Phylidonyris novaehollandiae), a nectarivorous passerine that is a key pollinator species. We predicted that rainfall-related mechanisms would be driving local adaptation of morphological traits, such that in areas of lower rainfall, where nectar is less available, more insectivorous traits – shorter, deeper bills, longer tarsi, and longer wings – would be favored. The study populations differed in phenotype across the Eyre, Yorke, and Fleurieu Peninsulas and Kangaroo Island despite high gene flow (single continuous population) and sex-biased dispersal . We tested the role of rainfall in shaping the observed phenotypic differences, and found strong support for our predicted relationships: birds in areas of higher rainfall had higher condition indices, as well as longer bill-head length, deeper bills, and shorter tarsi. Bill depth in males in high-rainfall sites showed signals of stabilizing selection, suggesting local adaptation. In addition to these local indications of selection, a global pattern of directional selection toward larger size for bill-head length, bill-nostril length, and wing length was also observed. We suggest this pattern may reflect an adaptive response to the relatively dry conditions that South Australia has experienced over the last decade. We conclude that rainfall has shaped aspects of phenology in P. novaehollandiae, both locally, with different patterns of stabilizing and directional selection, and globally, with evidence of adaptive divergence at a landscape scale.Identifying environmentally driven changes in traits that serve an ecological function is essential for predicting evolutionary outcomes of climate change. We examined population genetic structure, sex-specific dispersal patterns, and morphology in relation to rainfall patterns across an island and three peninsulas in South Australia. The study system was the New Holland Honeyeater ( Phylidonyris novaehollandiae), across an environmentally variable landscape in southern South Australia.Climate change has a worldwide impact upon biodiversity and is considered responsible for rapid changes in the phenology and distribution of many plant and animal species  show that long distance dispersal is not common, with 99.1% of birds moving <10 km from location of banding , allowing for comparison between sites experiencing high- and low-rainfall conditions. The climate system in South Australia is sometimes referred to as having unpredictable “boom and bust” conditions, namely high- or low-rainfall periods, respectively  test the role of genetic drift on morphological variation; and (5) test for signals of selection on morphological traits, using body condition as a proxy for fitness.In this study, we have two main aims: (1) to examine population genetic structure and sex-specific dispersal, and (2) to examine variation in morphological foraging traits in relation to rainfall. For the study of morphology in relation to rainfall, we (3) examine morphological variation in P. novaehollandiae at six sites in southern South Australia. Each site represented one of three rainfall categories: low (<32 mm), moderate (32–44 mm), and high (>44 mm)  and Yorrell  are dominant tree species found throughout thickly vegetated areas; E. diversifolia is often accompanied by understorey species including Wallowa , Coastal Velvet-bush , Felted Wallaby Bush (Beyeria lechnaultii), Dwarf Hop Bush (Dodonaea humilis), and Shiny Ground Berry (Acrotriche patula).We studied (>44 mm)  mean mon(>44 mm) c. Vegeta(>44 mm) , Ford an(>44 mm) , 1982, W(>44 mm) , Kleindo(>44 mm) , Schlotf(>44 mm) , Galliga(>44 mm) , Oorebee(>44 mm) , Myers e(>44 mm) . VegetatP. novaehollandiae are (is) predicted to be most influenced by rainfall: 2 months toward the end of the dry season , when nectar is expected to be least abundant, and 2 months during the wet season , when nectar is expected to be most abundant. Rainfall data were obtained from the Australian Bureau of Meteorology (http://www.bom.gov.au/index.shtml) using the nearest meteorological station to each site : “Low Rainfall 1” : Warooka meteorological station; “Low Rainfall 2” (Port Lincoln): North Shields meteorological station; “Moderate Rainfall 1” (Pelican Lagoon Conservation Park): Cape Willoughby meteorological station; “Moderate Rainfall 2” (Sandy Creek Conservation Park): Rosedale meteorological station; “High Rainfall 1” (Cox Scrub/Scott Conservation Parks): Kuitpo Forest Reserve meteorological station; and “High Rainfall 2” (Newland Head Conservation Park): Parawa meteorological station.For each site, we calculated mean monthly rainfall from across the study period using data from 4 months when survival and/or reproductive success in P. novaehollandiae should be most responsive to rainfall stimuli. Each bird was banded with an aluminum identification band, measured for morphological characteristics, and sampled for blood that was stored on FTA paper for DNA analysis  were sexed using the molecular genetic method of Kahn et al. (N = 71) had sex assigned using a univariate morphology-based sexing method  and SM (N = 117), who measured birds in all six study sites. Variation in measurement between researchers for all traits was not significant , and was less than the variation across sites. A second test of measurement error tested morphological variation across sites for each researcher separately, which mirrored the findings for both researchers combined, indicating a low degree of measurement error.At the time of banding, we measured seven morphological traits: (1) bill length from the tip of the bill to the back of the head (bill-head length); (2) bill length from the tip of the bill to the anterior extreme of the nostril (bill-nostril length); (3) bill depth measured at the base of the bill (bill depth), (4) bill width measured at the base of the bill (bill width); (5) length of the flattened wing (wing length); (6) tarsometatarsal length (tarsus length); and (7) body mass (mass). All traits, excluding mass, were measured to the nearest tenth of a millimeter using callipers. Mass was measured to the nearest tenth of a gram using scientific scales. All measurements were made by SK . No significant interaction effect was observed between sex and either study site or year, indicating that morphological variation between sexes did not change across study sites or years. Examination of trait means for each sex indicated that males had larger measurements for all traits  with sex as a fixed factor, as well as study site and year, showed that morphological variation differed significantly between the sexes , calculated as the standardized residuals of an ordinary least-squares linear regression of mass against tarsus length , expected and observed heterozygosities , and the inbreeding co-efficient (FIS) were calculated for each locus by site and globally for each locus using genepop v4 , although 12 of these pairs were found to depart from linkage disequilibrium at only one site. We followed recommendations by Kaeuffer et al. . The rLD for each locus pair was <0.55 (P < 0.05), indicating a probable distance of greater than 3 cM between loci . We investigated the effect of these loci on our analyses by comparing results obtained with and without them. Results were consistent in all cases and we concluded that the observed departures from Hardy–Weinberg equilibrium for these loci would most likely not be strong enough to significantly bias results. Therefore, keeping these violations in mind, we retained all loci for further analyses, assuming that the extra information in these loci outweighs any potential biases they may add.We carried out tests of linkage disequilibrium for each locus by site using ion Rice , signifir et al.  and estiion Rice , two locP. novaehollandiae (McFarland In birds with a resource defense mating system (Greenwood FST). The vAIc test is ineffective at detecting biased dispersal when dispersal frequencies are high, but performs better than the FST test when dispersal frequencies are low. These tests are one-sided, based on the principle that genotypes of the more philopatric sex will be more similar (relative to the dispersing sex) in the population in which they were sampled. We tested the hypothesis that, in P. novaehollandiae, males are the more philopatric sex.Goudet et al.  recommenP. novaehollandiae indicates a limited capacity for dispersal (c.a. 110 km max.), shorter than the distances between some of our study sites  on log of distance is expected (Rousset FST/(1–FST) for each sex. We calculated pairwise distance measures as the shortest distance across land between two points (km), assuming dispersal between Kangaroo Island and mainland South Australia only across the smallest sea divide that separates the two (between the eastern tip of Kangaroo Island and the southern tip of Fleurieu Peninsula). Log of distance was subsequently computed from these distances. We evaluated the correlation between log of distance and pairwise FST/(1−FST) for each sex using 1 × 107 randomizations. We accounted for non-independence of distance correlations inherent with matrix data by using Mantel tests  computation. The number of clusters, K, is a fixed parameter set by the user – the model probabilistically assigns individuals to a cluster in a way that minimizes departure from Hardy–Weinberg equilibrium at each locus while conforming to the set value of K. The model assumes that loci within clusters are in Hardy–Weinberg equilibrium and linkage equilibrium. The procedure to find which value of K best fits the data consists of running multiple MCMC replicates for varying K and inferring the most likely value from the approximation of their posterior probabilities. Based on mark-recapture data of P. novaehollandiae  for all further runs as they make the algorithm more sensitive to subtle structure  and MCMC length (1 × 105 iterations), we ran 10 MCMC replicates for K = 1−8. Although there is some debate about the best method for inferring clusters   see . In male.05) see ; in fema.05) see . We used.05) see . Birds h2 = 0.30, P < 0.001) and females : birds in areas with higher rainfall had higher mean condition index values , there were 192 (5.8%) missing values. The number of alleles per locus ranged from 6 to 18 (mean 13.5), expected heterozygosity ranged from 0.67 to 0.91 (mean 0.83), and allelic richness ranged from 4.45 to 14.83 (mean 10.15).P = 0.081), but male FST was significantly lower than female FST within sites (P = 0.038). Comparing our results with those of exhaustive sampling by Goudet et al. , then the dispersal rate in males should be no more than 53%.We found evidence for male-biased philopatry: male vAIc was not significantly greater than female vAIc within sites  and log of pairwise distance showed significant positive correlations in both males  and females , with a substantial proportion (>75%) of the neutral genetic variation between sites being explained by distance between sites.Mantel tests examining the correlation between pairwise K were maximal for K = 1 under the standard admixture model. As population differentiation appears to be low for these data, we also implemented the locprior model – a structure model that incorporates site information in the inference by using a modified prior distribution for clustering that allows the distribution of cluster assignments to vary by site  to our data, allowing for correlated allele frequencies, we investigated convergence and mixing properties of chains, and sensitivity of the data to alternative hyper-parameter priors, as previously described in the Methods for the standard model. All chains converged with mixing within 2 × 104 MCMC iterations and results were consistent between runs; therefore, we chose a relatively conservative burn-in of 5 × 104 MCMC iterations, which we fixed for all further runs, and we reverted to the default priors for all runs . Exploration of the data for consistency between replicate runs indicated a chain length of 1 × 105 MCMC iterations was most appropriate. Using our optimized burn-in length (5 × 104 iterations) and MCMC length (1 × 105 iterations), we ran 10 MCMC replicates for K = 1–8. Averaged over the 10 MCMC replicates for each value of K, the logarithm of probability of the data was maximal for K = 3, different from the estimate of K = 1 made using the standard model. The three clusters generally represented spatial groups, with individuals from sites within close proximity clustering together; most individuals from “Low Rainfall 2” clustered together, as did most individuals from “Low Rainfall 1,” while the remaining individuals formed a third cluster. Two facts support the findings of the locprior model over those of the standard model: (1) the clusters make biological sense by conforming to patterns of geographic isolation; and (2) cluster membership indicates shared ancestry between clusters, expected under high gene flow. Therefore, population estimates based on the locprior model should most accurately represent the true populations. We believe, the most likely reason for the standard structure model failing to detect structure was the strong signal of isolation by distance, which can swamp clustering estimates , expected and observed heterozygosities , and the inbreeding co-efficient (FIS) were calculated for each locus by cluster using genepop v4. We carried out tests of linkage disequilibrium for each locus by cluster; after Bonferroni correction, significant departure from linkage disequilibrium was detected for one locus pair in the largest cluster (P < 0.01). The estimated correlation co-efficient (rLD) for this locus pair, calculated using Linkdos, indicated that the loci were most likely separated by a distance of greater than 3 cM . We tested for Hardy–Weinberg equilibrium within each cluster; after Bonferroni correction, two loci, Pn4 and Pn5, differed significantly from Hardy–Weinberg equilibrium in the largest cluster, both showing heterozygote deficiency (P < 0.01).We carried out standard molecular genetic analysis for microsatellite loci within the three clusters identified by FST among sites ranged from 0.002 to 0.047. Eight of 15 pairwise comparisons were significantly different from 0 (FST among sites ranged from 0.000 to 0.032. Two pairwise comparisons showed negative FST (effectively 0) and one showed an FST of 0. Six pairwise comparisons were significantly different from 0  exceeded neutral genetic variation (FST) for all tested morphological variables in both males and females.In males, t from 0 . In femat from 0 . A geogrP. novaehollandiae, across one island and three peninsulas in South Australia. There was sex-biased dispersal: males were the philopatric sex and females, the dispersing sex. Finally, despite high gene flow, we found phenotypic differences between sites, which correlated with rainfall as predicted. Birds in low-rainfall areas had shorter bill-head length, deeper bills, longer tarsi, and lower body condition. Despite these relationships, a signal of stabilizing selection was detected only in bill depth, and only in males in high-rainfall sites.This study found a single continuous genetic population structure at a landscape scale in a key pollinator species, r ≥ 0.75), which we expected with the relative absence of physical barriers (after accounting for the sea divides) coupled with the limited dispersal capacity of P. novaehollandiae across the study area (Higgins and Peter FST) is consistent with the observed pattern of IBD. Under IBD, allele frequencies are expected to vary gradually with distance, and genotypes from the most remote sites are expected to be most differentiated. Accordingly, we found that pairwise comparisons including either “Low Rainfall 1” or “Low Rainfall 2,” our two most remote sites, were the only sites with significant FST, which is also reflected in the number and composition of clusters found by structure using the more powerful locprior model. The contrasting finding from the standard structure model and the locprior model probably reflects insensitivity of structure to an underlying population model of IBD with inadequate spatial sampling of populations in a “stepping stone” distribution  observed in both sexes was particularly strong  were lower than the original estimates , as expected. Subsequently, the large values that we observed for PST for all morphological variables, relative to FST in males and females, indicate a negligible influence of genetic drift in the divergence of morphological traits of P. novaehollandiae in our study area . Under drier conditions, less nectar is expected to drive a subsequent shift toward insectivory in P. novaehollandiae. This shift in foraging is predicted to be followed by adaptive morphological change toward shorter but deeper bills, and longer wings. While there appears to be directional selection in favor of longer winged birds in our study area, longer billed birds also appear to be favored. While a shorter, deeper bill provides increased crushing force and access to a greater diversity of prey . The mode and direction of selection in each trait for males and females were similar. With the exception of bill depth and width, selection was always directional toward larger size. Considering the broad scale of this pattern, it is unlikely that it is predominantly explained by local conditions, but rather is shaped by a large-scale mechanism, such as long-term global climate change. During the study period, the climate in South Australia was relatively dry (y Bowman , a longey Bowman  such as ae Paton . ContrarP. novaehollandiae in South Australia, both locally and globally, with a consistent pattern of adaptive divergence at a landscape scale. The conclusions of this study hinge largely upon the assumption that nectar availability is directly correlated with rainfall. Although we provided support for this assumption from the literature (Porter P. novaehollandiae diet, would test some of the arguments presented in this study. Despite these shortcomings, this study provides important baseline data about a key pollinator system in South Australia. For example, this is the first study to show high gene flow at a landscape scale in P. novaehollandiae, and to provide genetic evidence for sex-biased dispersal in the focal species. In addition, we have provided some evidence for the adaptive capacity of this key pollinator species. Notwithstanding this indirect evidence for adaptive shifts in phenotype, P. novaehollandiae is declining in parts of South Australia (Szabo et al. On the basis of combined evidence of our data, we conclude that rainfall has shaped aspects of phenology in e Porter , we did


Dendroctonus ponderosae Hopkins), an irruptive bark beetle that has caused unprecedented damage to lodgepole pine forests in western North America and is poised to expand its range across the boreal forest. We sampled MPB populations across British Columbia and Alberta and used phylogeographic methods to describe lineage diversification, characterize population structure, investigate expansion dynamics, and identify source populations of the outbreak. Using 1181 bp of mitochondrial DNA sequence from 267 individuals, we found high haplotype diversity, low nucleotide diversity, and limited lineage diversification. The overall pattern was consistent with isolation by distance at a continental scale, and with reduced diversity and population structure in the northerly, outbreak regions. Post-Pleistocene expansion was detected, however more recent expansion signals were not detected, potentially due to the size and rapid rate of range expansion. Based on the limited genetic structure, there were likely multiple source populations in southern British Columbia, although the magnitude of the demographic expansion and rate of spread have obscured the signature of these source populations. Our data highlight the need for caution in interpreting phylogeographic results for species with similar demographics.Irruptive forest insect pests cause considerable ecological and economic damage, and their outbreaks have been increasing in frequency and severity. We use a phylogeographic approach to understand the location and progression of an outbreak by the MPB ( Ceratitis capitata) was Latin America. As well, Stenotus rubrovittatus) in Japan, necessitating monitoring of nonoutbreak areas to prevent additional crop damage. Here, we use a phylogeographic approach to illuminate the outbreak dynamics of mountain pine beetle  and cytochrome c oxidase II (COII) genes as well as the complete tRNA-LEU using two sets of primers (C1-J-1718 (F): GGA GGA TTT GGA AAT TGA TTA GTT CC, C1-N-2611 (R): GCA AAA ACT GCA CCT ATT GA and C1-J-2193 (F): CCA GGG TTT GGT ATA ATT TCT T, C2-N-3174 (R): TTA GAG GGG AAG ACC TAT CTT GT; 2, 0.2 mM each dNTP, 0.25 µM each primer, and 1U Taq DNA polymerase (NEB). Thermocycling conditions consisted of an initial denaturation at 98°C for 30 sec, followed by 35 cycles of 95°C for 10 sec, 45°C for 30 sec, and 72°C for 60 sec, and a final extension at 72.0°C for 5 min. For primer pair C1-J-2193 and C2-N-3174, 15 ng DNA was used and the annealing temperature was 62°C. Primers and unincorporated dNTPs were removed using ExoSAP-IT  following manufacturer's instructions prior to sequencing. Purified PCR products were sequenced bidirectionally with Applied Biosystems Big Dye Terminator v 3.1 cycle sequencing kit  using an Applied Biosystems 3730 DNA Analyzer. Sequence data were analyzed and assembled with Seqman . Final assembled sequences were checked manually.Partial mtDNA sequence was generated from portions of cytochrome http://www.atgc-montpellier.fr/phyml/; D. jeffreyi Hopkins, sister species to D. ponderosae  and nucleotide (π) diversity for each subpopulation, and regressed these on latitude and longitude, where we expected diversity to decrease toward the north and to the east as MPB has recently expanded into central Alberta. All above calculations were performed in Arlequin 3.5.1.2 . We aligned our sequences with those from R2= 0.39, t32=–3.41 × 10–3, P= 7.47 × 10–5), but haplotype diversity was unrelated to either latitude or longitude . There was a greater than 10-fold difference in nucleotide diversity between the southern and northern beetle populations . The signal of expansion decreased to the north with no evidence of expansion in the south. However, when we analyzed the population groups based on their histories, we found a significant signal of expansion only for the central  and no significant signal for the southern  or northern groups . Genetic differentiation was highly correlated with geographic distance for the combined data  and low nucleotide diversity (0.001–0.011) . Nucleotulations . There wxpansion . Only twOur assessment of mitochondrial diversity in MPB across British Columbia and Alberta indicates poorly resolved phylogenetic structure with limited genetic differentiation. Across the sampled range, we observed high haplotype diversity, but found over a 10-fold difference in nucleotide diversity between the southern and central/northern populations, resulting in a significant correlation with latitude. When combined with the data from Limited phylogenetic structure of MPB is not surprising given their population demographics. MPB development is completed most often in one year and under certain conditions in two years  and foll2 bark  relative to all of our datapoints including its nearest neighbors . This low diversity is a signature of a very recently founded population (Grosmania clavigera, a fungal associate of MPB, and found two distinct lineages in the northern Rocky Mountains. However, more recent studies did not find any lineage diversification in a comprehensive treatment of the region (The magnitude of the recent outbreak, in terms of both the rapid population size increase and the geographic spread, has the potential to swamp phylogeographic signal. For example, pulation , and in e region , suggestThis study provides information on MPB dynamics, but we have also highlighted potential drawbacks in using phylogeographic analyses for highly dispersive species with large fluctuations in population size. This is clearly an avenue that deserves more detailed attention through model-based phylogeographic inference  as the u


Data on spatial genetic patterns may provide information about the ecological and behavioural mechanisms underlying population structure. Indeed, social organization and dispersal patterns of species may be reflected by the pattern of genetic structure within a population.Capreolus capreolus) population in Trois-Fontaines (France) using 12 microsatellite loci. The roe deer is weakly polygynous and highly sedentary, and can form matrilineal clans. We show that relatedness among individuals was negatively correlated with geographic distance, indicating that spatially proximate individuals are also genetically close. More unusually for a large mammalian herbivore, the link between relatedness and distance did not differ between the sexes, which is consistent with the lack of sex-biased dispersal and the weakly polygynous mating system of roe deer.We investigated the fine-scale spatial genetic structure of a roe deer (Our results contrast with previous reports on highly polygynous species with male-biased dispersal, such as red deer, where local genetic structure was detected in females only. This divergence between species highlights the importance of socio-spatial organization in determining local genetic structure of vertebrate populations. The fine-scale genetic structure of populations, i.e. the non-random spatial distribution of genetic variation at a local scale, is strongly influenced by species-specific social structure, dispersal patterns and mating system , living alone or in small family groups, deviates from this general pattern, exhibiting a low level of polygyny To date, most studies of fine-scale genetic structure have focused on polygynous and social species displaying male-biased natal dispersal  the social structure of roe deer results in a positive correlation between relatedness and spatial proximity at a fine spatial scale, as has been observed in previously studies of ungulates and (2) the lack of a sex-bias in natal dispersal and the low level of polygyny in roe deer results in similar patterns of spatial genetic structure for the two sexes.We sampled 643 roe deer from an enclosed population located at Trois-Fontaines, a forest of 1360 ha in North-Eastern France  that has been intensively monitored by Capture-Mark-Recapture (CMR) since 1976  to avoid the sampling of doe-fawn pairs prior to weaning and potential dispersal. The individuals were captured between 2002 and 2008 using drive netting and we recorded the geographic coordinates of the forest plot (mean plot size of 8 ha) where each individual was caught.Bos taurus, OarFCB304 in Ovis aries and NVHRT48 in Rangifer tarandusenescan and Genotyper software (Applied Biosystems) (see Genomic DNA was extracted from ear tissue using a DNeasy Tissue Kit (Qiagen). DNA was then amplified by polymerase chain reaction and genotyped with a multiplex panel of 12 microsatellites icro-Checker v.4.0.7 enepopWe first used Ma priori defined distance classes; for each class, random permutations of spatial locations of individuals (10000 permutations) were then used to assess deviations of the relationship coefficient from 0. Deviation from 0 means that individuals within a given distance class are significantly more  or less  related than at random. These analyses were performed with SPAGeDi v.1.2 r from Li et al.To assess the spatial genetic structure of male and female adult roe deer, we performed spatial autocorrelation analyses of their genotypes: we estimated a relationship coefficient among pairs of individuals belonging to the same sex and to the same icro-Checker. A total of 585 genotypic profiles were analyzed, of which 272 were females and 313 males. The mean number of alleles per locus was 7, ranging from 2 to 20.No genotyping error was detected by MIS was 0.023 and heterozygote deficiency per locus was not statistically significant after FDR-control, but global heterozygote deficiency  occurred (p = 0.0005). There was linkage disequilibrium between some pairs of loci after FDR-control (25 out of 66 – results not shown). The loci OarFCB304 (9 pairs out of the 25 significant pairs) and BM757 (6 pairs out of 25) seemed to occur the most frequently in linkage disequilibrium of loci pairs.The values of heterozygosity expected under Hardy-Weinberg equilibrium (corrected for sampling bias) and observed heterozygosity for all loci were respectively 0.675 and 0.659. Global Fr) between pairs of females and pairs of males were −0.0005±0.002 and −0.009±0.005 (mean±SE), respectively. The maximum relatedness coefficient within a distance class was 0.044±0.004 for females (between 320–480 m) and 0.069±0.009 for males (between 160–320 m). Positive r-values  occurred in the autocorrelation analysis up to 480 m for both females , the tests were certainly powerful enough to detect even a low level differentiation in the population.The population genetic analyses revealed significant heterozygote deficiency and linkage disequilibrium. These results differ from those previously reported from the same population, but including fawns only et al.Ovis aries), mostly driven by female philopatry. Similarly to roe deer, the wild boar (Sus scrofa) displays a low level of polygyny, with female philopatry and the formation of matrilines (clans), but male-biased dispersal. The patterns of spatial autocorrelation in wild boar were also observed to be different in the two sexes  In line with most studies on large mammals, but in contrast to our results, previous studies on ungulates have revealed differing patterns of spatial genetic structure in the two sexes, with spatial genetic structure generally observed among females, but not males . In red In conclusion, for roe deer, the low level of polygyny Figure S1Spatial auto-correlograms of three different estimators in relation to distance. Spatial auto-correlograms for females (red lines) and males (blue lines) are represented for different estimators: Moran's I (A), Queller and Goodnight (B) and Lynch and Ritland (C). Error bars represent the standard error around the estimated genetic correlation coefficients. The permuted 95% confidence intervals  around the null hypothesis of a random distribution of individuals  are also shown.(1.39 MB TIF)Click here for additional data file.


Ustilaginoideavirens is becoming a destructive disease throughout major rice-growing countries. Information about its genetic diversity and population structure is essential for rice breeding and efficient control of the disease. This study compared the genome sequences of two Uvirens.  isolates. Three SNP-rich genomic regions were identified as molecular markers that could be used to analyze the genetic diversity and population structure of Uvirens.  in China. A total of 56 multilocus sequence types (haplotypes) were identified out of 162 representative isolates from 15 provinces covering five major rice-growing areas in China. However, the phylogeny, based on sequences at individual SNP-rich regions, strongly conflicted with each other and there were significant genetic differences between different geographical populations. Gene flow between the different geographical populations and genetic differentiation within each geographical population were also detected. In addition, genetic recombination and genetic isolation resulting from geographic separation was also found.Rice false smut caused by the fungal pathogen  Ustilaginoideavirens (Cooke) Takah (Anamorph). Uvirens.  was recently placed in Clavicipitaceae as the new name Villosiclavavirens (Teleomorph) [Uvirens.  could invade rice coleoptiles and roots at the young seedling stage and the stamen filaments at the earlier booting stage [Uvirens.  mycelia, that colonized in young seedlings, can extend to the spikelets and form the ball-like symptom. At rice booting stage, Uvirens.  invades rice through small gap at the apex of spikelet before heading [Uvirens.  also contaminates rice seed and straw by producing antimitotic cyclic peptides that are poisonous to both humans and animals [False smut (green smut) is one of the most destructive diseases of rice. It is caused by the ascomycete fungus eomorph) ,2, whicheomorph) ,4. It wang stage -7. Howev heading  but does animals ,9. False animals –12. The  animals .Uvirens.  populations. Information on the genetic diversity of Uvirens.  populations will help create efficient strategies to reduce the yield loss caused by this pathogen. Zhou et al. [Uvirens.  populations from two provinces in North China using amplified fragment length polymorphism (AFLP) markers and discovered that the genetic variation in Uvirens.  populations was small in these areas. However, the samples used for the analysis were collected from only two provinces. The genetic diversity and population structure of Uvirens.  in the majority of the rice-growing areas across China are unknown.Previous studies on rice false smut mainly focused on its occurrence, pathogen detection, toxin analysis, pathogen life cycle and disease control ,14–19. Hu et al.  analyzedHistoplasma capsulatum [Candida albicans [Aspergillus fumigatus [Cryptococcus neoformans [Magnaporthe grisea [Methods based on molecular markers have been developed in order to analyze genetic diversity in fungal species. These markers include: allozyme, AFLP and microsatellites. DNA sequence-based markers have been widely used over recent years due to the reduced cost of DNA sequencing. Since an individual gene contains limited information, multi-locus sequence typing (MLST) has become a mainstream method for both molecular systematics and population genetics analyses ,22. MLSTpsulatum , Candidaalbicans , Aspergiumigatus  and Crypoformans . Most ofe grisea .Uvirens.  can persist on seeds or in soil for several years [Uvirens.  isolates from China on a large geographical scale will improve the design of efficient management strategies for rice false smut. In this study, three SNP-rich genomic regions were identified as novel molecular markers that could be used to study the population structure of Uvirens. . The genetic diversity and population structure of Uvirens.  in China were examined by comparing sequences located in SNP-rich genomic regions of 162 Uvirens.  isolates collected from five major rice-growing areas in China.Chlamydospores of al years . The accEthics statement: No specific permissions were required for sample collection on the locations mentioned in this manuscript and the fungus used in this study is not an endangered or protected species.Uvirens.  isolates were recovered from five rice cropping regions . The SNP sites of each selected genomic locus were identified by aligning the sequences at each selected locus in 10 isolates from 10 different provinces using the DNAMAN software . The putative functions of SNP-rich DNA fragments were deduced from BLAST alignment against sequences in GenBank (http://www.ncbi.nlm.nih.gov/) and gene prediction using GlimmerHMM software [In order to identify the genomic regions where SNPs are highly enriched, the genome sequences (unpublished data) of the two on 4.0.6 . Isolatesoftware .2), 200 µM dNTP, 0.4 µM of each primer and 1 U of Pfu DNA polymerase (TaKaRa). The PCR was performed in a thermocycler  under the following conditions: denaturation at 94°C for 2 min, followed by 30 cycles of denaturation at 94°C for 20 s, annealing at 56°C for 30 s and elongation at 72°C for 1 min. The PCR products were isolated by electrophoresis and purified for bi-directional sequencing.SNP-rich DNA fragments were amplified using specific primers . Each reDNA sequences were aligned using Clustal X 2.1 software . The resIA and rd) and by analyzing the allelic combinations. The number of haplotypes and nucleotide diversity were calculated using DnaSP software (version 5.10) [FST values were calculated using Arlequin 3.5.1.2 [Population genetics analysis was performed within and between populations (i.e. the above mentioned five rice cropping regions) using concatenated DNA sequence data set at three genomic loci. For within-population genetic analysis, we calculated the haplotype diversity and analyzed linkage disequilibrium  using MultiLocus 1.3 . Evidencon 5.10) . For cro 3.5.1.2 , based o 3.5.1.2 . Populat 3.5.1.2 ,41 with  3.5.1.2 . PrincipUvirens. , we explored new DNA markers that contained multiple SNP sites. First, two Uvirens.  isolates (cce007 and csc086) were chosen to do comparative analysis between their genome sequences. The assembled genome of cce007 is 38.8 Mb with a contig N50 length of 29.3 kb and the assembled genome of csc086 is 35.5 Mb with a contig N50 length of 27 kb (data not shown).In order to analyze the genetic diversity and population structure of Uvirens.  isolates, a total of 149669 SNP sites were found within 31.67 Mb matched genomic DNA between two isolates. As shown in Uvirens. . For the other genomic regions, their PCR products could be obtained in some isolates but not in other isolates. The presence of SNP sites at the primer regions in some isolates might affect the efficacy of PCR application.By comparing the genome sequences of two Magnaportheoryzae. The nucleotide sequences of the three markers from the representative isolate, cce003, were deposited in GenBank under the following accession numbers: JX483852 for Marker 1, JX483853 for Marker 2 and JX483854 for Marker 3. Sequence variations in these markers among the Uvirens.  isolates are shown in These DNA markers were named as Marker 1 (775 bp with 30 SNPs), Marker 2 (712 bp with 25 SNPs), and Marker 3 (594 bp with 70 SNPs) . Gene prUvirens.  isolates from 15 provinces, covering the five major rice-growing areas of China, were used to investigate the genetic diversity and population structure of the fungus . The 162 isolates could be grouped into more than 20 clades, based on the combined sequences at the three SNP-rich markers, and the isolate numbers for most clades were less than five  between genetic and geographic distances was detected with a correlation coefficient (r) of 0.09 , but not with latitude gradients .When two populations were compared, significant genetic differentiation could be found in most of the compared pairs, based on st China . AMOVA ast China , which ist China  and modest China . Further of 0.09 . FurtherUvirens.  isolates and this was further confirmed by the sequence analysis of 10 isolates from 10 different provinces. Most of the DNA sequences used for MLST in other fungi are housekeeping genes [Uvirens.  isolates in the future. As far as can be ascertained, this is the first report on the DNA-sequence-based analysis of the genetic diversity and population structure of Uvirens. .Sequence-based markers are important in both molecular systematic and population genetic analyses because they can provide unambiguous information on defined loci and enable the direct assessment of variability . In thisng genes –26. HoweUvirens.  populations in China have at least 56 multilocus sequence types (haplotypes). This means that Uvirens.  isolates in the field are genetically diverse. However, the correlation between virulence and genotype is unknown. Although several haplotypes, such as haplotype 1 and 17, were widely distributed over the rice-growing areas, the genetic differentiations between isolates from geographically distant rice-growing areas were higher than isolates between adjacent rice-growing areas. In addition, the Uvirens.  isolates collected from similar regions displayed considerable DNA composition stability. These results suggested that, naturally, Uvirens.  in China might not be spread over long distances. The wide distribution of some Uvirens.  haplotypes might be due to the transportation of rice seeds contaminated with smut balls containing chlamydospores.Analysis and comparison of the sequences of these SNP-rich regions revealed that Uvirens.  populations are high in most of rice-growing areas. The 162 isolates could be grouped into more than 20 clades. The high genetic diversity in Uvirens.  might be caused by genetic recombination through sexual cross between mating compatible strains in fields. In laboratory condition, Uvirens.  has sexual stage during which fruiting bodies emerge from sclerotia and ascospores are released [Uvirens.  populations. The genetic diversity of Uvirens.  in Northeast is relatively lower than other areas. Similar to this, an AFLP-based analysis showed that the genetic variation Uvirens.  populations of Liaoning province were low [Uvirens.  in Northeast suggests that sexual reproduction in this region might be less active than other regions. Climate conditions might affect sexual reproduction of Uvirens. .This study demonstrates that the genetic diversities of Table S1U. virens isolates.Geographical distributions and haplotypes of (XLS)Click here for additional data file.Table S2Sequence differences among 56 haplotypes found at the three loci with enriched SNP sites.(XLS)Click here for additional data file.Figure S1Allelic combination showing evidence of recombination.(DOC)Click here for additional data file.


Creontiades dilutus (Stål), the green mirid, is a polyphagous herbivorous insect endemic to Australia. Although common in the arid interior of Australia and found on several native host plants that are spatially and temporally ephemeral, green mirids also reach pest levels on several crops in eastern Australia. These host-associated dynamics, distributed across a large geographic area, raise questions as to whether (1) seasonal fluctuations in population size result in genetic bottlenecks and drift, (2) arid and agricultural populations are genetically isolated, and (3) the use of different host plants results in genetic differentiation. We sequenced a mitochondrial COI fragment from individuals collected over 24 years and screened microsatellite variation from 32 populations across two seasons. The predominance of a single COI haplotype and negative Tajima D in samples from 2006/2007 fit with a population expansion model. In the older collections (1983 and 1993), a different haplotype is most prevalent, consistent with successive population contractions and expansions. Microsatellite data indicates recent migration between inland sites and coastal crops and admixture in several populations. Altogether, the data suggest that long-distance dispersal occurs between arid and agricultural regions, and this, together with fluctuations in population size, leads to temporally dynamic patterns of genetic differentiation. Host-associated differentiation is evident between mirids sampled from plants in the genus Cullen (Fabaceae), the primary host, and alternative host plant species growing nearby in arid regions. Our results highlight the importance of jointly assessing natural and agricultural environments in understanding the ecology of pest insects. Many insects that damage agricultural crops have invaded the resources provided by agriculture across wide areas and this has generated alternative predictions as to their evolutionary trajectories. Although the provision of novel resources by agriculture might promote host adaptation Via , it has Stenotus rubrovittatus (Hemiptera: Miridae), native to Japan, and Queensland fruit fly Bactrocera tryoni (Diptera: Tephritidae) show strong geographic differentiation , and this has been associated with a shift from wild hosts to maize (Zea mays) within the last 9000 years since domestication , between its native range in California, and crops that it seasonally invades in Canada  have allowed an empirical evaluation of its effects  in arid regions of Australia, and novel hosts  that it has invaded within the last 200 years. We sampled mirids across both arid and agricultural regions in Australia, covering most of the geographic distribution of this endemic species and including the major host plants. We genotyped microsatellites from samples spanning two seasons and sequenced a mitochondrial COI fragment from green mirids collected over 24 years. Given the ecology of green mirids outlined below, and the challenges posed by both agricultural and arid environments, we structured our analyses according to the following three questions: (1) Do seasonal fluctuations in population size in both arid and agricultural regions result in genetic signatures of bottlenecks and drift? (2) Does long-distance dispersal occur between arid and agricultural populations? (3) Is genetic differentiation associated with the use of multiple host plants in the (arid) native range? We found that genetic patterns in C. dilutus are temporally dynamic, consistent with spatial and temporal heterogeneity in its arid range. Long-distance dispersal between arid and agricultural populations is evident from the data, and host-associated differentiation was found between the primary host plants and alternative hosts in arid regions. Together, these results highlight the importance of considering ecological and evolutionary processes across the distribution of an organism.In this study, we examine dispersal, fluctuations in population size, and the use of multiple host plants in Creontiades dilutus is a mirid bug that is endemic to Australia and has become a major pest of several agricultural crops . Host plants persist longer in winter (June–August), but only if sufficient rain falls. With the exception of “flood years,” when host plants may be unusually widespread and persistent, suitable hosts generally require two or more successive rain events to thrive. These rain events usually occur only locally and typically the hosts are spatially patchy, with large areas of barren land between. Inland temperatures are close to optimal for mirid development during winter, allowing a generation time of around 25 days  is a facultative winter reproductive phenomenon , observed (Ho), and unbiased expected (UHe) heterozygosities were computed using Genalex  in Genalex  and the allele frequency mode shift analysis were performed using the program BOTTLENECK ) against geographic distance (Rousset We tested for the presence of isolation by distance (IBD) to explore gene flow in relation to the temporal and geographic aspects of the sampling strategy. Initially, this analysis was restricted to the sampling period of July to August 2007 . Subsequently, all samples were analyzed to assess the temporal stability of the August 2007 pattern, these additional samples represented the same agricultural crops sampled in January 2007. The presence of an IBD effect was investigated by regressing ENA corrected genetic distance   from the proportion of individuals in each population sample that are assigned to other populations with high probability  had a nucleotide diversity (π) of 0.00055, and a haplotype diversity of 0.278. Tajima's D was −2.26 (P < 0.01) indicating an excess of low frequency polymorphisms likely due to population expansion. This pattern was similar in 1983 (n = 16), where nucleotide diversity was 0.00021, haplotype diversity 0.125, and Tajima's D −1.16, but not significant (P > 0.10), and in 1993 (n = 25), π = 0.00026, haplotype diversity 0.153, and Tajima's D −0.69, also not significant (P > 0.10).Genetic diversity was low for the mitochondrial COI sequences, with one haplotype dominating each of the three temporal samples. The 2006/2007 samples . Four of 32 population samples, three from inland and one from agriculture, showed genetic signatures indicating a recent bottleneck in the allele mode shift analysis . In addition, three of the four populations showed indications of admixture, and neither of the Wilcoxon tests was significant .A total of 105 alleles weresscored across all loci and all populations, once the null allele prone locus mirsat-3E had been removed Table , when avFST values using null allele corrected and non-corrected data; 2) removal of the two loci that had the greatest effect on HWE (mirsat1A1 and mirsat2G8), and then comparing results across the 6 locus and 8 locus data sets; and 3) selection of analyses that are more robust to low frequencies of null alleles and small deviations from HWE (see methods for details). Evaluating the effects of null allele/HWE deviations using these three methods revealed that the low frequencies of null alleles inferred in some population samples for some loci did not dramatically affect the overall signal in the data, and all results shown are for the 8-locus data set. Tests for genotypic linkage disequilibria returned no significant associations between pairs of loci for any of the 32 population samples after sequential Bonferroni correction for multiple comparisons.Deviations from HWE were inferred in all loci for some populations, and the presence of null alleles was also inferred Table . HWE devΦPT = 0.188, P = 0.001). The global FST estimates were similar with or without the elimination of null alleles, with the uncorrected data returning only a slightly higher estimate . Pairwise FST's ranged from 0.0019 to 0.329 (mean = 0.112), with 374 of 528 comparisons of genotypic differentiation being significant after sequential Bonferroni correction . In contrast, when all sampling events were included in the analysis (January 2007–March 2008), no isolation by distance effect was evident . The inclusion of these additional samples represented the same host plants that were sampled in agricultural regions during the July–August 2007 collections.The Mantel test of correspondence between geographic distance and genetic differentiation revealed a significant isolation by distance pattern when the analysis was restricted to the broad-scale geographic sampling of July to August 2007 . Admixture was evident in several populations, as several individuals had mixed posterior probabilities of assignment to each of the other two clusters. The proportion of admixed individuals and their cluster assignment shifted between January 2007 and July 2007 in both Biloela and Emerald, and between January 2007 and August 2007 in Narrabri ; however, host plant species were not sampled consistently across the whole of the sampling area . Genetic differentiation in relation to host plant species was therefore evaluated across two sites where several species could be sampled at each. At both of these, some degree of genetic differentiation was associated with plants in the genus Cullen relative to all other host plants sampled, although this was more pronounced at the Simpson Desert sites than at Milparinka in western New South Wales . The Evanno et al.  and alternative plant species, it was mostly the presence of rare alleles that was responsible for this pattern. We suggest that rare alleles on the primary hosts  could be a consequence of higher abundance of C. dilutus relative to their abundance on alternative host species. These points are expanded and justified below.Our combined analyses of genetic patterns in green mirid populations indicate that they are likely shaped by changes in population size Fig. , admixtuC. dilutus collected from crop hosts and those collected from non-crop hosts  or pesticide use (in agriculture) affected the temporal stability of patterns of genetic differentiation between mirid populations. We found no significant difference in microsatellite diversity between sts Fig. . Genetictus Fig. . None ofC. dilutus populations was higher than generally reported for pest insect species surveyed within an agricultural context , and other populations. The Structure analysis also clearly differentiated populations at these three sites from others. Although no heterozygote excess was detected at these sites (indicative of a recent bottleneck), we suspect that a combination of spatial heterogeneity and founder effects could contribute to strong genetic drift (and therefore high genetic differentiation) given that our study was conducted during a dry period when patches of host plants were separated by large areas of barren land. Elevated FST's are predicted (even when migration rates are high) under spatiotemporally dynamic population models if environmental heterogeneity contributes to a large variance in local population size  and change in predominant mitochondrial haplotype over the last 13 years was striking. Mitochondrial DNA is expected to suffer a more extreme loss of alleles than nuclear markers during demographic bottlenecks due to the uniparental inheritance of the plastid and the reduced effective population size of its genome  is thought to be unlikely based on prevailing wind directions and because positive evidence of its existence has never been found  in the structure analysis  and surviving. Further temporal samples at these inland sites would allow temporal estimates of effective population size across primary and secondary host plants, and the relative absence (and perhaps even loss) of rare alleles on alternative hosts needs to be investigated directly if these patterns are to be understood mechanistically.Previous quantified sampling showed that dum Fig. , althougCreontiades dilutus shows evidence of widespread dispersal in both the mitochondrial and microsatellite data sets examined here, despite this species using different plant resources , having seasonally inverse abundance between inland and agricultural regions, and presumably experiencing different selective pressures in these regions of Australia. The change in the most prevalent mitochondrial haplotype over 24 years is consistent with successive population contractions and expansions, likely in relation to fluctuations between dry periods and wet periods in the arid regions of Australia. Dispersal appears to be the major mechanism by which C. dilutus is able to survive on the ephemeral resources in this region, and the data provide no indication that large numbers of these bugs persist through dry periods by diapause. The spatiotemporal dynamics and changing gene frequencies outlined above contrast with the lack of genetic differentiation found in the same agricultural regions over several years for the highly dispersing H. armigera   appear to have not only facilitated the colonization of new agricultural habitats but also maintain gene flow across large distances.Our results highlight the importance of assessing evolutionary and ecological processes across the distribution of an organism that uses both native and human-altered habitats simultaneously. If our analyses had been restricted to either agricultural areas or localized parts of the arid range of this species, our interpretations might be quite different. For example, broader geographic analyses of le) Bush , to inclle) Bush . In C. d


Leopoldamys neilli, a cave-dwelling murine rodent living in limestone karsts of Thailand, and compared the molecular signature of mitochondrial and nuclear markers.Historical biogeography and evolutionary processes of cave taxa have been widely studied in temperate regions. However, Southeast Asian cave ecosystems remain largely unexplored despite their high scientific interest. Here we studied the phylogeography of L. neilli and the relative role of vicariance and dispersal were investigated using ancestral range reconstruction analysis and Approximate Bayesian computation (ABC).We used a large sampling (n = 225) from 28 localities in Thailand and a combination of mitochondrial and nuclear markers with various evolutionary rates (two intronic regions and 12 microsatellites). The evolutionary history of L. neilli. These deep vicariant events that occurred during Plio-Pleistocene are related to the formation of the Central Plain of Thailand. Consequently, the western, central, northern and northeastern groups of populations were historically isolated and should be considered as four distinct Evolutionarily Significant Units (ESUs).Both mitochondrial and nuclear markers support a large-scale population structure of four main groups  and a strong finer structure within each of these groups. A deep genealogical divergence among geographically close lineages is observed and denotes a high population fragmentation. Our findings suggest that the current phylogeographic pattern of this species results from the fragmentation of a widespread ancestral population and that vicariance has played a significant role in the evolutionary history of L. neilli evolutionary history at different levels of resolution. The complex genetic structure of Leopoldamys neilli is supported by congruent mitochondrial and nuclear markers and has been influenced by the geological history of Thailand during Plio-Pleistocene.Our study confirms the benefit of using several independent genetic markers to obtain a comprehensive and reliable picture of  Subterranean habitats and their singular fauna have fascinated biologists and biogeographers for centuries. Historically, there has been a long debate over the hypotheses regarding the evolution of subterranean animals and particularly the relative role of vicariance and dispersal and the influence of potential isolating barriers to explain biogeographic patterns of these taxa The modes of speciation of subterranean animals have also been debated and several models proposed: the Climatic Relict Hypothesis (CRH) 2Due to their discontinuous distribution and the peculiarity of their fauna, limestone karsts constitute an interesting model to explore these evolutionary processes. Limestone karsts are sedimentary rock outcrops consisting of calcium carbonate sculpted by solutional erosion into the typical landscape made of cone-shaped or sheer-sided hills riddled with caves and sinkholes Leopoldamys neilli , a murine rodent species endemic to limestone karsts of Thailand, within the Indo-Burmese biodiversity hotspot. The species was discovered in karsts of the Saraburi province, central Thailand and has also been recorded in few locations in northern and western Thailand  genetic diversity for this species et al.A preliminary study of the phylogeography of L. neilli populations genetically structured throughout Thailand according to mitochondrial and nuclear markers? (ii) Is the phylogeograhic structure of mtDNA representative of the evolutionary history of L. neilli? Are molecular signatures of mitochondrial and nuclear markers congruent? (iii) When did the divergence between L. neilli and L. edwardsi occur and what can we infer about the speciation process of L. neilli? (iv) Where were L. neilli ancestors distributed? What are the relative roles of vicariance, dispersal and potential isolating barriers in the evolutionary history of L. neilli? (v) According to its distribution and genetic structure, which conservation status should be given to this species?Using a much larger sampling and various nuclear markers, the present study was conducted to address the following questions: (i) How are http://www.ceropath.org/references/rodent_protocols_book) were evaluated by the ethics committee of Mahidol University in Bangkok.Sample collection was carried out in collaboration with researchers located at the Thailand Institute of Scientific and Technological Research (TISTR) and directors of Wildlife Research Stations  having the required permits. This study is part of the “CERoPath project” , ANR Biodiversity ANR07 BDIV012. CERoPath protocols , R3111 , R4222 , R4296 , R4370 ) For phylogenetic analysis, six samples of b gene (cytb) and the cytochrome oxidase I gene (COI), were used in this study in addition to two nuclear fragments; the intron 7 region of the β-fibrinogen gene (bfibr) and the intron 1 region of the X-linked glucose-6-phosphate dehydrogenase gene (G6pd). Sequences of cytb (n = 115), COI (n = 115) and bfibr (n = 65) were available from a previous study Two mitochondrial markers, the cytochrome All haplotype/allele sequences have been deposited in Genbank database  were discarded as burn-in values. A 50% Majority-rule consensus tree was generated in PAUP 4.0b10 Phylogenetic reconstructions were performed using the maximum likelihood (ML) and Bayesian inference (BI) approaches. Analyses were first run independently on mitochondrial and nuclear loci and then on combined datasets  and cytb/COI/bfibr/G6pd). The most suitable model of DNA substitution for each locus and dataset was determined using MODELTEST 3.0 Median joining networks were performed with NETWORK 4.5.1.6 Haplotype (h) and nucleotide (π) diversities of the main lineages identified by phylogenetic analysis were estimated for the four loci independently using ARLEQUIN. Tables of nuclear allele frequency were computed with GENEPOP 4.0.11 ST for the mitochondrial dataset and FST for the nuclear dataset using ARLEQUIN.The net genetic distance between lineages was computed in MEGA 4.1 FST/[1−FST]).A Mantel's test performed in ARLEQUIN on mitochondrial and nuclear datasets independently was used to assess the hypothesis of isolation by distance (IBD) among the four groups of populations by comparing pairwise geographic distance (log transformed) with pairwise  at 7 Myr Otomyini/Arvicanthi split at 5.7 Myr Batomys granti (AY324458) (Phloemyini) and Phloemys cumingi (DQ191484) (Phloemyini), Apodemus mystacinus (AF159394), Apodemus sylvaticus (AB033695), Apodemus flavicollis (AB032853), Otomys angoniensis (AM408343) (Otomyini) and Arvicanthis niloticus (AF004569) (Arvicanthi) were added to our dataset to calibrate the tree and constrain the age of some nodes. Analyses were performed under the TN93+G substitution model (previously estimated by MODELTEST), a strict molecular clock and a constant size coalescent population model. These priors were selected because they better fit the data than any other molecular clock and population models according to the Bayes factors calculated to compare the models 8 were performed, sampling every 1×104 generations. Convergence of the chains to the stationary distribution was checked using TRACER 1.5 http://www.bioportal.uio.no).Divergence times of Fis was calculated for each population and adjusted for multiple tests using a Bonferroni's correction with FSTAT. The proportion of null alleles (NA) at each locus and for each population was estimated with FREENA Genetic diversity was assessed by calculating expected (He) and observed (Ho) heterozygosities with ARLEQUIN and confirmation of Hardy-Weinberg equilibrium (HWE) was tested using GENEPOP for each locus separately and over all loci for each sampling site. The allelic richness (AR) was calculated using the rarefaction procedure implemented in FSTAT 2.9.3.2 5 and MCMC values of 1×106. We used CLUMPP 1.1 5 MCMC iterations were performed for K from 1 to 28 with a spatial coordinates uncertainty of 1 km. The posterior probability of population membership for each individual was determined using a burn-in of 3×104 iterations. A visual output of the STRUCTURE and GENELAND results was generated using DISTRUCT STRUCTURE 2.3.1 FST using ARLEQUIN.Genetic differentiation among clusters containing more than three individuals was quantified by computing pairwise FST/[1−FST]) and (RST/[1−RST]) using ARLEQUIN and SPAGeDI 1.3 IBD among the four groups of populations and within groups including at least four clusters (northeast and west) was tested by comparing pairwise geographic distance (log transformed) with pairwise (L. neilli distribution range. VIP uses a phylogenetic tree (ML tree of the combined dataset) and direct geographic information data to search for the disjunctions/barriers among sister groups.We used VIP (Vicariance Inference Program) L. neilli populations and investigate the relative role of vicariance and dispersal in the evolutionary history of this species, we used a dispersal–extinction–cladogenesis (DEC) model of range evolution implemented in LAGRANGE L. neilli, we constrained successively the area of this node to each combination of one, two, three or four areas (except for the combination centre+north because these two areas are not adjacent) and compared the global likelihood scores obtained for each area or combination.To infer ancestral areas of L. neilli combining our mitochondrial and microsatellite datasets. Several biogeographic scenarios were compared to test whether the current groups of populations originated from the fragmentation of a common ancestral population or from one of the current groups and whether admixture events occurred among populations. According to the phylogenetic trees and Bayesian clustering analysis, four groups of populations were used  and the origin of each group was tested independently in a four-step analysis using 40 scenarios  and FST as two-sample summary statistics.Approximate Bayesian computation implemented in the software DIYABC 1.0.4.45beta cenarios . For eac5 datasets were simulated to build a reference table. To check if the combination of scenarios and prior distributions of their parameters were able to generate datasets similar to the observed one, a principal component analysis (PCA) was performed on the first 10000 simulated datasets of the reference table in the space of summary statistics. To determine the most likely scenario, the normalized Euclidean distances between each simulated dataset of the reference table and the observed dataset was then computed and 1% of the closest simulated datasets were used to estimate the relative posterior probability  of each scenario with a logistic regression For each scenario, 5×10Fragments of 900 and 713 bp were obtained for cytb and COI genes, respectively. No stop codons, indels or heterozygous sequences were observed in these mitochondrial coding genes, supporting the mitochondrial origin of the sequences amplified. A total of 37 cytb haplotypes and 25 COI haplotypes were identified among our datasets. The cytb gene contained 158 (231 in the dataset including outgroup sequences) variable sites, whereas the COI gene contained 85 (138) variables sites.For the bfibr gene, fragments of 740 bp were obtained and no indels were observed in the alignment. The G6pd sequences obtained varied in length from 678 to 690 bp and the final alignment included three indels of 1, 2 and 10 bp. A total of 31 bfibr alleles and eight G6pd alleles were identified among our datasets. The bfibr gene contained 27 (49) variables sites, whereas the G6pd gene contained 17 (29) variables sites.Recombination was detected via a PHI test (p = 0.004) and GARD/SBP tests at position 386 for the bfibr gene. G6pd sequences did not present evidence of recombination, according to the PHI test (p = 1.0) and GARD/SBP tests. Therefore, we performed all phylogenetic trees including the bfibr gene with alignment containing only positions 1–385 of the bfibr gene.L. neilli is represented by L. edwardsi haplotypes and that all analyzed individuals belong to the L. neilli monophyletic group. The ML and Bayesian analysis gave congruent results. The haplotypes of L. neilli clustered into ten well-supported and geographically well-structured lineages: Loei/Khon Kaen , Petchabun (PET), Chaiyaphum (CHAI1-2), Chiang Rai (CHR), Nan (NAN), Phrae (PHR), Centre1 , Centre2 , Uthai Thani (UT), and Kanchanaburi (KAN1-2-3-4-5-6-7-8). These results are congruent with those obtained in Latinne et al.Mitochondrial loci were first analyzed separately; they yielded low-supported but congruent phylogenies (data not shown). They were then combined in a single matrix. The mitochondrial phylogenetic tree  clearly Leopoldamys species; however, intraspecific relationships within L. neilli were less clear than for mitochondrial dataset. Only one lineage is well-supported (BS = 81%–BP = 1.0) and includes all samples from western Thailand .Due to the low variability of our nuclear genes they were combined in a single matrix. For both phased and unphased datasets, the nuclear phylogenetic tree  supporteThe ML and Bayesian trees combining nuclear and mitochondrial datasets  recovereThe median joining network of mitochondrial haplotypes confirmed the results obtained in the phylogenetic trees and retrieved the same ten haplogroups . They arHaplotype diversities within lineages are quite low , rangingThe table of nuclear allele frequency showed a high genetic heterogeneity among lineages . Exact tST and FST values confirmed a high population differentiation , Centre1 and Centre2 lineages . Pairwise ΦST and FST values were particularly high between western  and central  lineages (from 0.96 to 0.99 for ΦST and from 0.66 to 0.84 for FST). These results are confirmed by the high genetic divergence  observed among lineages  and nuclear datasets . This result is consistent with no relationship between geographic distance and genetic distance.The Mantel's test was nonsignificant for both mitochondrial (L. neilli and L. edwardsi was estimated to occur at around 3.8 Myr. The TMRCA of L. neilli was dated around 2.4 Myr. The TMRCA of (centre+north+northeast) was estimated around 1.3 Myr while the TMRCA of (north+northeast) was dated around 0.9 Myr.Divergence time estimates are given in Fis) was significant at population level. Only one pair of loci (mLn04 and mLn12) showed significant linkage disequilibrium in two populations  after Bonferroni's correction.Analysis of microsatellite genetic diversity showed that observed heterozygosity ranged from 0.43 to 0.93, and allelic richness  ranged from 1.41 to 1.79 . Tests fΔK method described by Evanno et al.ΔK was found at K = 11 with a second peak at K = 4  and within the west  and northeast  groups.IBD was not detected among the four groups of populations using both VIP identified three main barriers within the studied area . The oldL. neilli tree. The most likely area of L. neilli ancestral population encompasses the four regions (west+centre+north+northeast) (relative probability = 1) (L. neilli are significantly different (more than 3-log likelihood units between the most likely area and the remaining likelihood scores). Several vicariant events progressively fragmented this large population and isolated the four regions. The ancestral range of the node containing the central, northern and northeastern lineages includes the three regions (centre+north+northeast) (relative probability = 1). Finally, the ancestral range of the node containing the northern and northeastern lineages encompasses these two regions (north+northeast) (relative probability = 1).LAGRANGE estimated the likelihoods of all possible ancestral range reconstructions for each node of the ity = 1) . The likThe PCA performed on the first 10000 simulated datasets of the reference table in the space of summary statistics revealed that the observed dataset is clearly surrounded by simulated datasets, indicating that our model was able to produce datasets similar to the observed one (data not shown).W>NNE>NC>NN)  of each step clearly indicated that all of the four groups originate from a common ancestral population and not from one of the current populations and that admixture events did not occur . These rE>NC>NN) .L. neilli.Three kind of molecular markers with various evolutionary rates and different modes of inheritance were used in this study and depicted an exhaustive picture of the phylogeographic structure of et al.L. neilli. The exact tests of genic differentiation among lineages also confirmed a high genetic heterogeneity of nuclear allele frequency at a finer scale.Due to their slow evolutionary rate and their higher coalescent time, the use of nuclear DNA sequences in phylogeographic studies has been limited. However the present study confirms the interest to combine several nuclear introns to gain more resolution. Indeed the intron 7 of the bfibr gene used alone in Latinne L. neilli are observed within Thailand . Moreover they also detected more recent isolations among populations and indicated a strong finer structure within each of these groups: ten well-supported and geographically well-structured lineages of Thailand , 6A.ΔK method  coupled with the high FST values and genetic distances among them revealed a high genetic divergence and low gene flows among lineages. This high degree of genetic differentiation is confirmed by the old divergence times estimated among lineages as the TMRCA of L. neilli was approximated around 2.4 Myr. Such deep genealogical divergence among geographically close lineages is quite surprising and denotes a high population fragmentation related to the patchy distribution of limestone karsts and a close association of L. neilli with this habitat. Similar phylogeographic patterns are rare among rodents but have been observed for a few other rodent species strongly dependent on rocky and karstic habitats in various regions of the world, such as the Laotian rock rat Laonastes aenigmamus in Central Lao PDR Dinaromys bogdanovi in the western Balkans Neotoma magister in northeastern America L. neilli. These results confirm the endemicity of L. neilli to limestone karsts previously described in the literature The congruent patterns of mitochondrial and nuclear markers indicated a strong population fragmentation for L. neilli and L. edwardsi was estimated to occur during Pliocene, around 3.8 Myr. Several splits subsequently occurred within L. neilli population throughout Quaternary. Due to the potential inaccuracy of temporal estimates based on molecular data across different evolutionary scales Leopoldamys in Indochina during Plio-Pleistocene. The oldest occurrence of Leopoldamys  has been reported in cave deposits from Late Pliocene to Early Pleistocene in the Kanchanaburi and Saraburi provinces, Thailand L. edwardsioides, a predecessor of L. edwardsi, from Early Pleistocene have also been identified in southern China The divergence between L. neilli and L. edwardsi is a matter of debate. Two main alternative models of speciation of cave animals have been proposed in the literature: the Climatic Relict Hypothesis (CRH) The evolutionary context of the divergence between L. neilli and L. edwardsi that occurred a long time before Pleistocene environmental instability and the present parapatric/partially sympatric distribution of these two sister species in northern and northeastern Thailand Leopoldamys ancestor could have invaded cave ecosystems to exploit the novel habitat or food resources and this ecological shift between the two populations could have been the driving force for genetic divergence and speciation L. neilli, we can only propose some hypotheses on this point but we suggest that this ecological shift could affect the reproductive behaviour of these rodents and we suspect L. neilli to use caves for breeding and nursing as young individuals and babies have been observed within caves during our survey. The ecological specialization of L. neilli to limestone karsts could also be associated to the particular xerophytic flora of karsts or to specific food resources available in caves such as insects.The old divergence between L. neilli and L. edwardsi followed by a more recent sympatry cannot be excluded. The two species could have evolved independently in different regions of Southeast Asia during Pliocene and subsequently dispersed throughout Indochina to establish the current parapatry in northern Thailand. Additional data about the past (and present) distribution of L. neilli, L. edwardsi and their ancestor are needed to infer the speciation process leading to the origin of a cave-dwelling rodent.However, an allopatric speciation of The relative role of vicariance (the fragmentation of populations due to isolating barriers) and dispersal  to explain biogeographic patterns of cave and karstic endemic taxa have also been widely discussed during decades and the debate is still ongoing L. neilli has been tested using ancestral range reconstruction analysis and ABC methods and they clearly supported the vicariant hypothesis. All four of the current groups of L. neilli originate from a common ancestral population widely distributed within Thailand during Pliocene and whose effective population size was several times higher than those of the current groups. Then the formation of the Central Plain of Thailand during Quaternary has played a significant role in progressively fragmenting the ancestral population and shaping the phylogeographic pattern of this species. The progressive burying and disappearing of limestone karsts of the Central Plain and of the main river valley basins would constitute a barrier to L. neilli dispersal and lead to the vicariant events isolating populations. The western populations were the first to have been isolated around 2.4 Myr, followed by the central populations around 1.3 Myr and finally the northern and northeastern groups around 0.9 Myr.The role of these barriers in the evolutionary history of L. neilli and even short distances of lowland areas among karsts seem to constitute important dispersal barriers. The high FST values for both mitochondrial and nuclear datasets between western and central populations  have been defined in Latinne et al.First, populations of L. neilli seems to be highly specific and the species is closely associated with limestone karsts. This habitat is currently strongly threatened through quarrying, hunting, and urbanization L. neilli is thus strongly dependent on the preservation of karstic habitats.Second, the habitat requirement of L. neilli is intensively trapped for human consumption in some regions of northeastern Thailand. This could threaten the long-term preservation of the species in this region.Third, L. neilli under the phylogenetic species concept Therefore, we strongly recommend classifying this species at least as “Near Threatened” on the IUCN Red List. If the four ESUs that we described were assessed separately, they would qualify as “Vulnerable” because of their small geographic ranges and population sizes. Particular attention should be paid to the western lineages, highly divergent for both mitochondrial (more than 7% of net K2P distance for cytb) and nuclear genes, which could be considered as a distinct sub-species of We also suggest, following the recommendations of Vermeulen & Whitten Leopoldamys neilli supported by both mitochondrial and nuclear markers. The evolutionary history of this species has been influenced by the geological history of Thailand during Plio-Pleistocene and vicariance has played a significant role in shaping the phylogeographic pattern of L. neilli. The western, central, northern and northeastern groups of populations were historically isolated and they evolved independently. To our knowledge this is the first phylogeographic study of karst endemic taxa in this region. Further phylogeographic studies of other co-distributed taxa will allow the determination of whether the phylogeographic pattern recovered for L. neilli and the strong isolation of western populations could be generalized to other species living in Thai limestone karsts and whether they share a similar evolutionary history.This study reveals a complex genetic structure for Figure S1Map of localities sampled during our survey of rodent diversity in Thai limestone karsts.(TIF)Click here for additional data file.Figure S2 of the Bayesian clustering analysis with STRUCTURE. (A) Plot of the likelihood of the mean Ln Pr (X|K). (B) Values of ΔK calculated according to Evanno et al. (2005).(TIF)Click here for additional data file.Figure S3Comparison of the posterior probabilities of all scenarios for each of four steps of our ABC analysis.(TIF)Click here for additional data file.Table S1Sampling locality, samples used for sequencing for each dataset (n) and haplotype/allele distribution.(DOC)Click here for additional data file.Table S2GenBank Accession numbers for cytb, COI, bfibr and G6pd haplotypes/alleles of L. neilli and outgroup sequences (L. edwardsi and L. sabanus).(DOC)Click here for additional data file.Table S3Primers and PCR conditions used in this study.(DOC)Click here for additional data file.Table S4Nuclear allele frequency among the main lineages of L. neilli.(DOC)Click here for additional data file.Table S5Pairwise genetic divergence (net K2P distance) for the cytb dataset among lineages of L. neilli.(DOC)Click here for additional data file.Table S6Pairwise FST values based on microsatellite dataset among clusters  containing more than three individuals.(DOC)Click here for additional data file.Table S7Estimations of the posterior distribution of the effective population sizes for the best scenario of each step revealed by the ABC analysis.(DOC)Click here for additional data file.


AbstractSphaerosyllis hystrix Claparède, 1863. A detailed morphometrical analysis of the Israeli specimens in comparison to specimens of Sphaerosyllis hystrix and Sphaerosyllis boeroi Musco, Çinar and Giangrande, 2005 supported the description of the former as a new species, Sphaerosyllis levantinasp. n. Individuals of Sphaerosyllis hystrix formed a very heterogeneous group with strong character variations in the analysis and the presumed cosmopolitan distribution of the species is discussed based on literature records.Examination of polychaete specimens from Haifa Bay  revealed several individuals exhibiting morphological characteristics similar to  Sphaerosyllis Claparède, 1863 (Annelida) is one of the most species-rich genera of the syllid subfamily Exogoninae. At present, ca. 48 species are considered valid within Sphaerosyllis after the recent split of the group into the three genera Sphaerosyllis, Prosphaerosyllis and Erinaceusyllis  images were dehydrated, critical point dried , sputter-coated with gold  and examined under a JEOL JSM-6390LV at the Department of Biology, University of Crete. Specimens are deposited in the invertebrate collection of the Smithsonian National Museum of Natural History, Washington D.C., USA (USNM) and in the Tel Aviv University Zoological Museum, Israel (TAU).Specimens were collected on 11 Oct. 2009 in Haifa Bay,  from fine to medium sands in shallow waters (10 m). Sediment samples were taken with a Van-Veen grab  32×35 cm, volume 20 l, penetration 20 cm. The sediment was preserved in buffered formalin 10% for 3–7 days, then sieved through a 250 µm mesh sieve and subsequently stored in 70% ethanol. Specimens were examined under an Olympus SZx12 stereomicroscope and an Olympus BX50 microscope. Illustrations in pencil were made by means of a drawing tube, subsequently scanned, imported into a graphic program (GIMP), re-drawnSphaerosyllis boeroi: 3 individuals; Sphaerosyllis hystrix: 21 individuals; Sphaerosyllis levantina sp. n.: 6 individuals) were analysed. Twenty-five variables were measured: I. body length, to account for size-dependencies of other characters; II. number of chaetigers; III. length of blade of dorsalmost falciger of a) anterior, b) midbody, c) posterior chaetigers; IV. length of blade of ventralmost falciger of a) anterior, b) midbody, c) posterior chaetigers; V. ratio of length of blades of dorsalmost to ventralmost falciger in a) anterior, b) midbody, c) posterior chaetigers; VI. ratio of length of blades of falcigers in anterior to posterior chaetigers for a) dorsalmost; b) ventralmost falciger; VII. Ratio of length of dorsalmost falciger to body length in in a) anterior, b) midbody, c) posterior chaetigers; VIII. Ratio of length of ventralmost falciger to body length in in a) anterior, b) midbody, c) posterior chaetigers; IX. maximum length of serration of falcigerous blades in a) anterior, b) midbody, c) posterior chaetigers ; X. presence of a subdistal spine in dorsalmost falcigerous blades of in a) anterior, b) midbody, c) posterior chaetigers.A total of 30 individuals belonging to three species . Three individuals of Summary statistics  were calculated for each species PageBreakMultidimensional Scaling (MDS) was subsequently employed to display the similarities of the different individuals. To test for significance of differences between species a PERMANOVA  was performed  into account, Gower’s similarity coefficient  was choserformed . A Princhttp://www.R-project.org).Multivariate statistical analyses were performed with PRIMER V6, correlation of the Principal Component Scores were calculated with the R package .The description of the new taxon was prepared in a Virtual Research Environment (Scratchpads) allowing for rapid and simultaneous publication of the results in print as well as electronically in a semantically enhanced form . This puurn:lsid:zoobank.org:act:9CEE8F90-9596-49F6-AA22-BB79C0E816D9http://species-id.net/wiki/Sphaerosyllis_levantinaSphaerosyllis levantina, Haifa Bay, coll. B. Galil 11.10.09 [Holotype]”. Paratypes USNM 1160541–1160573: 33 individuals, TAU-AN 25006: 10 individuals; Haifa Bay, Israel, Eastern Mediterranean Sea, Station ALA-IL-7, coll. 11.10.2009, depth 10.5 m; Labels: “Sphaerosyllis levantina, Haifa Bay, coll. B. Galil 11.10.09 [Paratype X]” (where X=1–43). All material preserved in 96% Ethanol.Holotype (USNM 1160540) ALA-IL-7, Haifa Bay, 10.5 m depth. Label: “.Sphaerosyllis boeroi Musco, Çinar, and Giangrande, 2005 . Sphaerosyllis hystrix , Chalkida, Aegean Sea, Greece: 1 specimen  , Chalkida, Aegean Sea, Greece: 6 specimens  , Chalkida, Aegean Sea, Greece: 4 specimens  , Thessaloniki, Aegean Sea, Greece, 1 specimen ) , all deposited the in Zoological Museum of the Aristotle University of Thessaloniki, Greece.32°54.533N, 35°04.071E).Eastern Mediterranean Sea, Levantine Basin, Israel, Haifa Bay , levantina being a neo-Latin adjective meaning “pertaining to the region where the sun raises”; feminine declination in accordance with the genus name .Israeli Coast .Fine to medium sands.Sphaerosyllis levantina sp. n. is similar to Sphaerosyllis minima Hartmann-Schröder, 1960 in having blades of falcigers with strong serration throughout the body. However, Sphaerosyllis minima has a stronger dorso-ventral gradation of the blades of falcigers  than Sphaerosyllis levantina sp. n.  and the ventral cirrus is longer than the parapodial lobe in Sphaerosyllis minima, whereas is is half as long as the parapodial lobe in Sphaerosyllis levantina sp. n. Sphaerosyllis capensis Day, 1953, Sphaerosyllis taylori Perkins, 1981, and Sphaerosyllis sandrae Álvarez and San Martín, 2009 are similar to Sphaerosyllis levantina sp. n. in the shape and serration of the blades of the falcigers, but Sphaerosyllis capensis has all antennae positioned in line , Sphaerosyllis taylori shows no dorso-ventral gradation of the falciger blade length  andPageBreakSphaerosyllis sandrae has smooth falcigerous blades posteriorly and parapodial glands with hyaline material . All the above species differ from Sphaerosyllis levantina sp. n. by lacking a subdistal spine on the blades of the falcigers. The only Sphaerosyllis species known to possess this spine are Sphaerosyllis hystrix Claparède, 1863, Sphaerosyllis parabulbosa San Martín and López, 2002 and Sphaerosyllis boeroi Musco, Çinar & Giangrande, 2005. Sphaerosyllis parabulbosa clearly differs from Sphaerosyllis levantina sp. n. by having minute dorsal cirri and antennae, by the presence of a subdistal spine only on blades of the posterior falcigers and by smooth blades of posterior falcigers. Sphaerosyllis boeroi differs from Sphaerosyllis levantina sp. n. in having much longer blades of the falcigers which show a more pronounced dorso-ventral gradation  than those of Sphaerosyllis levantina sp. n. (Sphaerosyllis levantina sp. n.) and by the dorsalmost falcigers being serrated only proximally. Sphaerosyllis hystrix, according to the literature, has a subdistal spine only on the blades of the anterior dorsalmost falcigers. However, in the examined material of Sphaerosyllis hystrix from the Aegean Sea 8 out of 21 specimens also possessed a subdistal spine in posterior falcigers. Sphaerosyllis hystrix can nevertheless be distinguished from Sphaerosyllis levantina sp. n. by having smooth or finely serrated posterior falcigers , even when the spine is present  (Sphaerosyllis hystrix (anteriorly 1.5 times longer than posteriorly), whereas they are of similar length throughout the body in Sphaerosyllis levantina sp. n. , whereas the pharynx of Sphaerosyllis levantina sp. n. is wider than ¾ of the width PageBreakof the proventricle. An identification key to the Mediterranean Sphaerosyllis species is provided at the end of this manuscript. sp. n.) , 3, 6. FSphaerosyllis hystrix occurring in her samples from Israel. Based on her description and illustrations, the animal identified as Sphaerosyllis hystrix sensu Sphaerosyllis levantina sp. n. because of the similar characters of falcigers andpapillation. However, the description does not report the characteristic subdistal spine on the blades of the posterior falcigers. In addition, Westheide’s (1974) description of Sphaerosyllis hystrix from the Galápagos Islands differs from both Ben-Eliahu’s specimen and the present material by the absence of parapodial glands . The Spearman’s correlation of the Principal Component scores with the measured character values of the individuals revealed that the length of the dorsalmost falcigerous blades in allPageBreakPageBreak body parts , as well as the ratio of the anterior to posterior ventralmost falcigerous blade are the most important characters discriminating between the three species  .The results of the Principal Component Analysis show that the first principal component (PC1) account for 77.4% of the variability, the second (PC2) for 16.4% and the remaining 3 PCs for 5.1%  cannot be sustained. Subsequent analyses of the differences between species through pairwise tests reveals significant differences between species .The PERMANOVA analysis results in a Sphaerosyllis –like many of the small-sized Exogoninae genera– has a difficult and often confused taxonomy and biogeography. Among the potential causes contributing to the current confusion the following could be cited: a) lack of detail in older (before ca. 1970) species descriptions; b) difficulties of observing certain characters in fixed material  is included among those species with an alleged cosmopolitan distribution, since it has been recorded from most European coasts including the Mediterranean Sea, the north-western coasts of America  descmaterial , presencmaterial  and varimaterial . These f America , the Gal America , China  and have never been re-described based on new material, they are tentatively included in the key below, but their identity remains questionable.The three species