations. It concluded that two investigated pheasant populations have same genetic origin and probably we can assume them as subpopulations of one population Figures .Genetic diversity is a major issue of conservation biology recognized by the IUCN . Within  Therefore, during the past few years, the genetic diversity of many threatened mammals, birds, fish, insects, and plants has been investigated . RecentlPhasianus colchicus) occurs widely in forested regions in northern Iran. Management of populations for sport hunting has centred on the south Caspian region, where special management areas have been set aside and stocks manipulated. Hunting of this bird is illegal in all seasons and some of its important residences such as the national park of Golestan, the preserved area of central Alborz, Miankaleh, Samkandeh, and Arasbaran are among the preserved areas. Unfortunately, the illegal hunting and destruction of its environment have exposed this species to a serious danger. The common pheasant  or re-introduced after extinction from the species' native range by releasing individuals from ex situ populations back into the wild. Pheasant propagation programs and subsequence releasing of young pheasant to their habitat could be possible practical way to increase number of Iranian pheasant conservation. Result of this study supported the hypothesis that captives Azerbaijan pheasant could be good resource for richness of genetic resource of Iran. It concluded that two investigated pheasant populations have same genetic origin and probably we can assume them as subpopulations of one population. These results demonstrate high genetic similarity of Iranian and Azerbaijan pheasant using RAPD markers. The reason for a difference in the polymorphism level for present populations and other similar species seems particularly due to different genetic makeup, species adaptation, natural selection, history of birds, migration, and mutation, even more by technical staff, which may influence the analysis. The data reported here open the opportunity to search for proper conservation strategy forincreasing population of pheasant in Iran. Analyses using more birds and more DNA markers will be useful to confirm or to reject these findings.


Isophya stysi Cejchan (Orthoptera: Tettigoniidae), an endemic Orthoptera species to the Carpathian Basin, using an enriched genomic library procedure. The polymorphism of these loci were tested in two populations of I. stysi, and the number of alleles per locus varied from 4 to 16. The expected and observed heterozygosities ranged from 0.612 to 0.925 and from 0.625 to 1.000, respectively. The interspecific applicability of these microsatellites was evaluated by amplification in 20 related species: Isophya camptoxypha, Isophya sicula, Isophya ciucasi, Isophya pienensis, Isophya harzi, Isophya kraussii, Isophya zubovskii, Isophya rectipennis, Isophya modesta, Isophya longicaudata, Isophya dobrogensis, Isophya hospodar, Isophya speciosa, Isophya modestior, Poecilimon fussii, Poecilimon affinis, Polysarcus denticauda, Barbitistes constrictus, Leptophyes discoidalis, Phaneroptera falcata. All primer pairs for the 10 loci yielded successful amplifications in at least one other taxon from the Isophya genus. This set of microsatellite loci would be useful for genetic studies in I. stysi and other species of the genus Isophya.Ten microsatellite loci were isolated and characterized for Stys's bush-cricket,  Isophya (Orthoptera: Tettigoniidae) is one of the largest of the Palearctic Orthoptera, comprising a large number of species with a high degree of morphological similarity  and requires strict conservation measures. In order to ensure effective conservation management, the genetic diversity of the populations of this endangered species needs to be evaluated. Microsatellite DNA is an optimal molecular marker for studies of genetic diversity in natural populations as it can assess population structure . Adaptor-ligated DNA fragments ranging from 400 to 1000 bp were selected, and enriched using 3′ biotin-labelled CA and GA repeat oligos bound to streptavidin coated magnetic beads . The DNA fragments were then ligated into the pJET 1.2 vector (Fermentas UAB) and transformed into DH5α Escherichia coli competent cells for cloning. The enriched genomic library was screened for repetitive sequences, and 73 clones containing inserts with microsatellite motives were selected. These were further sequenced using the LICOR 4300L Genetic Analyzer. The similarity of the flanking regions and the microsatellite length were determined with SciRoKo 3.4 .All 25 primer pairs were initially tested for amplification using 8 individuals of 2 , 50 mM KCl, 0.08% (v/v) Nonidet P40, 1.5 to 2.5 mM MgClGT 3.2 software package .The PCR program used consisted of 5 minutes denaturating at 95° C, followed by 30 cycles of 30 sec at 95° C, 30 sec at the annealing temperature . Genomic DNA was extracted from the middle leg of each individual using the Nucleospin Tissue kit .The degree of polymorphism of the 10 selected loci was tested in two populations of The null alleles frequencies were estimated by a maximum likelihood algorithm as implemented in FreeNA , IST3 only in the Ceahlau Mountain population, and 1ST 18 and 1ST 23 only in the Nuc-§oa-ra population. Null alleles were estimated as present in IST15 (estimated frequency f.e. = 0.028), IST21 (f.e. = 0.059), and IST24 (f.e. = 0.019) loci in the Ceahlau Mountain population, and in IST23 locus (f.e. = 0.073) in the Nuc-§oa-ra population. These results, together with the relative small sample size , may explain the deviation observed in some of the Hardy-Weinberg equilibrium tests. No significant linkage disequilibrium was found between loci pairs in tests performed across all populations.Significant deviation from the HardyWeinberg equilibrium was observed in 3 out of 20 possible single exact locus tests (p = 0.01), with a moderate pairwise FST value of 0.056. The genetic differentiation between the two populations can be explained by the geographical distance between them , which can determine a low level of geneflow.The molecular variance analysis, calculated using GenAlEx 6.4, showed significant differentiation between the two populations , two species of the genus Poecilimon , and four species from different genera  . IST2 and IST5 loci amplified in all Isophya species tested and IST15 locus amplified in 12 species. Eight of the microsatellite loci  . DNA samite loci  amplifiecal data .I. stysi may prove to be very useful in population genetic studies on other species of the genus Isophya, but their potential for cross-species amplification is limited outside the genus. These novel polymorphic loci should be a useful tool to study the genetic diversity and structure of I. stysi populations and to develop better conservation measures for this endangered species.These data show that the microsatellite markers isolated for


Aspen naturally grows in large, single-species, even-aged stands that regenerate clonally after fire disturbance. This offers an opportunity for an intensive clonal forestry system that closely emulates the natural life history of the species. In this paper, we assess the potential of genetic tree improvement and clonal deployment to enhance the productivity of aspen forests in Alberta. We further investigate geographic patterns of genetic variation in aspen and infer forest management strategies under uncertain future climates.Genetic variation among 242 clones from Alberta was evaluated in 13 common garden trials after 5–8 growing seasons in the field. Broad-sense heritabilities for height and diameter at breast height (DBH) ranged from 0.36 to 0.64, allowing 5–15% genetic gains in height and 9–34% genetic gains in DBH. Geographic partitioning of genetic variance revealed predominant latitudinal genetic differentiation. We further observed that northward movement of clones almost always resulted in increased growth relative to local planting material, while southward movement had a strong opposite effect.Aspen forests are an important natural resource in western Canada that is used for pulp and oriented strandboard production, accounting for ∼40% of the total forest harvest. Moderate to high broad-sense heritabilities in growth traits suggest good potential for a genetic tree improvement program with aspen. Significant productivity gains appear possible through clonal selection from existing trials. We propose two breeding regions for Alberta, and suggest that well-tested southern clones may be used in the northern breeding region, accounting for a general warming trend observed over the last several decades in Alberta. Populus tremuloides Michx.) currently represents approximately 42% of the combined conifer and deciduous annual harvest of 23 million cubic meters within the province of Alberta, making it an important local economic resource The western boreal hardwood forests cover approximately 60 million hectares, mainly in northern Alberta Aspen naturally grows in large, single-species, even-aged stands that regenerate clonally after fire disturbance from root suckers From a genetic perspective, viable tree improvement programs rely on high within-population genetic diversity for selection, and high heritability of desired traits. While previous research on aspen genetics is limited in scope, low to moderate broad-sense heritabilities of wood properties were estimated from naturally occurring aspen clones Traits of economic interest such as growth, survival, and insect and disease resistance are controlled by multiple genes as well as environmental factors. To distinguish environmental effects from genetic differences, genotypes are tested in common garden trials, where environmental conditions are the same for all genotypes . This allows for partitioning of the genetic variation from the overall phenotypic variation. Subsequently, the heritability of measured traits can be calculated, which represents the proportion of the total variance in a phenotypic trait that is controlled by genes. Heritability estimates can then be used to predict genetic gain from selection In addition to estimating genetic gain from selection, knowledge about geographic structure of genetic variation is important in the development of a tree improvement program. Reforestation material needs to be well adapted to the growing conditions of the planting site, and using planting stock for reforestation that originates within a restricted geographic area can minimize loss of productivity and forest health issues because of maladaptation Recognizing the potential for an aspen tree improvement program, an industrial cooperative, the Western Boreal Aspen Cooperative (WBAC) was formed in 1995. Their initial tree improvement strategy for Alberta was originally described by Li Mature trees of desirable phenotypes (plus-trees) were collected from throughout northern Alberta and north-eastern British Columbia , and werRoots were returned to a lab and rinsed to remove soil, followed by a few minutes of a 0.5% bleaching soak for sterilization. Subsequently, 1.5 m root segments were cut into smaller pieces of approximately 30 cm length, placed into horticultural flats, covered with vermiculite and drenched with fungicide. Horticultural flats were periodically irrigated to maintain moisture content until root sprouts appeared after approximately 10 days. Once root sprouts reached 1.5 cm, they were harvested with a scalpel over a period of approximately six weeks from the horticultural flats. The sprouts were inserted into pellets of peat growth media and placed in a growth chamber at 20°C and high humidity until they rooted within one to two weeks. Rooted sprouts subsequently were transferred into larger styroblocks and grown in a nursery setting under shade for two weeks before being moved to full light in July. In the fall of the first growing season dormant root propagules (stecklings) were placed into cold storage for field planting in the subsequent spring.Aspen propagules generated in the years 1999, 2001 and 2002 were planted on at least three sites established in the same year , Table 1Stecklings were planted with a spacing of approximately 2.5 meters by 3.0 meters, and a double row of border trees from leftover clonal planting material was planted around all trials. Test sites were fenced to prevent animal browse, and were managed for vegetation control comprised of a combination of chemical and mechanical means over the first three years with subsequent maintenance as needed. Measurements of height and diameter at breast height (DBH) were recorded after eight, six, and five growing seasons for the 1999, 2001, and 2002 trial series, respectively. Clonal trial series collected and established in different years had minimal or no overlap in terms of clonal material and were therefore analyzed separately.Prior to statistical analysis, all data were examined with box plots and line plots to identify errors in measurements, data entry, or unusual growth patterns over multiple years. All trees were measured in one- or two-year intervals, and we removed all growth increments that represented a Tukey outer-fence outlier. Also, measurements identified as Tukey inner-fence outliers in one year that returned to within the interquartile range of the clone in the subsequent year were set to missing values, which was possible without detrimental effects on the statistical power of the analysis, because all treatments were replicated in multiple-tree row plots. Subsequently, individual tree data from row plots were averaged to be used as experimental units in the statistical analysis. Analysis of variance and variance component estimation was carried out separately for each site with PROC MIXED of the SAS statistical software package G is the total genetic variation represented by the variance component due to clone effects and VP the phenotypic variation, represented by the variance component due to clones plus the residual error. Block effects were not included in the denominator. To derive standard errors for heritability estimates, standard errors of variance components were generated with the COVTEST option of PROC MIXED Broad-sense heritabilities were determined separately for each clonal experiment, and calculated as:2:MVpart package v1.2–6 for the R programming environment Breeding region delineations were carried out with a variance partitioning approach that used geographic predictor variables to partition genetic variation observed in common garden trials Multivariate regression trees (MRT) are based on the same principles as Classification and Regression Trees (CART), but extended to more than one response variable Prior to a formal variance partitioning analysis, we visualized the results of clonal trials at multiple test sites by means of box plots and scatter plots. We focus on the 2002 trial series because it contained the largest selection of clones  and 3, bCollections in the northern forest management areas Slocan Forest Products and Daishowa-Marubeni International  tend to perform below average at all sites, and especially poorly at the most southern Site 31 . The locRank order changes of clones among pairs of test sites are shown in The 1999 and 2001 trial series reveal similar patterns. In the 1999 series  and S2 lBased on the regional split presented above, identifying two breeding regions for Alberta separated at approximately 56°N latitude, we separately analyzed northern clones at the northern site (Slocan and DMI sources planted at Site 10) and southern clones at the southern planting sites . Variance components and heritabilities for these putative breeding populations are provided in Broad-sense heritabilities for height and DBH for the southern breeding region and southern collections by Weyerhaeuser, Millar Western Forest Products, Alberta-Pacific Forest Industries Inc., Ainsworth Lumber, and Slave Lake Pulp Corporation were variable among individual trials . GeneralThe northern collections by Slocan Forest Products and Daishowa-Marubeni International at the northern Site 10 yielded somewhat lower broad-sense heritabilities between 0.13 and 0.54, for height and DBH. Standard errors in this trial series were generally higher and the number of clones included in the trials was generally lower. Nevertheless, two experiments . However, for an informal assessment, we can rank clones across different genetic tests by expressing height and DBH in units of standard deviations from a test mean of zero. For the following assessment we therefore make the assumption that the average genetic worth of different clonal trial series is the same, and that the effectiveness of plus-tree selections was similar in different years.Clonal deployment of planting stock in Alberta requires at least 18 clones in a deployment population for reforestation on public lands The conceptual basis for the delineation of breeding regions is that local sources are optimally adapted to local environments and should therefore not to be moved too far from the collection location to avoid mal-adaptation. Our results suggest that this assumption may not apply for aspen in Western Canada. Instead, a northward movement of planting material almost always resulted in increased growth and a southward movement had a strong opposite effect. Northern sources  generally underperformed at southern sites , whereas more southern sources  generally performed better or on-par with local material at the northern site , and S3.We should note, however, that the observation of local non-optimality is not uncommon, and may have a number of natural causes besides adaptational lag If southern clones from the 1999 to 2002 series, tested at the northern site (10) were used in the northern breeding region, the top 18 clones would comprise six northern sources and 12 southern sources, yielding a 16% genetic gain in height. The additional 11% gain may be associated with an increased risk, depending on the underlying cause of the observed non-optimality. If adaptational lag is the underlying cause, the use of well tested southern sources in the northern breeding region would be a sensible climate change adaptation strategy. If reduced growth of local sources is caused by tradeoffs required by survival adaptations, those genetic gains could be associated with some risks. For example frost damage may occur under rare, unseasonal cold events. After only 5–8 growing seasons in the field, we found no indication of such tradeoffs. Survival was generally high, and survival of southern clones transplanted to the northern test sites (99%) was nearly identical to that of local sources at the northern trials . Nevertheless, a cautious approach to seed transfer would avoid long-distance relocations, exceeding 2–3° latitude.Two breeding regions for aspen in Alberta, north and south of 56°N latitude are supported by our results. It should be noted, however, that none of the clones sampled in Alberta were collected north of 59°N latitude and this should therefore be a prudent limit for the northern breeding region. Similarly no material was tested south of 52°30′N, which would serve as a limit for the southern breeding region. The northern breeding region may include an elevation limit, since we detected a genetic difference along an elevational gradient in the 2002 clonal trial series. However, given observed warming trends in Alberta this limit should mainly be viewed as a restriction against including higher elevation sources for low elevation deployment, not vice versa. Data from this and a previous study further suggests that eastern and western regions of Alberta at the same latitude and similar ecosystem types do not require separate breeding regions for lack of genetic differentiation.Broad-sense heritabilities estimated from multiple trials suggest that substantial genetic gains are possible through clonal selection as part of an aspen tree improvement program. Broad-sense heritabilities for height and DBH ranged from 0.36 to 0.64 on good sites. In a first round of selection for the southern breeding region, 15% genetic gain in height and 34% in DBH could be achieved immediately through deployment of clones, prior to the first generation of breeding. For the northern breeding region, genetic gains are predicted to be smaller (5% for height and 9% in DBH), because of lower heritabilities and fewer clones currently available for selection. However, including southern sources that have been tested at the northern site would also result in a 16% genetic gain for the northern breeding region as well. Local non-optimality observed in this study and recent climate change trends observed in Alberta suggests that in general transfer of any genetic material toward lower elevation and towards the south should be avoided, while upward and northward transfer of genetic material should be part of a tree improvement strategy to achieve enhanced forest productivity in the future.Figure S1Range of aspen clone means for 8-year height and DBH at multiple test sites . The total numbers of clones representing each management area are: SLN, 10; SLS, 4; DMI, 25; AIN, 11; SLP, 14; MW, 15; WEY, 32; and ALP, 7). For abbreviation of forest management areas, refer to (PDF)Click here for additional data file.Figure S2Rank changes of clones among pairs of sites for the 1999 clonal series. Scatter plots above the diagonal show 8-year DBH at two sites, and scatters below the diagonal show height. Note that in each scatter plot, only clones that were planted at both sites can be shown. The map above shows the location of test sites (triangles) and collection sites (circles).(PDF)Click here for additional data file.Figure S3Range of aspen clone means for 6-year height and DBH at multiple test sites . The total numbers of clones representing each management area are: DMI, 23; AIN, 14; MW, 9; and WEY, 66). For abbreviation of forest management areas, refer to (PDF)Click here for additional data file.Figure S4Rank changes of clones among pairs of sites for the 2001 clonal series. Scatter plots above the diagonal show 6-year DBH at two sites, and scatters below the diagonal show height. Note that in each scatter plot, only clones that were planted at both sites can be shown. The map above shows the location of test sites (triangles) and collection sites (circles).(PDF)Click here for additional data file.


The results showed that carboxyl groups and sulfate esters were presented in SNC after hydrolysis with H2SO4, while no sulfate esters were detected in SNC during HCl-hydrolysis. TEM results showed that, compared to H2SO4-hydrolyzed sample, a wider size distribution of SNC prepared by HCl-hydrolysis were observed. Zeta-potentials were −23.1 and −5.02 mV for H2SO4- and HCl-hydrolyzed SNC suspensions at pH 6.5, respectively. Nevertheless, the zeta-potential values decreased to −32.3 and −10.2 mV as the dispersion pH was adjusted to 10.6. After placed 48 h at pH 10.6, zeta-potential increased to −24.1 mV for H2SO4-hydrolyzed SNC, while no change was detected for HCl-hydrolyzed one. The higher zeta-potential and relative small particle distribution of SNC caused more stable suspensions compared to HCl-hydrolyzed sample.Surface chemical compositions of starch nanocrystals (SNC) prepared using sulfuric acid (H Recently, there has been growing interest in starch nanocrystals (SNC) for abundant availability of starch, comparatively easy processability, biocompatibility, biodegradability, and non-toxicity, comparing with inorganic nanoparticles 2SO4) or hydrochloric acid (HCl) at 25–55°C for various periods of time. However, hydrolysis of starch by H2SO4 was more widely adapted because of its high efficiency comparing with HCl 2SO4 hydrolysis resulted in more stable suspensions than HCl hydrolyzed samples SNC are crystalline platelets resulting from the disruption of the semi-crystalline structure of starch granules by hydrolysis of amorphous parts. The preparation methods by different acid hydrolysis have been extensively studied 2SO4 have not been explicitly reported except for some hypotheses. These proposals mainly included the formation of sulfate ester groups on the surface of the SNC A homogeneous dispersion state of SNC was a key step required for high mechanical performances of rubber or other material properties 2SO4- and HCl- hydrolysis was, thus, analyzed and its effects on zeta-potentials of SNC suspensions were examined. This will provide basic information of SNC hydrolyzed with HCl and H2SO4 and explain the reason of their different dispersity.In this work, the change of surface chemical compositions of SNC after H2SO4, HCl, sodium hydroxide (NaOH), potassium chloride (KCl) and uranyl acetate were purchased from Sinopharm Chemical Reagent Co., Ltd.  and of analytical grade. Potassium bromide (KBr) was of spectroscopic grade and bought from Sinopharm Chemical Reagent Co., Ltd. . Milli-q water was used in all experiments.Waxy maize starch (WMS) was kindly donated by Tianjin Tingfung Starch Development Co., Ltd. . H2SO4 solution and placed at 40°C for 3 and 7 d under stirring at the speed of 200 rpm. For the HCl-hydrolyzed SNC, 20 g starch powders were mixed with 400 mL 2.2 N hydrochloric acid solution at 40°C for 7 d with constantly stirring at the speed of 200 rpm. The suspensions were washed by successive centrifugations in distilled water until pH was constant. The resultant suspensions were redispersed using Ultra Turrax T20 (IKA) at 13,500 rpm for 3 min to avoid aggregates and stored at 4°C with several drops of chloroform. The SNC concentrations were determined by weighting freeze-dried powders of the homogeneous dispersion of SNC (10 mL) and expressed as weight percentage relative to the volume of the water phase. The concentrations of H2SO4- and HCl-hydrolyzed SNC suspensions were 3.50% (w/v) and 2.08% (w/v), respectively. Different concentrations of SNC suspensions were derived by diluting the “stock” homogeneous dispersion of SNC.SNC were prepared by acid hydrolysis of waxy maize starch according to the method by Angellier et al. with minor modification hν = 1486.6 eV) X-ray source of 150 W at 15 kV. The kinetic energy of photoelectrons was determined with a hemispheric analyzer set to pass energy of 160 eV for wide-scan spectra and 20 eV for high-resolution spectra, respectively. During all measurements, electrostatic charging of the samples was avoided by means of a low-energy electron source working in combination with a magnetic immersion lens. Later, all recorded peaks were shifted by the same value to set the C1s peak to 284.8 eV. Quantitative elemental compositions were determined from peak areas using sensitivity factors experimentally and the spectrometer transmission function. Spectrum background was subtracted according to Shirley XPS experiments were carried out by means of a Multifunctional imaging electron spectrometer . The spectrometer was equipped with a monochromatic Al Kα (2SO4-hydrolyzed (for 3 d and 7 d) SNC powders were collected between 4000 cm−1 and 500 cm−1 on a FT-IR spectrophotometer  with 256 scans at a resolution of 4 cm−1. The SNC powders (3 mg) were ground with spectroscopic grade KBr (200 mg) powders and then pressed into 1 mm pellets. A blank disc was used as the background.The FT-IR spectra of the lyophilized and HCl- and HZeta-potentials were determined in presence of 1 mM KCl at 25°C using a commercial Malvern Zetasizer Nano ZS 90. The pH of the suspension  was adjusted using dilute HCl or NaOH. The SNC suspensions were placed for 2 h and 48 h before measurement. Measurements were carried out in quintuplicate for error analysis. The refractive index of fluid and particle used were 1.33 and 1.53, respectively.2SO4 were sonicated at 4°C for 15 min. A drop of suspension was spread onto the copper grids coated with carbon support film. Prior to complete drying, a drop of 2% (w/v) uranyl acetate negative stain was added. After 1 min, the liquid in excess was blotted with filter paper and the remaining film was allowed to dry for observation.TEM observations were performed using a JEOL JEM-2100 (HR) at an acceleration voltage of 80 kV. SNC suspensions  hydrolyzed by HCl and HStatistical analysis was performed using ORIGIN 7.5 . Data were expressed as means ± standard deviations and analyzed by a one-way analysis of variance (ANOVA). P value ≤0.05 was regarded as significant throughout the study.2SO4-hydrolyzed starch for 3 d  and 7 d  were displayed in H2SO4,3 did not show significant change in the first 3 d, whereas it increased from 0.43 to 0.61 after hydrolysis for 7 d. The relative slow increase of O/C ratio in the first 3 days might result from the incomplete exposed internal reducing ends, since waxy maize starch could not absolutely crack. As the hydrolysis process extending, more and more internal reducing ends were exposed, leading to the increase of the O/C ratio. However, no significant changes of O/C ratio were detected between H2SO4- and HCl-prepared samples after hydrolyzing for 7 d.The general XPS spectra of WMS and HH2SO4,7 showed that the peak intensity corresponding to oxygen atoms became almost two times higher than the one corresponding to carbon atoms. This increase indicated the disruption of starch granules and the increase of reducing ends. The surface lipids that washed away by acid hydrolysis were contributed to the relative increase of oxygen atoms 2SO4. No surface nitrogen was observed after acid hydrolysis, this result was in agreement with the findings reported by Thielemans, et al XPS spectra of SNCH2SO4,3, and the relative content of C1 peak decreased simultaneously. The increase in C4 peak might be ascribed to the adsorption of formic and levulinic acids derived from the hydrolyzed products of glucose 2SO4-hydrolyzed cellulose microcrystal suspensions HCl,7 was lower than that for SNCH2SO4,7, the reason is probably that the concentration of H2SO4 used in the experiment was much higher than that of HCl.The C 1 s peak in the XPS spectra was deconvoluted for each sample into several peaks . The difH2SO4,3, SNCH2SO4,7, and SNCHCl,7 were shown in HCl,7. However, it was detected for SNCH2SO4,3 and SNCH2SO4,7 samples. The S 2p spectra at 168 eV were composed of S 2p3/2 and S 2p1/2 peaks, and both peaks represented sulfur in its highest oxidation state, i.e., O-SO3− or C-SO3−. Nevertheless, no C-S bond was presented in the C 1 s peaks for SNCH2SO4,3 and SNCH2SO4,7  Breakage of α-D-(1-4) and α-D-(1-6)-glycosidic bonds was the dominant reaction, which resulted in disappearance of amorphous and semicrystalline layers of the WMS, leaving the acid-resistant fraction named SNC; (2) Glucose was further degraded to formic and levulinic acids, and adsorption of these acids to the surface of SNC made them negative-charged ; (3) ForHCl,7 and SNCH2SO4,7 suspensions at pH 6.5 and 10.6 placed for 2 and 48 h were shown in HCl,7 suspension, the final pH was 6.5 with complete washing by distilled water. However, it was difficult to wash SNCH2SO4,7 till neutrality due to presence of carboxyl groups and sulfate esters (H2SO4,7 suspension was adjusted to 6.5 with dilute NaOH in this work. Owing to the presence of sulfate esters and carboxyl groups, zeta-potential of SNCH2SO4,7 reached −23.1 mV, which was much lower than that of the SNCHCl,7 suspension. No changes of zeta-potentials were detected for the two samples after 48 h at pH 6.5. Zeta-potentials decreased to −32.3 and −10.2 mV for SNCH2SO4,7 and SNCHCl,7, respectively, while the dispersion pH was adjusted to 10.6 and placed for 2 h. After 48 h, zeta-potential for SNCH2SO4,7 increased to −24.1 mV, while no changes were found for SNCHCl,7. These results revealed that degradation of sulfate esters under alkaline condition contributed to the increase of the zeta-potential of SNCH2SO4,7 suspension.Zeta-potentials of SNCe esters . Therefo2SO4-hydrolyzed SNC suspensions. It could be seen from the picture that SNC derived from HCl-hydrolysis for 7 d showed a wide particle distribution, which was in the range of 50–300 nm. The low acid concentration used in the experiment might result in the heterogeneous SNC distribution. On the contrary, a homogenous SNC distribution in the range of 50–150 nm was obtained after H2SO4-hydrolysis for 7 d.2SO4-hydrolyzed sample. This could be ascribed to the relatively low zeta-potential of SNC suspension obtained by HCl-hydrolysis. Compared to the SNCHCl,7 suspension, the higher zeta-potentials and the relative smaller particle distribution of SNCH2SO4,7 generated the more stable suspensions.In addition, the aggregation behavior of HCl-hydrolyzed SNC was more serious than H2SO4-hydrolyzed SNC were different from the HCl-hydrolyzed samples. Adsorption of formic and levulinic acids forming from the acid hydrolysis of glucose were probably contributed to the presence of carboxyl groups in HCl- and H2SO4-hydrolyzed SNC, while sulfate esters in H2SO4-hydrolyzed SNC were derived from the esterification reaction between hydroxyl bonds and H2SO4. The higher zeta potential for SNCH2SO4,7 and the samller particle distribution limited flocculation and generated more stable suspensions comparing with the HCl-hydrolyzed sample. These results suggested that a more stable suspension was obtained by H2SO4-hydrolysis due to the higher zeta potential and much smaller and narrower particle size distribution.Surface chemical compositions of H


Parasites are evolutionary hitchhikers whose phylogenies often track the evolutionary history of their hosts. Incongruence in the evolutionary history of closely associated lineages can be explained through a variety of possible events including host switching and host independent speciation. However, in recently diverged lineages stochastic population processes, such as retention of ancestral polymorphism or secondary contact, can also explain discordant genealogies, even in fully co-speciating taxa. The relatively simple biogeographic arrangement of the Galápagos archipelago, compared with mainland biomes, provides a framework to identify stochastic and evolutionary informative components of genealogic data in these recently diverged organisms.Mitochondrial DNA sequences were obtained for four species of Galápagos mockingbirds and three sympatric species of ectoparasites - two louse and one mite species. These data were complemented with nuclear EF1α sequences in selected samples of parasites and with information from microsatellite loci in the mockingbirds. Mitochondrial sequence data revealed differences in population genetic diversity between all taxa and varying degrees of topological congruence between host and parasite lineages. A very low level of genetic variability and lack of congruence was found in one of the louse parasites, which was excluded from subsequent joint analysis of mitochondrial data. The reconciled multi-species tree obtained from the analysis is congruent with both the nuclear data and the geological history of the islands.The gene genealogies of Galápagos mockingbirds and two of their ectoparasites show strong phylogeographic correlations, with instances of incongruence mostly explained by ancestral genetic polymorphism. A third parasite genealogy shows low levels of genetic diversity and little evidence of co-phylogeny with their hosts. These differences can mostly be explained by variation in life-history characteristics, primarily host specificity and dispersal capabilities. We show that pooling genetic data from organisms living in close ecological association reveals a more accurate phylogeographic history for these taxa. Our results have implications for the conservation and taxonomy of Galápagos mockingbirds and their parasites. Parasites represent evolutionary hitchhikers on their hosts with evolutionary histories of each lineage often running in parallel e.g. ,2]. When. When2]]A theoretical framework relating co-phylogenetic patterns with population genetic processes was defined by Rannala and Michalakis , who forDue to their relative isolation from mainland biota, restricted surface area and low probability of multiple colonization events, oceanic islands like Galápagos provide a convenient model to study co-phylogenetic patterns in hosts and parasites. Founding populations of island colonists are typically small in size and can only carry a limited number of gene alleles leading to rapid coalescence. Consequently the effects of selection and genetic drift quickly lead to genetic differentiation and the formation of new species .Mimus spp.) in particular, have played a prominent role in research on island speciation. It was Charles Darwin's observation on the distinctiveness of Galápagos mockingbirds from their mainland relatives that provided much of the founding evidence for the idea that species evolve through time . Ancesta priori expectation of them having a shared evolutionary history, improves confidence in the inferred phylogeographic patterns. Parasites, and host specific ectoparasites in particular, can predict the population structure of their hosts , and, anda prsts  it is possible that B. galapagensis occasionally occurs on the Small Ground Finch as stragglers. The lack of genetic structure in B. galapagensis might be explained by phoretic transfer to the Small Ground Finch and the inter-island migration of this species, followed by recurrent phoretic transfer back to mockingbirds.Comparative studies of genetic differences at a very low (intra-specific) evolutionary levels are rare in lice [for exceptions see -83] but ery rare . Hippoboiginosa) ,84 whichiginosa) ,85. AlthBrueelia also show very little intra-population variability, which is compatible with low population sizes of the parasite recovered during collecting. Despite large numbers of mockingbirds inspected for lice, Brueelia was absent on many islands, especially on larger islands of the archipelago and in those instances when Brueelia was present, its abundance and prevalence were very low in comparison to Myrsidea . We found that co-phylogeographic patterns inferred on the level of single gene genealogies  are considerably impacted by differential distribution of ancestral polymorphism. In contrast, extremely low genetic variability and lack of co-phylogeographic congruence was found in Brueelia. These differences may be explained by life history traits in Brueelia such as their dispersal capabilitity, abundance, and lower levels of host specificity. A more accurate picture of the phylogeographic history of these lineages, congruent with the geological history of the islands and with available nuclear data was obtained through a joint analysis of data for the three co-evolving groups. We show that pooling genetic data for several organisms living in close ecological association improves the inference of phylogeographic histories in recently diverged species.Using mitochondrial DNA sequences and nuclear data we studied 400 samples of recently diverged Galápagos mockingbirds and 229 specimens of 3 species of their parasites  for the COI gene.Click here for file*BEAST chronograms. Provided are multi-species chronogram (a) and chronograms for Mimus (b), Analges (c) and Myrsidea (d) datasets. Tip labels on the multi-species tree are the same as in Figure Click here for fileZipped archive containing KML files for each of the taxon phylogenies. These data files should be viewed in Google Earth [http://www.google.com/earth].Click here for fileHost-parasite phylogenies mapped with Jane software. Results for Mimus-Analges (a) and Mimus-Myrsidea (b) associations are shown. Hollow circles mark co-speciations, solid circles mark duplications. Host switches are marked by arrows and losses by dashed lines. Host trees are in black, mapped parasite histories are in blue. Yellow nodes indicate another location of equal cost exists, red nodes mark the solution with the lowest cost. Taxon labels were assigned as species and island abbreviations as shown in Figures Click here for file


ManitobaN = 144; South DakotaN = 653) and a smaller phenotypic analysis from Kansas (KansasN = 44), we compared mean levels of pigmentation, genetic variation and heritability in three pigmentation traits among populations of the common garter snake, Thamnophis sirtalis, along a north-south gradient, including a postglacial northern population and a putative southern refuge population. Counter to our expectations, we found that genetic variance and heritability for the three pigmentation traits were the same or higher in the postglacial population than in the southern population.Global climate change is expected to trigger northward shifts in the ranges of natural populations of plants and animals, with subsequent effects on intraspecific genetic diversity. Investigating how genetic diversity is patterned among populations that arose following the last Ice Age is a promising method for understanding the potential future effects of climate change. Theoretical and empirical work has suggested that overall genetic diversity can decrease in colonial populations following rapid expansion into postglacial landscapes, with potential negative effects on the ability of populations to adapt to new environmental regimes. The crucial measure of this genetic variation and a population's overall adaptability is the heritable variation in phenotypic traits, as it is this variation that mediates the rate and direction of a population's multigenerational response to selection. Using two large full-sib quantitative genetic studies ( One of the primary challenges facing contemporary ecological and evolutionary research is predicting the potential effects of global climate change on populations across broad latitudinal ranges. Of recent concern has been the theoretical prediction that populations expanding into northern climates will experience a rapid loss of genetic diversity The primary hypothesis for how genetic diversity is patterned across the postglacial landscape states that populations founded after a postglacial expansion should have less genetic diversity as a result of population bottlenecks and/or new selection pressures, resulting in reduced effective population sizes and thus less genetic diversity relative to their historical populations outside the glacial barrier Although these studies provide a compelling picture of a population's genetic diversity following postglacial colonization, the true metric of a population's adaptive ability is not neutral genetic diversity per se, but rather heritable genetic variation. This is because heritable genetic variation mediates the rate and direction of population-scale responses to selection Thamnophis sirtalis, in South Dakota, United States and Manitoba, Canada. The Manitoba population is located in the Interlake near the town of Vogar. The South Dakota population is located at Lake Traverse on the South Dakota-Minnesota border, approximately 600 km due south of the Manitoba site, and represents the southern extremis of the Laurentide ice sheet, which retreated approximately 12,000 years before present To better understand the consequences of postglacial colonization on heritable genetic variation, we quantified levels of genetic variation in pigmentation traits T. sirtalis has found a lack of concordance between neutral molecular trees and those based on color pattern, further suggesting a role for selection in pigmentation evolution We focused on pigmentation phenotypes for our study because garter snake color traits are scored easily and are thought to be of adaptive importance in natural populations Thamnophis sirtalis because it is one of only a few reptile species to colonize extreme northern latitudes T. sirtalisb locus We chose The primary question we address within this multiple population framework is: Does heritability of adaptive pigmentation traits in the common garter snake decline at northern latitudes? The results of our study suggest that heritability of the three pigment traits is not significantly lower in the postglacial population. The data presented herein further shed light on the effect of rapid colonization on heritable variation in natural populations.P<0.0001) for blotch area and 0.910 (P<0.0001) for pigment area, both of which are as high, or higher, than repeatability values considered acceptable in previous quantitative genetic studies of garter snakes Pearson product-moment correlation coefficients for between-observer scores for this sample were 0.926 , pigment area  or ratio . Furthermore, no sex-by-population effect was detected for any trait . Because we saw no difference in trait means between sexes, males and females were pooled in all subsequent analyses. All phenotypic color estimates are in units of blotch-to-scale area, i.e. the proportion of integument that was white or red relative to the immediately adjacent dermal scute.Adult phenotypic data was collected from both male (n = 139) and female (n = 44) snakes at the Manitoba site. At the South Dakota site, females were far more numerous than males on the date of collection and the sample was therefore skewed towards females . The Kansas sample had approximately equal representation of sexes . Blotch area and pigment area were normally distributed for all populations. Distribution of the ratio values tended to be skewed to the right, but were nonetheless amenable to testing given the procedures used, which are generally robust to minor deviations from normality. Two-way ANOVAs with main effects of sex and population revealed no differences between sexes for blotch area . However, the Kansas population had higher means for both blotch area and pigment area than the South Dakota sample  but the composite ratio of the two pigmentation measures was significantly smaller in the Kansas population  relative to the South Dakota population and not significantly different from the Manitoba population .All trait means for adults were significantly higher in the South Dakota population relative to the Manitoba population . Kansas neonates had significantly higher blotch area  and pigment area than the Manitoba population  but the blotch area/pigment area ratios were not significantly different . Compared to the South Dakota population, Kansas neonates had significantly higher blotch area  but pigment area was not significantly different  nor was the blotch area/pigment area ratio .We generated a second set of phenotypic data from 144 offspring from 25 dams originating at the Manitoba site and 653 offspring from 50 dams from the South Dakota site. For the neonate snakes all pigmentation phenotypes were smaller relative to those measured on the adults . Again, We obtained genetic variance and heritability data from two populations: 144 offspring from 25 dams originating at the Manitoba site, and 653 offspring from 50 dams from the South Dakota site. Because only small numbers of litters were available in the Kansas population, we were unable to generate usable estimates of the genetic variances and heritabilities for this population. Genetic variances and heritabilities were estimated using a full-sib ANOVA on the neonate offspring from each population. The genetic variance for blotch area was 409.62±131.64 s.e. for Manitoba and 360.07±80.96 s.e. for South Dakota; genetic variance for pigment area was 284.44±129.69 s.e. for Manitoba and 162.35±41.64 s.e. for South Dakota; genetic variance for blotch area/pigment area ratio was 0.0466±0.021 s.e. for Manitoba and 0.0067±0.002 s.e. for South Dakota . HeritabA common issue in the comparison of genetic variance estimates among populations using a quantitative genetic design is achieving statistical precision that allows the genetic variance and heritability to be bounded away from zero, and also allows those parameters to be bounded away from the population to which it is being compared T. sirtalis on either side of the boundary of the Laurentide ice sheet, which retreated approximately 12,000 years ago In the present study we evaluate levels of heritable variation in pigmentation between two geographically distinct populations of T. sirtalis also display patterns of among-population variation consistent with a phenotypic cline  at birth than adults from the same population, and furthermore were found to increase in red pigmentation during their early development. The South Dakota and Kansas neonates were also found to have reduced pigmentation relative to the adult samples from their respective populations. Although suggestive, neither the Rye T. sirtalis, we cannot completely rule out the admixture hypothesis.We know of two sources of evidence that argue against the population admixture hypothesis. Rye explored the hypothesis that Manitoba populations were hybrid products of eastern and western clades An alternative explanation for increased genetic variation in postglacial populations is the rearrangement of genetic architectures in postglacial populations T. sirtalis in Manitoba has experienced a more favorable environment since colonization, with a concomitant decrease in natural selection and increase in heritability. 2. New selection pressures. New selection regimes in a novel environment have exposed hidden variation or favored new allelic combinations.Where the strength of selection itself is reduced, heritabilities can be higher than under more stringent selection regimes. For example, documented correlations between quality of environment and heritability suggest that heritabilities are lower in poor environments, e.g., where selection is stronger Although genetic diversity is expected to decrease with distance from the founding population during a stepping-stone colonization event, other factors can rescue genetic diversity. For example, high gene flow between neighboring populations can maintain allelic diversity Thamnophis sirtalisT. sirtalis has been performed in other regions Although at present we are unable to convincingly support or refute any explanation for an increase in genetic variance of three color traits in the Manitoba population, our results suggest that, although genetic diversity at some loci may be reduced in postglacial populations of Policy on the Humane Care and Use of Laboratory Animals, the US Department of Agriculture's (USDA) Animal Welfare Act & Regulations , and the United States Government Principles for the Utilization and Care of Vertebrate Animals Used in Research, Teaching and Testing. The work was approved by the respective Institutional Animal Care and Use Committees (IACUCs) at Oregon State University ; Kansas State University ; and Black Hills State University .All work was conducted in strict accordance with the US Public Health Service (PHS) 25 gravid female snakes were collected from a single hibernaculum near Vogar, Manitoba . A sample of 173 adult snakes  was also collected at the Manitoba site for phenotypic scoring. 50 gravid females were collected from a single hibernaculum on the shore of Lake Traverse near Sisseton, South Dakota . Two additional gravid females were collected from a single location in Douglas County, Kansas  see map, , as wellFemale snakes were housed in dedicated facilities at Oregon State University (Manitoba sample) and Black Hills State University (South Dakota sample) and were reared under identical environmental and feeding regimes We used a standardized scoring system, described in Two personnel were involved in scoring the pigmentation traits; therefore we conducted between-observer repeatability tests using 50 adult snakes from the South Dakota population. 50 adult snakes from the South Dakota population were scored independently by MFW and JM. Scorings were separated by a period of several months, but both measurements occurred during the stable portion of the pigmentation ontogeny that occurs when snakes are adults. Pearson correlation coefficients were calculated in SAS v 9.2 using the PROC CORR procedure to assess the repeatability of scoring between observers.Trait means and standard errors for each population were obtained using PROC MEANS in SAS v 9.2. Trait means among populations were compared by a one-way ANOVA using PROC GLM in SAS v 9.2, followed by multiple comparisons. For the quantitative genetic analysis, we analyzed litter data from only the populations with a large number of litters, that is the Manitoba (25 litters and 144 offspring) and South Dakota populations (50 litters and 653 offspring). Population-specific genetic estimates (i.e. genetic variance and heritabilities) and their associated standard errors were calculated using h2boot Figure S1System for scoring size and pigment saturation of dorsolateral blotches in T. sirtalis. Images on left show among-individual variation in both the size of blotches and the extent to which red pigment saturates the blotches. Images on right focus on a portion of a blotch at one scale row. Top image receives a pigment area score of “0,” middle image receives a score of “0.5,” and bottom image receives a score of “1.” Schematic drawing at bottom depicts how blotch length is measured. Adjacent scale is assigned a length of 10 units, and the blotch is assigned a length relative to the adjacent scale in intervals of 1 unit. In the depicted example, the blotch at that scale row would receive a score of 7. Blotch widths at all scale rows are summed over three adjacent blotches to give individual blotch area score. Pigment area at each scale row is obtained by multiplying the pigment score by the blotch length. Pigment scores at each scale row are then summed over the same three adjacent blotches to give the individual pigment area score.(DOC)Click here for additional data file.


Mugil cephalus represents an interesting case study. Several divergent allopatric mtDNA lineages have been described in this species worldwide, and three occur in sympatry in the NW Pacific.The study of speciation in the marine realm is challenging because of the apparent absence of physical barriers to dispersal, which are one of the main drivers of genetic diversity. Although phylogeographic studies using mitochondrial DNA (mtDNA) information often reveal significant genetic heterogeneity within marine species, the evolutionary significance of such diversity is difficult to interpret with these markers. In the northwestern (NW) Pacific, several studies have emphasised the potential importance of sea-level regression during the most recent glaciations as a driver of genetic diversity in marine species. These studies have failed, however, to determine whether the period of isolation was long enough for divergence to attain speciation. Among these marine species, the cosmopolitan estuarine-dependent fish M. cephalus. Three cryptic species of M. cephalus were identified within this region  using an assignment test on the microsatellite data. Each species corresponds with one of the three mtDNA lineages in the COI phylogenetic tree. NWP3 is the most divergent species, with a distribution range that suggests tropical affinities, while NWP1, with a northward distribution from Taiwan to Russia, is a temperate species. NWP2 is distributed along the warm Kuroshio Current. The divergence of NWP1 from NWP2 dates back to the Pleistocene epoch and probably corresponds to the separation of the Japan and China Seas when sea levels dropped. Despite their subsequent range expansion since this period of glaciation, no gene flow was observed among these three lineages, indicating that speciation has been achieved.Ten nuclear microsatellites were surveyed to estimate the level of genetic isolation of these lineages and determine the role of sea-level fluctuation in the evolution of NW Pacific M. cephalus inhabiting the NW Pacific, using a combination of microsatellites and mitochondrial genetic markers. The current genetic architecture of the M. cephalus species complex in the NW Pacific is the result of a complex interaction of contemporary processes and historical events. Sea level and temperature fluctuations during Plio-Pleistocene epochs probably played a major role in creating the marine species diversity of the NW Pacific that is found today.This study successfully identified three cryptic species in  In the marine environment, fluctuations in sea level, water temperatures and sea ice caused by glacial cycles are believed to have had a major influence on species distributions and the population connectivity of marine species -3. For iThe northwestern (NW) Pacific is characterised by marginal seas, which were particularly impacted by Plio-Pleistocene glacial cycles. The lowering of sea level caused the recurrent closure of the Japan Sea, the semi-closure of the South China Sea and the partial or full exposure of the East China and Yellow Seas . The cloMugil cephalus Linnaeus, 1758, is a euryhaline teleost distributed worldwide in coastal waters, lagoons, bays and estuaries between latitudes 42° N and 42° S  and the statistic ΔK, as described in ,50, respThere was congruence between the two molecular markers when the results of the assignment test were compared to the COI sequence data Figure . The resP < 0.001) and NWP1 (P < 0.001) and slightly lower at NWP2 than NWP1 (P < 0.05) for the ten microsatellite loci (HO was lower at NWP3 than the other two clusters). The genotypes at Mce-14 of NWP2 and Mce-10 of NWP3 were all homozygous.In each location sampled, individuals belonging to the same mtDNA lineages were grouped together to further investigate the genetic structure of the three Operational Taxonomic Units (OTUs). Genetic variability analysis showed that only two of the 30 combinations, loci Mce-3 and Mce-8 of NWP3, deviated significantly from Hardy-Weinberg proportions after Bonferroni correction . Pair-wise θ comparison indicated that NWP3 was the most differentiated , while level of differentiation was lower but still highly significant between NWP1 and NWP2 . In addition, no genetic heterogeneity was detected among samples belonging to any one of the three OTUs, with the exception of the Ariake Sea, which had some genetic heterogeneity among the samples of NWP1  Pacific . While it appears likely that the refugium of the NWP2 lineage was located in the South China Sea, the location of the NWP1 refugium is more difficult to assess. The closure of the Tsushima Straits . If the East China Sea experienced a post-glacial colonisation wave from the Japan Sea, it appears that in species such as Chelon haematocheilus and Eriocheir sensu stricto, older colonisation waves were also possible, which allowed the emergence of a specific East China Sea lineage in these species [Lastly, the East China Sea, which was exposed during the Pleistocene glaciations, seems to be a post-glacial contact zone for  species ,54.M. cephalus is a species complex rather than a unique panmictic unit. To date, however, this has not been proven, due to inadequate sampling schemes with samples separated by thousands of km, or the use of only maternally inherited markers [M. cephalus is indeed composed of at least three genetically divergent species in the NW Pacific. While COI inter-lineage divergence in M. cephalus exceeds intra-lineage divergence (0.4 to 0.2%) by a ratio greater than 10, which is an indicator of cryptic species according to the 10× rule [M. cephalus were significantly assigned (posterior probabilities <10% and >90%). Furthermore, the complete congruence between nuclear groups and mitochondrial lineages suggests that lineage sorting of ancestral polymorphisms has been completed. Lastly, further to the argument that the M. cephalus mitochondrial lineages are true species, the gene flow estimated with microsatellite loci appeared to be more limited among sympatric individuals belonging to different mtDNA lineages than between geographically remote individuals sharing the same mtDNA lineage. These results reveal the marked genetic isolation of the three M. cephalus species in the NW Pacific.It has previously been hypothesised that  markers ,31,55-5710× rule , the nucM. cephalus in the NW Pacific. While some individuals migrate over large geographic scales, others appear to be year-round residents of estuaries [M. cephalus juveniles in Taiwanese estuaries earlier in the year (end of autumn) was interpreted, therefore, as evidence of the existence of a resident M. cephalus population [If, as suggested earlier, the genetic differentiation of these cryptic species is related to past physical barriers to gene flow, the absence of genetic introgression suggests active reproductive isolation where they coexist sympatrically. Previous studies have reported various different reproductive behaviours in stuaries -64. Migrstuaries -61. The stuaries ,62-64. Tpulation .M. cephalus (offshore waters of Kaohsiung) in the spawning season (end of December), over three successive years, belonged to NWP1. The high GSI of these individuals suggested that they were spawning or would spawn soon. By contrast, M. cephalus collected in estuarine or coastal waters in the same period belonged to NWP1, NWP2 and NWP3, but all exhibited low GSI , it is evident that the population structure revealed using AFLP [No nuclear genetic heterogeneity was recorded within the cryptic u et al. , who docing AFLP  relied ming AFLP  describeing AFLP , a northM. cephalus species revealed in this study are able to disperse and migrate at sea, low genetic structuring is not surprising because large oceanographic current systems in the NW Pacific are expected to greatly facilitate larval dispersal over considerable distances. However, dynamic oceanographic systems can also profoundly restrict connectivity among groups, genetic heterogeneity is generally increased in marine zones with predictable oceanographic fronts [Finally, if all cryptic c fronts -67.M. cephalus species appears mainly to be shaped by three major oceanographic current systems, namely the South China, North China Coastal and Kuroshio Currents. The range of the NWP2 species appears to match the circulation pattern of the Kuroshio Current, which brings warm tropical water northeastward from the Luzon Straits, past both the west and east coasts of Taiwan and, finally, to Japan, where a branch of the current (the Tsushima Current) enters the Japanese Sea [The distributional range of the three NW Pacific nese Sea ,69. As tnese Sea , Figure nese Sea ,59,60.M. cephalus species in the NW Pacific Ocean, using both microsatellites and mitochondrial genetic markers. The genetic architecture and current distribution ranges of these species suggest a complex interaction of contemporary and evolutionary factors. Allopatric and adaptive speciation probably occurred during the Pliocene and Pleistocene epochs, when the Sea of Japan was periodically isolated and influenced by cold northern oceanographic currents. After the last glacial maxima, species range expansions were probably facilitated by changes in the oceanographic current structure and the species' temperature preferences. Currently, these species overlap in their distribution ranges, and it is likely that the genetic integrity of each is maintained by temporal and spatial isolation during the spawning period. This study emphasizes the importance of historical separations of marginal seas, in conjunction with fluctuating temperatures, in creating aquatic biodiversity in the NW Pacific. Isolation and demographic fluctuations are expected to constitute a powerful evolutionary force that increases speciation processes in the marine environment. These regional results also shed new light on the potential genetic diversity of M. cephalus worldwide.This study successfully identified three cryptic KNS carried out the molecular genetic studies, performed the statistical analysis and drafted the manuscript. BWJ and CCH participated in the collection of samples and biological data. WNT participated in the design of the study and obtained funding. JDD conceived the study, participated in its design and coordination, and helped to draft the manuscript. All authors have read and approved the final version of the manuscript.Table S1. Variable positions in the 627 bp mitochondrial COI gene segment of Mugil cephalus from 12 locations in the northwestern Pacific. Dots represent identical nucleotides relative to haplotype 1. Frequency of each haplotypes for each lineages  are also shown. Different color means the locations of the lineage specific nucleotides.Click here for fileTable S2. Genetic variability at ten microsatellite loci of Mugil cephalus collected in the northwestern Pacific for spatial and temporal genetic structure test. Table-wide significance levels were applied using the sequential Bonferroni technique [echnique .Click here for fileTable S3. Log probability and ΔK [y and ΔK  for eachy and ΔK .Click here for fileTable S4. The mean q-values and standard deviations (sd) for assignment test of 3 Mugil cephalus cryptic species  as implemented in STRUCTURE [TRUCTURE .Click here for fileTable S5. Genetic variability at ten microsatellite loci of Mugil cephalus among 3 cryptic species. Table-wide significance levels were applied using the sequential Bonferroni technique [echnique .Click here for fileFigure S1. Ethidium bromide stained 2% agarose gel showing the multiplex COI haplotype-specific PCR (MHS-PCR) for the rapid screening of three Mugil cephalus cryptic species in the NW Pacific. 1-13: unidentified individuals belonging either to lineage NWP1 (2), NWP2  or NWP3 . M: 100-bp DNA ladder.Click here for fileFigure S2. Phylogenetic relationships within Mugil cephalus recovered from 1140 bp of the cytochrome b sequences according to the neighbour-joining tree using Kimura 2 parameter distance. Leaves of the tree correspond to haplotypes of M. cephalus observed by Ke et al. [e et al.  (H1-H43)Click here for file


Euperipatoides rowelli in southeastern Australian montane forests that were not subject to Pleistocene glaciations, and thus likely retained more forest cover than systems under glaciation. Over a ~100 km transect of structurally-connected forest, we found marked nuclear and mitochondrial (mt) DNA genetic structuring, with spatially-localised groups. Patterns from mtDNA and nuclear data broadly corresponded with previously defined geographic regions, consistent with repeated isolation in refuges during Pleistocene climatic cycling. Nevertheless, some E. rowelli genetic contact zones were displaced relative to hypothesized influential landscape structures, implying more recent processes overlying impacts of past environmental history. Major impacts at different timescales were seen in the phylogenetic relationships among mtDNA sequences, which matched geographic relationships and nuclear data only at recent timescales, indicating historical gene flow and/or incomplete lineage sorting. Five major E. rowelli phylogeographic groups were identified, showing substantial but incomplete reproductive isolation despite continuous habitat. Regional distinctiveness, in the face of lineages abutting within forest habitat, could indicate pre- and/or postzygotic gene flow limitation. A potentially functional phenotypic character, colour pattern variation, reflected the geographic patterns in the molecular data. Spatial-genetic patterns broadly match those in previously-studied, co-occurring low-mobility organisms, despite a variety of life histories. We suggest that for E. rowelli, the complex topography and history of the region has led to interplay among limited dispersal ability, historical responses to environmental change, local adaptation, and some resistance to free admixture at geographic secondary contact, leading to strong genetic structuring at fine spatial scale.Phylogeographic studies provide a framework for understanding the importance of intrinsic versus extrinsic factors in shaping patterns of biodiversity through identifying past and present microevolutionary processes that contributed to lineage divergence. Here we investigate population structure and diversity of the Onychophoran (velvet worm)  Morphologically cryptic diversity is important in conservation biology because failure to recognise the presence of distinct genetic groups can result in the loss of unique lineages not considered by management actions . HoweverAn appropriate landscape setting for investigating factors that generate biodiversity is the Tallaganda comparative phylogeographic study system described in Garrick et al.  and subsThe amplitude of late Pleistocene glacial cycling in the Southern hemisphere was less pronounced than that in the northern hemisphere . Thus, rEuperipatoides rowelli is a member of the phylum Onychophora, a group of soft-bodied terrestrial invertebrates accepted as the sister clade to the Arthropoda [Euperipatoides rowelli is distributed over much of southern New South Wales, including Tallaganda, although morphological, chromosomal and genetic data indicate this range almost certainly includes multiple cryptic taxa [E. rowelli is saproxylic and a predator of small invertebrates. Behavioural studies indicate they have complex social organization with a female dominated hierarchy [The locally abundant thropoda ,20. Eupetic taxa ,21,22. Wtic taxa , and mortic taxa . Like moierarchy . Desiccaierarchy ,27. Euperipatoides rowelli is an important addition to the set of taxa studied at Tallaganda for at least two reasons. First, unlike the other invertebrate taxa studied in this system, there are detailed accounts of its life history, behaviour and dispersal [E. rowelli has being used as a model for evolutionary development, and will be the first Onychophora to have its full genome sequenced (http://www.arthropodgenomes.org/wiki/i5K) [ispersal ,25,28,29iki/i5K) ,31. Secoiki/i5K) . Since lE. rowelli to environmental and paleoclimatic factors affecting forest distributions at Tallaganda throughout the Pleistocene and earlier. We test the hypotheses that (1) major genetic groups will be present within E. rowelli at the catchment-scale (i.e. associated with watersheds) and (2) they will broadly correspond to microgeographic regions of the Garrick et al. [Onychophoran species diversity is likely to have been substantially underestimated ,22,32,33k et al.  model.Euperipatoides rowelli individuals were collected by hand from decaying logs throughout Tallaganda between 1989 and 2009, with overlapping sets of individuals used for microsatellite, mtDNA and colour pattern analyses. Samples were collected on public land, under permits issued to NN Tait and named associates, renewed annually, covering all years of collection, by Forest New South Wales State Forests (XX51212 covering Southern and Monaro Regions) and New South Wales National Parks . For each log, latitude and longitude were recorded, and it was classified into one of the microgeographic regions of Garrick et al. [k et al. . SamplinIndividuals from single logs were collected into containers with rotting wood to maintain humidity. Subsequently, individuals were weighed to the nearest milligram, and sex was determined according to presence or absence of male-specific crural papillae: individuals >25 mg without crural papillae were classified females based on dissection data , and indDNA was extracted from ~40 mg tissue per individual using a salting-out  or modifSequence variation in a 448 bp portion of the mitochondrial cytochrome c oxidase (COI) gene was assayed for 601 individuals from 57 sites  using a combination of Single Stranded Conformational Polymorphism (SSCP) assays  followinE. rowelli is under genetic control . Three colour pattern traits were scored for 1030 adults from 92 sites  and their geographic distributions subsequently compared with genetic data and the microgeographic-region model for Tallaganda [Common-garden culture experiments indicate that body colouration in llaganda . Traits a priori microgeographic-region model.Statistical analyses were performed to determine the presence of geographic structure in the microsatellite, mtDNA and colour pattern data separately, and to determine if these patterns were concordant among the three data types, and with the 6 and 3x106 MCMC generations respectively per run, with 20 replicate runs for each K-value. The admixture model and correlated allele frequencies were used with other settings left as default; location priors were not used. The ΔK method [To ensure microsatellite data were appropriate for analyses, linkage disequilibrium and departures from Hardy-Weinberg equilibrium were assessed using GENEPOP v4.0  with sitK method  was usedK method .ST - RST tests between groups were performed in SPAGeDi v1.3 [As the ΔK method detects the highest level of structure in hierarchically structured populations , each greDi v1.3 . Euperipatoides leuckartii (GenBank accessions: EU855390.1 and U62426.1), Ruhbergia bifalcata (AF337996.1) and Ooperipatus sp. (AF337994.1). Where nuclear microsatellite data were available for E. rowelli individuals with mtDNA sequence data, STRUCTURE clusters were overlaid on the phylogenetic tree, as was the presence of each haplotype in five major geographic regions delimited by common mtDNA haplotypes, which corresponded roughly to the a prior regions (Results). Tajima’s D [To estimate relationships among mtDNA haplotypes, a phylogenetic tree was inferred using MrBayes  under thjima’s D , Fu’s Fsjima’s D , ratio ojima’s D .To determine how the three colour-traits combined into multi-trait phenotypes, a log-linear model  was usedTwo approaches were used to determine correspondence of patterns among data types and to microgeographic region boundaries. First, Fisher’s exact tests  for two-Second, boundary overlap analysis  was usedStep 1: Boundary detection. We used categorical wombling [Step 2: Testing for directional overlap. The statistic O1 (mean geographic distance of BEs in Boundary 1 to the nearest BE in Boundary 2) was used to test the hypothesis of no association between / among boundaries. To generate null distributions of O1, 10,000 Monte Carlo randomizations were performed under complete spatial randomness permutation in BOUNDARYSEER v 1.3.14 (Biomedware). Because catchment boundaries are independent variables that may influence the location of contact zones, only the latter were randomized. We assessed significance of observed test-statistics relative to the lower tail at the 0.05 level.Boundary overlap analysis involves two steps: identification of geographic zones of rapid change in the value of one or multiple variables relative to neighbouring sites, and evaluation of whether boundaries coincide to a significant extent ,70. Stepwombling : each siwombling . Using a All individuals were successfully genotyped for all loci. Between three and 13 alleles were detected at microsatellite loci  diverged over timescales sufficient for microsatellite evolution (both P = 0.02). Conversely, divergence between groups 2 and 3 was explicable by recent genetic drift (P = 0.42). Nearly all pairwise pRST – RST tests were non-significant at lower hierarchical levels, with none significant after correcting for multiple tests, also indicating a drift timescale  of these occurred in very few individuals, but seven among common haplotypes. These common E. rowelli protein variants may not be strongly constrained by function: six residues  also vary among insects [MtDNA diversity was substantial; the 601 individuals yielded 66 unique haplotypes, with 154 variable sites of which 116 were parsimony-informative. Synonymous differences were far more common than non-synonymous (172 to 36), with 26 first-position, 15 second-position, and 113 third-position sites variable. Tajima’s D was not significantly different from zero when analysed across all sequences , nor was Fu’s Fs , providing no evidence of strong nucleotide selection or population growth. Haplotype frequencies ranged from 0.17 % to 26.47 %, with eight being common , and differed by 0.22 % to 15.40 % uncorrected  insects , and the insects .The inferred mtDNA phylogeny had strong structure: six distinct clades had Bayesian posterior probability values > 0.75, including a basal clade on a very long branch . For eaca priori microgeographic regions each had a single very common haplotype along with up to three other common haplotypes  was more closely related to southernmost sequences (AR-PSR-BR) than to neighbouring common haplotype 1 (ESR) . Rare ha228 = 0.94, P = 0.33 for three-way interaction, G2>29 > 239.17, P < 0.001 for all two-way interactions). Nearly all (> 97 %) individuals had one of eight common phenotypes, with the remainder being spread over 11 rare phenotypes. Colour pattern phenotype was much less spatially structured than were nuclear groups or mtDNA haplotypes: a given area was less strongly characterised by a single phenotype and the transitions between areas, especially in the northern half of the study area, were less sharp than for genetic markers  . When exE. rowelli. Major mtDNA clades , common mtDNA haplotypes (reflecting shorter-term processes of population differentiation dominated by genetic drift), and colour pattern phenotype-based contact zones all showed significant directional overlap with CB-2 , which runs east-west along the Ballalaba (ESR-Ba) and Jerrabatgulla (ESR-Je) watershed and bisects ESR, was the strongest predictor of contact zone boundaries seen in Euperipatoides rowelli at Tallaganda. Nuclear, mitochondrial and phenotypic data identified distinct lineages distributed generally within catchments or subcatchments despite continuous habitat.Strong, locally endemic genetic structure was seen for E. rowelli and other low-mobility inhabitants of Tallaganda [Planipapillus that radiated in the alpine region of Australia has less mtDNA COI sequence divergence than seen in E. rowelli in a single log [These patterns of biodiversity are markedly different to those seen elsewhere in other members of the Gondwanan Onychophoran family Peripatopsidae ,34,39. Illaganda . Few arengle log .E. rowelli has expanded into regions likely to have been treeless steppe during glacial cycling, presumably from nearby refugia.At Tallaganda, the broad association between the spatial distribution of genetic groups and catchment boundaries indicates a strong role for watersheds and associated environments in producing and / or maintain genetic structuring. However, this is apparently not the only causative factor, because some genetic breaks are spatially displaced from their putative geographic drivers. Genetic breaks that are spatially displaced from geographic features involved in population subdivision suggest a predominantly historical rather than contemporary role for those environmental elements in separating genetic groups ,73. A hiE. rowelli presently occupying the rest of Tallaganda (group 1). Only a single major Pleistocene refuge was predicted at Tallaganda by Garrick et al. [E. rowelli [E. rowelli lineages in AR-PSR-BR. This implies that the conditions for survival of viable populations under glacial contraction were less stringent than originally imagined in Garrick et al. [The spatial locations of refugia where biodiversity may be generated or maintained through environmental change are important to consider for understanding and managing biodiversity ,76. The k et al. , hypothek et al. , and twok et al. , which a rowelli . In addik et al. . More dek et al. ,78.The hierarchical nature of the genetic structuring in the present data and the large number of groups implies a complicated evolutionary history . Multi-sE. rowelli revealed that there was little geographic structure in deep nodes but much more at the tips. This contrasts with patterns in other organisms at Tallaganda, whose clades are mostly monophyletic among catchments [Euperipatoides rowelli may experience incomplete lineage sorting of mtDNA owing to large effective population sizes. Although haplotypes should sort up to four times faster than nuclear markers in diploid species with equal operational sex ratios [E. rowelli mtDNA effective population size is likely to be inflated by female-biased demographics, low variance in female reproductive success, and high female philopatry, given rare patch extinction [The mtDNA phylogeny of tchments ,13,16. Ttchments , and onetchments . Three ptchments , incompltchments , and limx ratios , E. rowetinction ,28,29,83tinction  found thE. rowelli at Tallaganda are likely maintained and enhanced by low contemporary gene flow. Onychophoran biology renders them extremely susceptible to desiccation outside logs [E. rowelli [E. rowelli from different catchments at Tallaganda: ESR-AR hybrids have variation in leg number spanning 13-16 pairs and lateral asymmetries, whereas 15 pairs of legs is otherwise highly conserved in Australian Onychophora. This implies genomic incompatibilities between E. rowelli of neighbouring Tallaganda regions sufficient to affect fundamental body plans [E. rowelli are subject to strong barriers to gene flow zones of contact: the two northernmost forms (HCR and ESR) appear to experience few adverse effects where they interbreed [The patterns of genetic structuring produced during the proposed complex history of ide logs ,27, so dide logs ,25,28. G rowelli ,85,86. F rowelli . Evidencdy plans ,24, consterbreed .a priori regions. The exception is that AR and PSR phenotypes are both ‘black’ with or without white spots yet the nuclear data indicate that black PSR and brightly spotted BR are more closely related to each other than either is to black AR. This implies that colour pattern phenotypes have been evolutionarily labile on less than the timescale taken to achieve drift-based differentiation of microsatellite loci. Scenarios include that the black pattern evolved independently in AR and PSR, that it swept faster than neutral markers from one form to the other, or that BR has secondarily acquired brightly-coloured spots. Any of these patterns might indicate a function for colouration [E. rowelli colour pattern forms seem very minor in regards to Crypsis; the species occupies the largely dark environment of rotting logs, and has poor vision including in the colour range of the characters in question [With one exception, colour pattern phenotype data were consistent with nuclear and mtDNA in identifying groups corresponding to the five ouration , althougquestion ,91.E. rowelli, as with most other organisms studied at Tallaganda [Very fine-scale endemism was observed within llaganda . The excllaganda ,17. Overllaganda ,92.File S1STRUCTURE microsatellite data file used, including location data (UTM grid 55H).(TXT)Click here for additional data file.File S2STRUCTURE main parameter file for analysis of (TXT)Click here for additional data file.Table S1Sampling locations for each data type.(DOCX)Click here for additional data file.Table S2Distribution of COI haplotypes by sampling location.(XLS)Click here for additional data file.Table S3Distribution of phenotypes by sampling location.(XLS)Click here for additional data file.Table S4Uncorrected pairwise differences between haplotypes.(XLS)Click here for additional data file.Table S5Microsatellite allele frequencies in nuclear genetic groups identified by STRUCTURE.(XLS)Click here for additional data file.