Tetrao urogallus L.) is a grouse species of open boreal or high altitude forests of Eurasia. It is endangered throughout most mountain range habitat areas in Europe. Two major genetically identifiable lineages of Western Capercaillie have been described to date: the southern lineage at the species' southernmost range of distribution in Europe, and the boreal lineage. We address the question of genetic differentiation of capercaillie populations from the Rhodope and Rila Mountains in Bulgaria, across the Dinaric Mountains to the Slovenian Alps. The two lineages' contact zone and resulting conservation strategies in this so-far understudied area of distribution have not been previously determined. The results of analysis of mitochondrial DNA control region sequences of 319 samples from the studied populations show that Alpine populations were composed exclusively of boreal lineage; Dinaric populations of both, but predominantly (96%) of boreal lineage; and Rhodope-Rila populations predominantly (>90%) of southern lineage individuals. The Bulgarian mountains were identified as the core area of the southern lineage, and the Dinaric Mountains as the western contact zone between both lineages in the Balkans. Bulgarian populations appeared genetically distinct from Alpine and Dinaric populations and exhibited characteristics of a long-term stationary population, suggesting that they should be considered as a glacial relict and probably a distinct subspecies. Although all of the studied populations suffered a decline in the past, the significantly lower level of genetic diversity when compared with the neighbouring Alpine and Bulgarian populations suggests that the isolated Dinaric capercaillie is particularly vulnerable to continuing population decline. The results are discussed in the context of conservation of the species in the Balkans, its principal threats and legal protection status. Potential conservation strategies should consider the existence of the two lineages and their vulnerable Dinaric contact zone and support the specificities of the populations.The Western Capercaillie ( Tetrao urogallus L.) is a grouse species of open boreal or high altitude forests of Eurasia and is endangered throughout most mountain range habitat areas in Europe The Western Capercaillie  and the boreal lineage , with different ecological adaptation and post-glaciation development southern lineage originally expanded throughout Europe or at least to Romania and Bulgaria, and that the boreal lineage expanded in Asia and North-Eastern Europe southern lineage during late Pleistocene glacial periods boreal lineage, as evidenced by a star-like phylogeny, low nucleotide and high haplotype diversity southern lineage remained localised at its proposed glacial refugium in Iberia (the Cantabrian Mountains and the Pyrenees) and the Balkans (Bulgaria and the southern Carpathians in Romania) Southern lineage populations have been discussed as glacial relicts and as potentially having a different post-glacial diversification from the boreal lineage, which occupies the rest of and, at the same time, the majority of the current capercaillie habitat range, including the Alps Extensive phylogenetic and phylogeographic studies of the Western Capercaillie have been made across Eurasia, in search of evidence of genetic diversification and conservation strategies through appropriate management on a local scale T. urogallus major and T. urogallus rudolfi) T. urogallus lineages in Balkans would probably necessitate implementation of different conservation management strategies for different populations in the region.Different habitat suitability indexes southern lineage individuals in the Balkans Recent findings on T. urogallus haplotypes discovered in this study, there were 28 variable sites , of which 13 were parsimony informative. For the combined dataset comprising all sampled T. urogallus haplotype sequences and T. urogallus and closely related Black-billed Capercaillie (Tetrao parvirostris) sequences retrieved from Genbank, the best model of nucleotide substitution, chosen according to AICc, was HKY+G. Estimated parameters were: nucleotide equilibrium frequencies of 0.2624, 0.2861, 0.1436 and 0.3079 for A, C, G and T respectively, Ti/Tv ratio of 6.2287 (kappa 11.9429) and gamma distribution parameter alpha of 0.115. ML and MSN methods produced complementary trees  the boreal lineage haplogroup was composed of 46 haplotypes, including all Alpine, Polish and Belarus haplotypes, 8 Dinaric haplotypes corresponding to 144  samples and 1 Rhodopes-Rila haplotype (T3) corresponding to 6  samples; (ii) the southern lineage haplogroup was composed of 22 haplotypes including 3 Dinaric haplotypes corresponding to 6  samples and 6 Rhodopes-Rila haplotypes corresponding to 55  samples.In a total of 319 successfully sequenced Western Capercaillie mtDNA CRI samples collected in Central and South-Eastern Europe , we idenry trees  and regrboreal lineage exhibited a star-like topology of the MSN tree radiating from a central haplotype (TuB39)   , which i44 link)  consisteThe highest haplotype diversity was found for the Alpine group of populations . NucleotST test using the Tamura-Nei model of nucleotide substitutions, the Rhodopes-Rila group appeared distinctly differentiated from both other population groups with statistically significant (p<0.05) pairwise FST values of 0.7867 for Rhodopes-Rila vs. Alps, 0.7988 for Rhodopes-Rila vs. Dinarides and 0.0548 for Alps vs. Dinarides population pairs. Similarly, the Exact test of population differentiation supported the separation of the three sampled geographic groups with statistical significance (p<0.05). The estimated level of differentiation between Alpine and Dinaric populations was an order of magnitude lower when compared to Alps vs. Rhodopes-Rila or Dinarides vs. Rhodopes-Rila, suggesting a high level of connectivity between Alpine and Dinaric populations in the past. The low level of population differentiation between Alpine and Dinaric populations was also supported by the number of shared haplotypes , representing 37.25% and 88.67% of samples for the Alpine and Dinaric groups, respectively. In contrast we found no shared haplotypes between the Alpine and Rhodopes-Rila groups of populations and a single shared haplotype for the Dinaric and Rhodopes-Rila groups (TuB25), representing only 1.33% and 1.64% of samples, respectively. Population group differentiation estimates were consistent with corrected average number of pairwise differences between population groups  and a majority of boreal lineage individuals outcompeting the southern lineage population during the post-glacial geographic and demographic expansion of the boreal lineage. Under this assumption, we tested only the boreal lineage portion  of the Dinaric population (Dinarides-A). The mismatch distribution for Dinarides-A was unimodal and smooth , so agreement with neutrality could not be rejected based solely on Tajima's D test boreal lineage, with the highest haplotype diversity (15 different haplotypes) of the sampled populations. Eight (53%) of the 15 Alpine haplotypes were novel, previously not described. It should be noted that haplotype TuB39  was not considered to be novel since it is assumed to be identical to haplotypes Tu7 boreal lineage according to the mtDNA data has been discussed as a result of its rapid post-glacial expansion to its current habitat range All sampled individuals from the Slovenian Alpine populations belong to the boreal lineage being distinctly dominant, with 96.00% of the samples. Tu11 is the single most dominant haplotype in the Dinarides, representing 68.67% of the samples. Such a prevalence of a single haplotype is in stark contrast to the neighbouring Alpine and Rhodopes-Rila populations, where the most dominant haplotypes represent only 24.51% (Tu11) and 36.07% (T2), respectively. The above-mentioned single-haplotype-prevalence and overall low genetic diversity/high homogeneity could be the result of severe population decline and isolation. Similarly low genetic diversities have been reported for isolated capercaillie populations in the Black Forest and Thuringia, where a severe reduction in numbers has occurred in recent decades The Dinaric populations are composed of representatives of both lineages, with the southern lineage individuals (6 different haplotypes) representing 90.16% of the sample. The multimodal and rough mtDNA CRI mismatch distribution and the presence of few frequent haplotypes suggest that the Rhodopian population is in demographic equilibrium (stationary) or declining. Similar demographic trends have been reported for Cantabrian and Pyrenean populations, which are also composed of both lineages ST values, the results of Monmonier's maximum difference algorithm and AMOVA support claims that the Bulgarian capercaillie is very likely a different subspecies  from the Alpine and Dinaric capercaillie . The results of these population differentiation tests are consistent with the proposed subspecies distribution The Bulgarian Rhodope and Rila mountains population is composed of both lineages, with southern lineage individuals in the Dinarides confirms our hypothesis that at least the SE Dinarides served as a glacial refugium for capercaillie during the last glacial maximum (LGM). Southern lineage individuals were discovered in the central and south-eastern parts of the mountain range  and appear to be randomly distributed, since we were unable to identify any local predominantly southern lineage demes. We hypothesize that southern lineage individuals could be considered to be descendants of the capercaillie population existing in the area during LGM and out-competed by the boreal lineage sometime during the period following the LGM and expansion of the latter lineage. On this assumption, the Dinarides can be identified as the north-westernmost contact zone of the two lineages in the Balkans. The historic demographic expansion of the boreal lineage in the region is supported by the results of demographic analyses in this study. Our findings extend the known species LGM habitat range in the Balkans Dinaromys bogdanovi) Clethrionomys glareolus) Triturus vulgaris) saxatilis and graeca subspecies of Rock Ptarmigan (Alectoris graeca) Oporornis tolmiei) The presence of southern lineage was more abundant in the Dinarides in the more recent past and that the current low abundance is the result of a severe population decline (population bottleneck or near-bottleneck conditions) with subsequent population recovery through limited numbers of surviving Dinaric individuals and boreal lineage migrants from the Alps. An alternative explanation of the presence of southern lineage individuals in the Dinarides is the possibility of a historic exchange of migrants from the Rhodope-Rila-Pirin massif through the Osogovo-Belasica mountain range in the east, and the West Vardar-Pelagonia mountain range and Šar Mountains in the central and western part of FYR Macedonia.It is plausible that the southern lineage haplotypes suggest that these two populations have been effectively isolated for a considerable time. It is plausible that the constant anthropogenic pressure, which perhaps started already in pre-antiquity, caused the destruction of suitable forest habitats in most of Macedonia and consequently the interruption of a Dinarides-Macedonia-Bulgaria capercaillie habitat corridor.The presence of capercaillie in Macedonia has not been factually confirmed since at least 1946 th and 18th centuries th century, forests were fragmented and reduced to 20% in the Alps and 30% in the Dinarides, with a significant reduction of Western Capercaillie populations th to early 19th centuries have also been reported for other alpine countries In addition to current population decline, historic records of land use suggest a possibility that severe population reduction conditions also existed for capercaillie in Slovenia between the 15Changes towards more sustainable land use practices introduced in the 19th century led to a gradual increase in forest cover and capercaillie population numbers in Slovenia, which reached a peak between 1910 and 1930 th to 18th centuries in the Dinaric region in a southeast direction from Slovenia, we propose a scenario explaining the present low level of genetic diversity, derived from mtDNA CRI data analyses for the Dinaric population. Mature forest habitats in the Dinarides are characterised by a higher proportion of deciduous species and much lower abundance of the essential bilberry Assuming a similar reduction of forest cover in the 15ST values and Monmonier's maximum difference algorithm results. Historic records The above-mentioned differences between the Alpine and Dinaric habitats are not reflected significantly in mtDNA CRI sequence diversification of respective populations, as evident from low pairwise Fsouthern lineage expanding throughout Europe, at least to Romania and Bulgaria, while the boreal lineage expanded in Asia and North-Eastern Europe. During the LGM, the lineages retreated to separate refugia: Iberia and the Balkans for the southern lineage, South Asia or Beringia for the boreal lineage. After the LGM, the boreal lineage expanded and replaced the southern lineage in most of Eurasia (perhaps being more competitive) with the latter surviving only in Cantabria, the Pyrenees and, to a lesser extent, in the Balkans – all at the extreme southern edge of the species habitat range. (ii) Rodríguez-Muñoz et al. southern lineage. They discussed the possibility of the southern lineage being broadly distributed in pre-glacial Europe, with distribution becoming fragmented with the advance of glaciation. The southern lineage may have survived in Mediterranean refugia, with new boreal lineage haplotypes evolving in the Balkans or Italian refugia and expanding throughout Eurasia following climate warming and the retreat of the ice sheet. The latter authors also predicted the existence of southern lineage haplotypes in the Balkans or Italy, a hypothesis confirmed by Duriez et al. Our phylogenetic analyses regrouped the haplotypes discovered in the studied populations into two distinct haplogroups (clades). The existence of two distinct capercaillie lineages in Eurasia has been demonstrated previously by microsatellite southern lineage. Of particular interest is that, according to ML and the MSN tree, the two lineages are linked via haplotypes found in Bulgaria  Strix aluco) boreal lineage evolved in colder boreal-like habitats of glacial Eurasia, and as such was better adapted to and more competitive in similar climatic conditions, offers a possible explanation of why the boreal lineage was more successful than the southern lineage in colonizing Eurasia during successive habitat changes following the retreat of glaciation  that the southern lineage appeared ancestral to the boreal lineage in 76% and that haplotypes originating in the Balkans (not exclusively T3-Tu44) represented 74% of the closest or one of equally probable closest interlineage haplotype pairs.Both MSN and ML trees in our study also suggest the ancestral position of the Additional samples from the Italian Alps, the Balkans and other parts of SE Europe, and the southern edge habitat range in Asia (Russia and Kazakhstan) should be analysed to gain more accurate and reliable results in relation to the geographic and evolutionary origin of lineages of Western Capercaillie. Furthermore, evidence is mounting that, in addition to traditionally recognised Mediterranean glacial refugia, smaller glacial refugia also existed further north in the Carpathians and the Carpathian Basin in Central and SE Europe boreal lineage evolved from the southern lineage in the Balkans during the period of major glaciations. Rodríguez-Muñoz et al. T. urogallus mtDNA CR Accurate calculation of the capercaillie lineage divergence time would provide a criterion for estimating the validity of the proposed scenario that the Analysis of a mitochondrial DNA control region, although a reliable genetic marker for inferring the long term demographic history, phylogeography and basic phylogeny of the studied species, also has its limitations. In particular, analyses of mtDNA CRI have limited power to detect more recent demographic and evolutionary events, such as recent bottlenecks, interlineage hybridisations and additional, sub-lineage, diversifications. Analyses of additional genetic markers, such as nuclear microsatellites, would be required to improve the significance and reliability of the estimates presented in this study, foremost on the level of population genetics and higher resolution phylogenetic analyses.Similar to most western, central and southern European capercaillie populations Principal threats to capercaillie in the region include: (i) human disturbance of habitat areas due to mountain tourism and traditional picking of wild berries, mushrooms and herbs, (ii) destruction of old forest habitats by forestry and farming operations, (iii) climate change and forest development to more deciduous and thus less suitable stands, (iv) high predator densities and (v) collisions with wire fences for the Slovenian Alps and Dinarides southern lineage individuals, similar to Cantabrian and Pyrenean populations southern lineage in the Balkans to the Dinarides. As evident from the distribution map  and that each should be considered to be a separate conservation Management Unit (MU). We propose that the three populations sampled in our study be considered to be distinct MUs, due to differences in habitat characteristics and different genetic lineage composition. An effort, however, should be made to coordinate conservation strategies for the Slovenian Alpine and the entire Dinaric population, since they once formed a connected metapopulation. Although Bulgarian populations are reportedly stable southern lineage core area in the Balkans.The first large-scale sampling of the Bulgarian population, to our knowledge, conducted in this study, revealed that it is composed predominantly of tion map , southerminimal viable capercaillie population size of 500 Due to the low genetic diversity revealed in this study and its isolated status, the current Dinaric population appears to be particularly vulnerable to further habitat fragmentation, loss of intra-population connectivity and population size reduction. Continuing population decline may lead to a further reduction in genetic diversity, with potentially severe consequences in terms of the fitness of the population and, ultimately, its survival. Loss of functional connectedness with the Alpine population is probably affecting both but will probably have a more negative long-term effect on the Dinaric population. Currently, the Dinaric population is estimated to be no more than 1400–1500 individuals General conservation measures in the region should focus on preservation of suitable habitat areas, sustainable forestry and agricultural practices and landscape management, effective enforcement of a hunting ban and poaching and predator control. A particular effort should be made to secure and possibly increase connectivity between local populations through adapted forest management ensuring revitalisation and expansion of habitat corridors (towards a more stable metapopulation structure). The re-establishment of the connectedness of the Alpine and Dinaric populations in Slovenia would be of particular importance for the long-term survival of the capercaillie in the Western Balkans region.southern vs. boreal lineage). Until the remaining Balkan populations are analysed , we propose that Balkan populations should be treated as mixed populations composed of individuals from both lineages. Furthermore, no Tetrao urogallus reintroduction efforts in the Balkans should include only the boreal lineage but should also include the southern lineage, which appears to be more flexible and better adapted to forest stands with a higher proportion of deciduous trees boreal lineage and the Cantabrian Mountains or Pyrenees for the southern lineage) could in fact decrease the fitness of populations under rescue by introduction of suboptimal traits. Further issues associated with reintroduction efforts include the risk of introduced individuals and their offspring outcompeting the endangered native population. It is thus vital to closely monitor the effects of introduced individuals on the population under rescue. Genetic rescue is probably the only viable solution to assure the survival of the endangered Western Capercaillie populations in the Balkans, such as the small and isolated population in the western Balkan Mountains  from sites covering the species habitat in the Slovenian Alps, Dinarides , and Rhodope and Rila Mountains (Bulgaria)  , includiulgaria) . Most saT. urogallus and 5 sequences of Black-billed Capercaillie (Tetrao parvirostris) Faeces samples were lyophilized and stored in sealed plastic bags with silicagel at −20°C. Tissue samples were preserved in absolute ethanol in sealed vials and stored at −20°C until analysis. For phylogenetic analysis, we also used 51 mitochondrial DNA control region I (mtDNA CRI) sequences retrieved from Genbank  and 25 mg (dry weight) of material used for extraction. Both tip and blood clot in the superior umbilicus of the feather shaft were used for DNA extraction Total DNA from faeces was extracted with the PowerSoil DNA isolation kit , DNA from feathers and tissue samples was extracted with the UltraClean Tissue and Cells DNA kit . Faeces samples (100 mg dry weight) were first cut into smaller pieces using sterile scissors and rehydrated with 2× volumes of DNA/RNA-free sterile dH2, 50 µM of each dNTP, 0.6 µM of each primer, 1.5 U of AmpliTaq® Gold (Applied Biosystems) and 1–5 µl of DNA extract. Reactions were performed using Applied Biosystems GeneAmp® PCR System 9700 thermocyclers with a polymerase activation step , 5 touch-down cycles , 45 amplification cycles  and a final extension step . To assess possible contaminations or other problems, each batch of PCR reactions also included a DNA extraction negative control, a PCR negative control containing DNA/RNA-free water instead of DNA extract and a positive control containing DNA extract from the same tissue sample from Bosnia and Herzegovina. PCR products were resolved in 1.5% agarose gels and visualized by ethidium bromide staining. PCR yielding non-target amplification products were separated as above, target products were excised from the agarose gel and purified with Wizard® SV Gel and PCR Clean-up System (Promega). Sequencing was performed commercially at Macrogen Inc.  in both directions using amplification primers. Sequencing chromatograms were manually checked and corrected using FinchTV v1.4.0 software (Geospiza Inc.).We targeted the first 435 nucleotides of the mitochondrial control region I (CRI), the most variable domain of the mitochondrial DNA (mtDNA) control region in grouses Sequences were aligned with ClustalX v2.0.12 HomOBSSΠHPresPres = 2·NeF·μ, NeF is the effective female population size, μ is the mutation rate per site per generation) and population growth force parameters – g For population-level analysis, we pooled the populations into three geographical groups: Alpine, Dinaric and Rhodopes-Rila . We usedSTPopulation pairwise genetic distances FST, exact ti/Tv ratio according to the jModelTest v0.1.1.In order to detect possible genetic barriers and their locations between or within the studied populations, we applied Monmonier's maximum difference algorithm i/Tv ratio according to jModelTest v0.1.1. For AMOVA, the sampled populations were assigned to two groups according to Barrier v2.2: the first including all sampled Alpine and Dinaric demes and the second including Rhodopes-Rila demes.We used Arlequin v3.11 for computing analysis of molecular variance (AMOVA), assuming Tamura-Nei distances with a T


Campostoma ornatum is a freshwater fish endemic to montane and semiarid regions in northwest Mexico and southern Arizona. Its wide range of distribution and the previously observed morphological differentiation between populations in different watersheds make this species a useful model to investigate the biogeographic role of the Sierra Madre Occidental and to disentangle the actions of Pliocene tecto-volcanic processes vs Quaternary climatic change. Our phylogeographic study was based on DNA sequences from one mitochondrial gene  and two nuclear gene regions  obtained from 18 to 29 localities, in addition to a morphological survey covering the entire distribution area. Such a dataset allowed us to assess whether any of the populations/lineages sampled deserve to be categorised as an evolutionarily significant unit.Studies of the phylogeography of Mexican species are steadily revealing genetic patterns shared by different species, which will help to unravel the complex biogeographic history of the region. C. ornatum. One is located in the northern river drainages  and another one is found in the southern drainages . The split between these two lineages took place about 3.9 Mya (CI = 2.1-5.9). Within the northern lineage, there was strong and significant inter-basin genetic differentiation and also several secondary dispersal episodes whit gene homogenization between drainages. Interestingly, three divergent mitochondrial lineages were found in sympatry in two northern localities from the Yaqui river basin.We found two morphologically and genetically well-differentiated groups within C. ornatum populations emerges, following the taxon pulse theory and mainly related with Pliocene tecto-volcanic processes. In the northern group, several events of vicariance promoted by river or drainage isolation episodes were found, but within both groups, the phylogeographic patterns suggest the occurrence of several events of river capture and fauna interchange. The Yaqui River supports the most diverse populations of C. ornatum, with several events of dispersal and isolation within the basin. Based on our genetic results, we defined three ESUs within C. ornatum as a first attempt to promote the conservation of the evolutionary processes determining the genetic diversity of this species. They will likely be revealed as a valuable tool for freshwater conservation policies in northwest Mexico, where many environmental problems concerning the use of water have rapidly arisen in recent decades.Our results indicate that there was isolation between the northern and southern phylogroups since the Pliocene, which was related to the formation of the ancient Nazas River paleosystem, where the southern group originated. Within groups, a complex reticulate biogeographic history for  At present, it is a thriving field of research aiming to disentangle the relative importance of glacial refugia, extrinsic environmental factors and species interactions, aided to the ability of locating species boundaries, phylogeographical breaks and hybrid zones . Congruently, most of the non-significant values corresponded to intra-basin comparisons , but some locations belonging in different drainages also had ΦST-values that were not significant . The ΦST-values for the K = 4 arrangement were all significant, with the highest value resulting from the comparison of Nazas-Aguanaval-Piaxtla with Yaqui2-Casas Grandes  and all other populations, including the Piaxtla River samples   and showed a unimodal distribution that closely fit the expected distribution (data not shown).Therefore, we analysed the demographic history of mtDNA lineages , rather than sampling sites or SAMOVA groupings. This way, we could infer which lineages had experienced expansions, instead of trying to infer demographic expansions in the contemporary populations containing haplotypes of mixed ancestry. Neutrality tests applied to each phylogroup revealed no deviations from neutrality due to population expansion. The only lineage with n) Table . The misC. ornatum, supporting prior preliminary investigations [cytb and S7 genes, a result indicative of a long history of isolation between these clades. Such an allopatric fragmentation dated back to the Pliocene , as revealed by the Bayesian coalescent analyses. The absence of a sharp differentiation between the western (Pacific drainages) and eastern (Atlantic or endorheic basins) lineages, together with the lack of an altitudinal gradient of haplotype diversity (data not shown) lead us to discard a recent (Upper Quaternary) origin for the observed phylogeographic pattern.Our genetic and morphological results unambiguously showed two well-differentiated evolutionary groups within igations . The firC. ornatum, rather than recent habitat fluctuations caused, for instance, by alternating glacial/interglacial stages. The genetic pattern we observed allowed us to infer common river piracy phenomena.In the light of this, and following the proposal of distinguishing between competing models of diversification for North and Mesoamerica , we can P = 5.2%) is an early Pliocene vicariant event that split a widespread common ancestor. According to our dating, the allopatric fragmentation between the northern and southern lineages took place around 3.9 Mya (CI = 2.2-5.9). This scenario is also supported by the high genetic diversity found in phylogroup V   [Gila, Cyprinella, Ictiobus and Cyprinodon genera [C. ornatum in the Aguanaval and Piaxtla Rivers is caused by a recent dispersal event from Nazas River, as revealed by the phylogenetic trees, the cytb network and ΦST results. This hypothesis is also in agreement with the biogeographic scenario postulated for semi-aquatic snakes of the genus Thamnophis [The existence of an ancient paleosystem related to the Nazas River has been put forward in prior literature in light of the distribution of its ichthyofauna and herpetofauna -46. Numeiformes) . The ancn genera ,48-50. TC. ornatum populations, following a clear pattern of taxon pulse episodes [A complex, reticulate biogeographic history in time and space emerged within the northern episodes . This moepisodes . At thisepisodes ,54.P = 2.3% ± 0.4% between Conchos and Fuerte Rivers populations, excluding the BCY and URI populations), pointing to a long evolutionary history in isolation  the position of the URI population in the phylogenetic tree . This result disagrees with the existence of the hypothesised ancient pluvial Lake Palomas [ca. 200 000 years ago). This event would have formed the current Guzman basin, which include the Mimbres, Casas Grandes, Santa Maria and Santa Clara River endorheic basins [All markers revealed that the Santa Clara population is the most divergent  in the phylogenetic tree Figure  and netw formosa ) and .C. ornatum populations. Haplotypes found in this northern basin are found in three different mitochondrial subnetworks   Figure  and thei) Figure , which w) Figure . This inΦST value, which may be indicative of recent gene flow. Faunal exchanges between the Yaqui River and surrounding areas have been previously reported [Concerning descriptors of genetic diversity, the mitochondrial subnetwork VI had the greatest number of haplotypes, segregating sites, haplotype diversity and average number of differences Table . In lighreported ,62. In freported .P = 1.5%, also suggesting a relatively long isolation. Interestingly, the Terapa population (TER), also belonging to the Yaqui River basin, shows a high amount of divergence with respect to other Yaqui populations (P = 1.3%). These results are, again, congruent with episodes of isolation in different tributaries of the Yaqui River, followed by a secondary dispersal from some isolated demes to other Yaqui tributaries. We postulate this hypothesis as the most likely explanation for the presence of those three divergent mitochondrial lineages at Cabullona and Agua Prieta.The Yaqui River system hosts three divergent haplogroups  that occur in sympatry at the Cabullona and Agua Prieta locations . One of those haplogroups clusters with haplotypes found in the Casas Grandes River forming subnetwork VII. Haplotype cab7845 falls within the second haplogroup (subnetwork VI), closely related with haplotypes form the Tomochic and Papigochic sites (Yaqui River). The third lineage (subnetwork V) is composed by only two haplotypes and shows a mean genetic divergence of P = 2.1% ± 0.3%) with other members of its monophyletic group  site, a small spring located at the Sonora River Basin, belong to a separated subnetwork (II) and show large genetic distances (sensu [C. ornatum corresponding to the following groups: (i) the southern lineage, (ii) the northern lineage and (iii) the Yaqui river basin. While acknowledging that the Yaqui belongs to the northern lineage, we define this third sub-hierarchical ESU due to the existence of three deeply divergent lineages at Agua Prieta. Here, the term sub-hierarchical does not infer subordination but rather a frame of reference on a phylogenetic continuum [C. ornatum throughout its range.In the light of our genetic and morphologic results, we hereby define three ESUs (sensu ) within ontinuum . At presC. ornatum with a complex, reticulate biogeographic history with repeated events of isolation and dispersal, which is consistent with the taxon pulse theory. This complex history could be related to the dynamic tecto-volcanic activity that has occurred in the region since the early Pliocene. Both genetics and morphology revealed a strong differentiation between the southern and northern populations of C. ornatum. In light of this, a taxonomic revision is necessary to clarify the taxonomic status of the two well-differentiated groups within Campostoma ornatum. The differentiation between the northern and southern taxa was likely due to the isolation of the paleosystem that connected the Nazas with other northern river basins during the Pliocene. In addition, we determined the presence of three very divergent mitochondrial lineages occurring concurrently in the northern Agua Prieta Stream (Yaqui river basin). We postulated that episodes of isolation and the subsequent admixture of lineages from different sources could explain the co-occurrence of these lineages at Yaqui. In addition, three ESUs are proposed that cover the full range of the Mexican stoneroller. The pattern obtained for Campostoma ornatum paves the way for a future comparative phylogeographical study aimed at disentangling the evolutionary factors that shape the genetic structure of this species and its populations in the northwest of Mexico.We found populations of Camposoma ornatum individuals were collected at 29 locations in northwest Mexico   Table . To confollowing , i.e., u2 piece of the pectoral fin using the High Pure™ PCR Template Preparation Kit , following the manufacturer's instructions. We amplified a 1140-bp fragment of the mitochondrial cytochrome b gene (cytb) in 285 individuals by Polymerase Chain Reaction (PCR) and the GLUf - THRr primer pair [Genomic DNA was extracted from a <0.25 cmmer pair .Taq DNA polymerase (5PRIME), 0.2 mM of each dNTP, 0.2 μM of each primer and 50-100 ng of DNA. Similar conditions were employed to amplify the first intron of the S7 ribosomal protein gene in 56 specimens , 1 U S7RPEX2R ) and appS7RPEX2R ).cytb started with an initial denaturing step at 95°C for 2 minutes, followed by 35 amplification cycles at 94°C for 1 minute, 56°C for 1.5 minutes, 65°C for 1 minute and a final extension step at 65°C for 7 minutes. PCR for S7 started with an initial denaturing step at 95°C for 2 minutes, followed by 30 amplification cycles at 95°C for 1 minute, 59.5°C for 1.5 minutes, 65°C for 2 minutes and a final extension step at 65°C for 7 minutes. PCR for RAG1 started with an initial denaturing step at 94°C for 2 minutes, followed by 40 amplification cycles at 94°C for 45 seconds, 56-58°C for 1 minute, 68°C for 2 minutes and a final extension step at 68°C for 7 minutes.PCR for Cytb PCR products were purified and bidirectionally sequenced at the DNA Sequencing Service . S7 and RAG1 PCR products were purified and bidirectionally sequenced on a 3130 × l Genetic Analyser  at SAI . New sequences were deposited in GenBank . For phylogenetic analyses, RAG1 alignments included sequence EU082549 from Durango (Nazas River basin). Mitochondrial alignments were straightforward and unambiguous. For the analysis of nuclear DNA, the sites where individuals contained heterozygous genotypes for the sampled nuclear loci and any heterozygous base pair positions were coded using standard degeneracy codes [DNA electropherograms were checked and aligned using CODONCODES 3.5.6 . Mitochondrial alignments were straightforward. Apart from the 285 individuals sequenced for the present work, phylogenetic analyses included three cy codes . Heterozcy codes , which ucy codes . We emplcy codes , runs coS), nucleotide diversity per gene (π), haplotype diversity (h) and the average number of nucleotide differences (k) were calculated using DNAsp 5.1 [R) was computed after rarefaction to three individuals [http://www.pierroton.inra.fr/genetics/labo/Software/) for all populations that included at least three individuals.Haplotypes and their frequencies, standard indices of genetic variation such as the number of segregating sites , Bayesian inference (BI) and maximum likelihood (ML) methods. Distance analysis was carried out as implemented in PAUP *4.0b10 [Campostoma oligolepis (accession number DQ324064) as an outgroup to root the tree. Using sequences from Campostoma pullum (EU082477) or Campostoma pauciradii (DQ324065) as outgroup did not significantly change any of the results shown in the present study.As DNA variation may arise through different molecular evolution models, we ran jMODELTEST 0.1.1  to selec *4.0b10  for 1000 *4.0b10 , and two *4.0b10 , "burn iCampostoma pauciradii (DQ324065) by forcing a connection limit of 130 steps. Similarly, 95% SP networks were calculated for S7 and RAG1 datasets, both using only homozygous positions  and the separated haplotypes inferred by PHASE.The phylogeny of the haplotypes was also inferred using the statistical parsimony (SP) criterion . The 95%Campostoma, we applied a rate of molecular evolution of 1.05% per million years, calibrated for the cytb in North American Phoxinini [We estimated divergence times among sets of mitochondrial sequences and their associated credibility intervals using the Bayesian coalescent approach as implemented in BEAST 1.4.6 . Markov hoxinini  and widehoxinini ,80. The hoxinini  to checkFST analogues (ΦST) based on the Tamura-Nei model of sequence evolution. Statistical significance of covariance components (and Φ-statistics) from AMOVAs and pairwise ΦST values was determined on the basis of the distribution of values obtained from 1000 permutations of the data.We tested for evidence of population subdivision under different criteria using an analysis of molecular variance (AMOVA) as implemented in ARLEQUIN v.3.5.1.2 . We alsok values from 2 to 29.We also assessed population structuring using the simulated annealing procedure as implemented in SAMOVA  1.0 . We usedcytb phylogroups was first explored using Fu's Fs [R2 [The demographic history of the  Fu's Fs  and R2 [s Fs [R2  statistiFs and R2 tests with the mismatch distribution of the pairwise genetic differences [We characterised the demographic expansions detected for all phylogroups, rejecting the null hypothesis under the ferences , as implODD conceived the study, collected the samples, performed phylogenetic analyses and drafted the manuscript. MV obtained the DNA sequences, performed the population genetic analyses, participated in the phylogenetic survey and contributed in the writing of the paper. RPR collected the samples, performed morphological and molecular dating analyses and was involved in writing the manuscript. NR carried out the molecular labwork and participated in the sequence alignment. ID conceived the study, participated in its design and coordination as well as was involved in the writing. All authors read and approved the final manuscript.Campostoma groupsInformative meristic characters for Northern and Southern . Morphological character values registered throughout the range of C. ornatum: counts in lateral line (LLS), predorsal (PrDS) and the circumferential scales (CircS).Click here for fileΦST-values according to phylogenetic groupingMatrix of population pairwise . Matrix of population pairwise ΦST-values according to phylogenetic grouping and obtained under the Tamura-Nei model of sequence evolution. All values were significant after correction for multiple testing.Click here for fileΦST-values by river basinMatrix of pairwise . Matrix of pairwise ΦST-values by River basin and obtained under the Tamura-Nei model of sequence evolution. All values are significant after correction for multiple testing.Click here for fileΦST-values by sampling localityMatrices of pairwise . Below diagonal: Pairwise ΦST values calculated under the Tamura-Nei model. Above diagonal: same calculations using haplotype frequencies. Significance was evaluated after 10000 permutations.Click here for fileΦST-values according to SAMOVA (K = 4) groupingsMatrix of population pairwise . Matrix of population pairwise ΦST-values according to SAMOVA (K = 4) groupings and obtained under the Tamura-Nei model of sequence evolution. All values were significant after correction for multiple testing.Click here for fileΦST-values according to SAMOVA (K = 13)Matrix of population pairwise . Matrix of population pairwise ΦST-values according to SAMOVA (K = 13) groupings and obtained under the Tamura-Nei model of sequence evolution. All values significant after correction for multiple testing.Click here for file


Rheum palmatum and R. tanguticum are important but endangered medicinal plants endemic to China. In this study, we aimed to (i) investigate the level and pattern of genetic variability within/among populations of those species; (ii) evaluate genetic differentiation between both species and its relationships and ascertain whether both species are consistent with their current taxonomical treatment as separate species; and (iii) discuss the implications for the effective conservation of two species.Both R. palmatum and R. tanguticum were collected, covering the entire distribution range of two species in China. The genetic variation within and among 30 populations was evaluated using inter-simple sequence repeat (ISSR) markers.Total 574 individuals from 30 populations of H) and Shannon's index (I) of both species were high at species level . But for both species, the genetic diversity was low at population level, and average within-population diversity of R. palmatum was H = 0.1438, I = 0.2151, and that of R. tanguticum was H = 0.1415, I = 0.2126. The hierarchical AMOVA revealed high levels of among-population genetic differentiation in both species, in line with the gene differentiation coefficient and the limited among-population gene flow . By contrast, only 6.52% of the total genetic variance was partitioned between R. palmatum and R. tanguticum. Bayesian analysis, UPGMA cluster analysis, and PCoA analysis all demonstrated the similar results. A significant isolation-by-distance pattern was revealed in R. palmatum , but not in R. tanguticum . Based on these results, effective conservation strategies were proposed for these two species. The small molecular variance between R. palmatum and R. tanguticum revealed that they had a common ancestor, and we considered that these two species might not be good species.Twelve selected ISSR primers generated a total of 175 fragments, 173 (98.86%) of which were polymorphic. The Nei's gene diversity ( Preserving genetic diversity of species is one of the primary goals of conservation planning, because long-term survival and evolution of species depend on the maintenance of sufficient genetic variability within and/or among populations to accommodate new selection pressures brought about by environmental changes Rheum Section Palmata A. Los. (Polygonaceae) is endemic to China, and comprises four described species  Maxim. ex Balf., including var. tanguticum and var. liupanshanense C. Y. Cheng et T. C. Kao, and R. laciniatum Prain) R. officinale are called “south rhubarb”, while that of R. tanguticum and R. palmatum are called “north rhubarb”.Because the rhubarb from R. tanguticum has the best quality, R. tanguticum has become endangered and was listed in the China higher plants endangered list due to the overexploitation and the limited distribution R. officinale and R. palmatum face great pressure exacerbated by the reduction of the wild resources of R. tanguticum, and both are treated as “threatened” species in China. In previous work, we have investigated the genetic diversity of R. officinaleR. tanguticum were reported but based on very limited samples  based on PCR technique, have a better reproducibility than randomly amplified polymorphic DNA (RAPD) In the present study, ISSR markers were employed to (1) investigate the level and pattern of genetic variability within/among populations of those species; (2) evaluate genetic differentiation between both species and its relationships and ascertain whether both species are consistent with their current taxonomical treatment as separate species; and (3) discuss the implications for the effective conservation of two species.R. palmatum and R. tanguticum  and RP13 , respectively. In R. tanguticum, the highest genetic diversity existed in population RT1  and the lowest in RT6 . The average within-population diversity of R. palmatum  was slightly higher than that of R. tanguticum , and the former also harbored more ISSR diversity at the species level .In general, ISSR variation within populations was very low in each species, and varied erratically across localities . In R. pR. palmatum and R. tanguticum, while most of this variation still resided among populations within species (93.48%) .The hierarchical AMOVA for all ISSR data set showed that only 6.52% of total genetic variance was partitioned between (93.48%) . By contR. palmatum in Mts. Qinling and five populations of R. palmatum in Mts. Daxue, and three populations of R. tanguticum in Mts. Min. To investigate the gene flow among populations in the same region, both Gst and Nm were analyzed for eight Mts. Qinling populations , five Mts. Daxue populations  and three Mts. Min populations . Both Gst and Nm was 0.470 and 0.563 for eight populations from Mts. Qinling, 0.436 and 0.646 for five populations from Mts. Daxue, and 0.351 and 0.927 for three populations from Mts. Min.,On some mountains, more than one population was sampled. For instance, there were eight populations of R. palmatum, and from 0.1015 (RT3 vs. RT5) to 0.3165 (RT1 vs. RT6) in R. tanguticum.POPGENE analysis revealed that Nei's unbiased genetic distances ranged from 0.0992 (RP19 vs. RP20) to 0.3324 (RP3 vs. RP21) in R. palmatum from Nanmulin county of Tibet formed a sole group, and the remaining populations of R. palmatum and R. tanguticum formed the other group, which can be divided into two subgroups. One subgroup included RP1 population in R. palmatum from Qinshui county of Shanxi province, and the other contained populations from RP2 to RP17 (16 populations), from RP19 to RP21 (3 populations), and all populations in R. tanguticum. The populations from a same species were not clustered in the same group or subgroup.The UPGMA tree based on Nei's unbiased genetic distance clustered all populations into two major groups . PopulatR. palmatum  and the only population from R. tanguticum var. liupanshanense (RT1 from Ningxia) occupied similar position along the axis 1. Three populations from Tibet  in R. palmatum also had similar genetic similarity. These results showed that both R. palmatum and R. tanguticum formed a conspicuous close-knit group.The two-dimensional PCoA analysis for all 574 individuals in both species accounted for 28.42% (axis 1) and 18.35% (axis 2) of total variance, respectively . In the K value with the highest value of ΔK for the 574 individuals was K = 2 .In the ISSR admixture analysis using STRUCTURE, the real as K = 2 . The proas K = 2  which reR. palmatum were positively correlated with their geographical distances with significance , indicating a significant isolation-by-distance pattern. However, no significant correlation between genetic distances and geographical distances was found for populations in R. tanguticum .The Mantel's test results showed that genetic distances among populations in R. palmatum: PPB = 41.42%, H = 0.1438, I = 0.2151; R. tanguticum: PPB = 41.90%, H = 0.1415, I = 0.2126), and relatively higher at species level , which is similar to the level of genetic diversity reported by Hu et al. on P. tanguticum  R. palmatum was higher than that of R. tanguticum, but it was slightly lower than that of R. officinale  based on isozyme analysis R. palmatum) usually have higher intraspecific genetic diversity than their narrowly distributed counterparts (i.e. R. tanguticum) Genetic diversity is one of the most important attributes to any population. Environments are constantly changing, and genetic diversity is necessary if populations are to continuously evolve and to adapt to new situations R. palmatum and R. tanguticum is probably related to its unique evolutionary history. Fossil records indicated the occurrence of Polygonaceae at least as far as the Paleocene Rheum and its sister groups was dated to the Miocene, about 22 million years before present, based on a molecular clock hypothesis Rheum are anthophilous R. palmatum and R. tanguticum have more opportunity to accumulate mutant. Moreover, the natural geographic distribution may affect the genetic diversity level for a species and the widespread species have higher level of genetic variability than narrowly distributed ones in general R. palmatum and R. tanguticum are widespread in China, especially for R. palmatum which is distributed from southern Shanxi to the central of Tibet The high intraspecific genetic diversity of R. palmatum  and R. tanguticum , and both were slightly lower than R. officinale  R. tanguticum revealed by ISSR  Rhodiola alsia (Crassulaceae) Torreya jackiiHigh level of among-population genetic differentiation was revealed in both R. palmatum, indicating that geographic isolation has significant effect on genetic structure in this species. Samples of R. palmatum in this study covered the whole distribution range. R. palmatum growths only in related higher altitude mountains , and this isolated distribution undoubtedly restricts gene exchange via seed or pollen dispersal. R. palmatum and R. tanguticum are pollinated by insects R. palmatum and R. tanguticum is 41% and 48% in the field, respectively R. palmatum (mN = 0.432) and R. tanguticum (mN = 0.507), which enhance the genetic differentiation among populations in each species. Gene flow among populations on the same mountain for both species was also restricted. FThe Nm of eight populations in R. palmatum from Mts. Qinling and five populations in R. palmatum from Mts. Daxue was 0.563 and 0.646, respectively, while the Nm of three populations in R. tanguticum from Mts. Min was 0.927. Wright determined that Nm>1 is sufficient to overcome the effects of genetic drift R. tanguticum, which also provides further evidence of the existence of genetic drift R. tanguticum, implying that gene flow might also have occurred among geographically distant populations. This possibility cannot be ruled out because this species has a long history of cultivation. It has been widely cultivated in Qinghai and Gansu provinces, and long-distance inter-regional exchange of seedlings or seeds may have happened due to anthropogenic activities.The high level of population genetic differentiation within a species may be explained by several factors, including geographic distribution, breeding system, genetic drift or genetic isolation of populations R. palmatum and R. tanguticum is called “north rhubarb”, which has the best quality. However, both species are morphologically similar in many characteristics , and it is very difficult to identify them. According to the description of Flora of China In China medicinal material markets, it is well known that the rhubarb derived from roots and rhizomes of STΦ = 0.0652) was detected between R. palmatum and R. tanguticum, which indicates that these two morphologically closely related species are genetically similar. The samples used in this study were collected from the whole distribution regions of two species, and thus were more representative than those in previous studies R. palmatum are nested with that of R. tanguticum   . In addiR. qinlingense Y. K. Yang, D. K. Zhang et J. K. Wu in the Sect. Palmata was reported, which was collected at Taibai county, Shaanxi province (Mts. Qinling) according to the diverse leaf characteristics, i.e. the depth of leaf division  R. qinlingense) and other populations was 0.3068 (RP18) and 0.1043 (RP8), respectively, with a mean of 0.2149. Cluster analysis showed strong correlations between RP7 and RP8, as well as RP4 and RP6. RP7 was not classified as one group based on ISSR markers. SinceRP7 did not stand alone in these analyses, it is necessary to deliberate when classifying it as a separate species.A new species R. palmatum, and RT9 in R. tanguticum, should be given priority for in situ conservation, and their habitats should be protected and the exploitation of wild resources be forbidden. If ex situ conservation is required in gardens, samples should be collected from as many populations as possible. Considering both R. palmatum and R. tanguticum have a bulk demands and a long history of utilization in China, and their wild resources have long been subject to excessive collection. It would be good if sufficient artificial plantations can be established and meet the market demands for these two species. Only in this way can the excessive collection of their wild resources be alleviated.The results of the present study showed that both species have high genetic diversity at species level, however, extraordinarily high among-population genetic differentiation existed in both species. As high genetic differentiation resided among the populations of each species, also each of the remaining populations could represent a large proportion of the genetic variation of these two species; therefore, great efforts should be made to preserve all the extant populations and their habitats in the field, especially for those populations with higher genetic diversity, e.g., RP1 and RP12 in R. palmatum nor R. tanguticum is in the list of state-protected plant species , Plants 1999 (5): 3–11; Regulations of the People's Republic of China on wild plants protection, http://www.people.com.cn/item/faguiku/zrzyf/U1020.html). Thus, no specific permits were required for the described field studies. During the samples collection, only a small fraction of a blade was collected to avoid causing any harm to the plants and their habitats.According to regulations of the People's Republic of China on wild plants protection, the permits are required only for those species included in the list of state-protected plant species when it is collected. Neither R. palmatum (21 populations) and R. tanguticum , including Hebei, Shanxi, Shaanxi, Gansu, Sichuan, Qinghai, Ningxia, and Tibet provinces in China. Fresh leaves from 18–20 individuals  were collected randomly in each population, depending on accessibility and population size. Each population was positioned by a GPS, and the detailed locations of the studied populations are listed in From 2008 to 2010, thirty populations were collected throughout the distribution of both Total DNA was extracted from the silica gel-dried leaves using the modified 2× CTAB procedure R. officinaleOne hundred ISSR primers synthesized by Sangon Biological Engineering Technology & Service (Shanghai), according to the primer set published by University of British Columbia, Canada (UBC set No. 9) were used for amplification to standardize the PCR conditions. Twelve of one hundred ISSR primers produced clear, reproducible and relatively high polymorphism bands were selected to amplify all 574 samples . The PCRPPB), Nei's gene diversity (H) I) STG) Nm). An estimate of Nm among populations was computed using the formula of Nm = 0.5(1−STG)/STGThe amplified fragments, with the same mobility according to their molecular weight (bp), were scored in terms of a binary code as present (1) or absent (0). Only those consistently reproducible bands were scored, and smeared and weak bands were excluded. The following genetic diversity parameters were calculated for each population and species using POPGENE version 1.32 Analyses of molecular variance (AMOVA) were performed on the Euclidean distances among ISSR phenotypes by using ARLEQUIN to deduce the significance of genetic divisions between both species as well as among populations within each species. Geographic distances were interpreted by the latitudes and longitudes with Mapinfo 8.0 Program. Mantel test of genetic and geographic distances was carried out to evaluate the correlation between the two data matrices using TFPGA software viz. hybridization), a Bayesian analysis of ISSR population structure was performed on the entire data set using the program STRUCTURE (version 2.3) K) in a sample and to assign individuals to one or more of these populations (K). The number of genetically distinct clusters (K) was set to vary from 1 to 21. The model was run for eight independent simulations for each K, used a burn-in length of 50,000 and a run length of 100,000 iterations. Other parameters were set to default values. Following the program's dominant marker settings, the “no admixture” model was used, and uncorrelated allele frequencies among populations were assumed. The most likely number of clusters was estimated according to the model value (ΔK) based on the second order rate of change, with respect to K, of the likelihood function, following the procedure described by Evanno et al. Finally, to test for genetic admixture across species boundaries (R. palmatum and R. tanguticum revealed by ISSR markers. High genetic differentiation among populations for both species was attributed to the geographical isolation and genetic drift. The observed genetic structure of populations in both R. palmatum and R. tanguticum implied that as many populations as possible should be collected when making conservation strategy. The small molecular variance between both species indicated that they had a common ancestor, and these two species might not be good species.In summary, genetic diversity was low at population level but high at species level for both


Porites panamensis, a hermatypic coral endemic to the ETP. Specifically, we test if levels of diversity and connectivity are higher among abundant than among depauperate populations, as expected by a geographically relaxed version of the Abundant Center Hypothesis (rel-ACH). Unlike the original ACH, referring to a geographical center of distribution of maximal abundance, the rel-ACH refers only to a center of maximum abundance, irrespective of its geographic position. The patterns of relative abundance of P. panamensis in the Mexican Pacific revealed that northern populations from Baja California represent its center of abundance; and southern depauperate populations along the continental margin are peripheral relative to it. Genetic patterns of diversity and structure of nuclear DNA sequences  and five alloenzymatic loci partially agreed with rel-ACH predictions. We found higher diversity levels in peninsular populations and significant differentiation between peninsular and continental colonies. In addition, continental populations showed higher levels of differentiation and lower connectivity than peninsular populations in the absence of isolation by distance in each region. Some discrepancies with model expectations may relate to the influence of significant habitat discontinuities in the face of limited dispersal potential. Environmental data analyses and niche modeling allowed us to identify temperature, water clarity, and substrate availability as the main factors correlating with patterns of abundance, genetic diversity, and structure, which may hold the key to the survival of P. panamensis in the face of widespread environmental degradation.The coral fauna of the Eastern Tropical Pacific (ETP) is depauperate and peripheral; hence, it has drawn attention to the factors allowing its survival. Here, we use a genetic seascape approach and ecological niche modeling to unravel the environmental factors correlating with the genetic variation of  Viable populations persist where reproduction and survival balance or exceed mortality and emigration. The highest abundance will generally occur in a region where conditions are close to the physiological optima of the species and biotic interactions allow the highest population growth rates  populations located near the center of abundance will show higher levels of connectivity and less genetic structure than marginal populations; (3) patterns of diversity and genetic structure predicted by the rel-ACH will be strongly influenced by the presence of significant habitat discontinuities acting as barriers to dispersal; and (4) temperature will be among the most influential environmental variables characterizing the habitat of the species. We found partial agreement with the rel-ACH predictions in the patterns of genetic diversity and structure of colonies of P. panamensis sampled from nine populations along the Mexican Pacific. We argue that these patterns are the result of the limited dispersal potential and discontinuity in habitat availability, the latter being strongly influenced by temperature, water clarity, and hard substrate availability.Here, we address hypotheses regarding the patterns of geographic variation of genetic diversity and structure of a dominant and endemic ETP hermatypic coral in the Mexican Pacific. namensis  is distrnamensis . Populatnamensis . Sea surnamensis . In lighP. panamensis and other zooxanthellate corals in the Mexican Pacific, we conducted surveys between July 2008 and July 2011 at nine localities known to possess live coral colonies of P. panamensis: Bahía de los Ángeles (BLA), Bahía Concepción (BC), Loreto (LO), Bahía de La Paz (LP), Cabo Pulmo (CP), Mazatlán (MZ), Islas Marietas (IM), Carrizales (CA), and Bahías de Huatulco (BH) (P = 0.004) but not for P. panamensis (P = 0.926), hence no standardization was necessary for the latter  . Except e latter .P. panamensis, we used the MaxEnt approach of ENM to map its probability of potential occurrence in the tropical Mexican Pacific and the Gulf of California (GC). MaxEnt is a machine-learning method particularly suited to model species distributions based on presence-only data  and logistic output. The geographic output presents the probability of occurrence of the species. The discriminatory power of the model was evaluated on the basis of the area under the curve (AUC) of the threshold independent receiving operating characteristic (ROC) analysis  resolution . Alkalinn = 2) from Isla María Madre . Sampled colonies were separated by at least 2–3 m to avoid inadvertently resampling the same clone. Coral fragments were suspended in preservation buffer   were collected from live colonies from August 2008 to November 2011  at the sn = 12 per locality), owing to its lower level of polymorphism  from a previous study of P. panamensis in this region. These samples were collected from August 2004 to December 2006 from five localities sampled in this study , and an additional site in the southern GC (Punta Arenas –PA–). See Paz-García et al.  and an anonymous single copy Open Reading Frame (ORF) were PCR amplified using primers ITS4 and ITS5  and nucleotide (π) diversities using Arlequin v.3.1  and the number of private haplotypes (pA) were standardized to n = 12 and n = 21 using the program ADZE v. 1.0  to n = 12 using a manual resampling procedure in combination with Arlequin v.3.1  and of Molecular Variance  as implemented in Arlequin v.3.1 , between central/peninsular  and continental/peripheral  regions (Φct), and among localities within regions (Φsc). In addition, we tested for differences between pairs of localities. ANOVA and AMOVA significance was evaluated through permutation tests and adjusted for multiple testing using the sequential Bonferroni correction  and gene flow (M = m/μ) using a model-based approach involving maximum likelihood Markov Chain Monte Carlo (MCMC), as implemented in MIGRATE v.2.1.3 (Beerli M (magnitude and direction) among populations, assuming a constant per-locus mutation rate (μ). We used 10 short-chain searches  and three long-chain searches  and a burn-in of 10,000 trees to ensure independence from initial conditions.One of the basic genetic predictions of the rel-ACH is that the central populations are more likely to share high levels of connectivity than peripheral populations; hence, we estimated mutation-scaled levels of effective population size (θ = 23 Beerli . MIGRATE3), euphotic zone depth (m) , total particulate organic carbon (mol/kg) , and primary productivity (gC/m2/year) (source: http://www.science.oregonstate.edu/ocean.productivity/). First, we reduced the dimensionality of the 19 variables into a new set that accounted for most of the observed variance with a principal component analysis (PCA) of their standardized  values. These principal components (PC) were then correlated with levels of sample size-standardized genetic diversity from each location. As environmental gradients from central to peripheral populations may generate a stepping stone pattern of dispersal in species with limited dispersal capability, we tested the existence of isolation by distance (IBD). In addition, we assessed whether genetic differentiation between pairs of sites correlated with environmental distance, and if the latter correlated with geographic distance, as expected in the presence of an environmental gradient. For this, we constructed matrices of geographical (km), environmental (Euclidean distances from the 19 standardized variables), and genetic (pairwise molecular Φst) distances between pairs of sites and performed all pairwise correlations, uncorrected and corrected for the third matrix, with partial Mantel test as implemented in IBD v.1.52  were used in parallel analyses to test the same fundamental hypotheses regarding the patterns of genetic diversity and structure predicted by the rel-ACH.The two sequenced (rDNA and ORF) and the five allozyme loci (P. panamensis is higher in northern (peninsular) than in southern  localities . These patterns reveal that in the Mexican Pacific, northern peninsular populations represent the center of abundance of the species whereas southern continental localities harbor peripheral depauperate populations. Admittedly, our sampling does not cover the entire range of the species, which extends south to Colombia. However, we expect this pattern of southern depauperate populations to hold based on published abundance patterns  and four environmental variables explained ∼77% of the variance: PAR (35.7%), nitrate concentration (16.5%), minimum temperature (15.4%), and bathymetry (9.6%); the remaining were of lesser importance. The results showed the most likely habitat for the species is found in the GC and a few hotspots along the continental Mexican Pacific, such as in the islands close to the entrance of the GC and in the Gulf of Tehuantepec  or relative abundance (P = 0.53), there were some notable parallels. For instance, BLA with the highest probability of occurrence (0.849) had one of the highest coral covers and MZ with a smaller probability of occurrence (0.544) showed consistently the lowest coral cover  = 1.17, P = 0.24), hence we opted to treat heteroplasmic colonies as independent haploid organisms.A few (15.3%) rDNA sequences revealed the presence of intragenomic polymorphisms: all instances involved the combination of only two sequence variants. Individual haplotypes from these organisms were completely resolved . To testn = 215 seqs. 555 bp) contained 18 variable sites, 13 parsimony-informative sites, and eight indels. A total of 35 haplotypes were found, only eight of which were shared among some localities  contained seven segregating sites, six of them parsimony informative. Fourteen ORF haplotypes were found in total, four of which were shared among some localities. The most widespread and prevalent occurred in 58% of the individuals (ME-1 and GDH-1) were di-allelic, two (GDH-2 and EST-1) were tri-allelic, and one (LGG-1) was penta-allelic  = 0.51, P = 0.61; ORF t95(0.05) = 1.40, P = 0.16; allozyme t102(0.05) = 0.52, P = 0.60), the most diverse localities were located in Baja California  ≥ 0.63; ORF: BLA, LO, and CP h ≥ 0.62; allozymes: BLA, BC, and LP He > 0.553; h(12) ≥ 0.63, three of four in ORF with h ≥ 0.62, and three of four in allozymes with He ≥ 0.553). Nucleotide diversity behaved similarly to haplotype diversity. Similar trends to h were found in haplotype richness of rDNA as LO, LP, and CP (A(12) = 4.65, 5.48 and 6.94) were the most diverse in Baja California, and IM (A(12) = 5.52) in the continent. Likewise, the highest haplotype richness of ORF were found in peninsular localities (BLA = 5 and CP = 7). In contrast, the total number of alloenzymatic alleles for all loci varied very little among localities (14 ≥ A ≥ 12)  .h and A for rDNA and ORF and He and A for allozymes). Even though the trends suggest a positive relationship of DNA sequence diversity with coral abundance (A(12)  .To test diversity patterns according to rel-ACH predictions, we correlated the percent live coral cover with levels of genetic diversity  B. Mean l = 0.02) E–F. Care = 0.02) A–D. NotaPorites panamensis exhibited significant population structure across the Mexican Pacific . Significant structure was also found between peninsular and continental regions in rDNA (Φct = 0.437 P < 0.001) and allozymes (Fct = 0.050 P = 0.02), ORF being marginally nonsignificant (Φct = 0.116 P = 0.058); and finally among populations within regions . Differentiation among localities in each region revealed a higher degree of genetic structuring among continental  than among peninsular  populations /4)  Wright , reveale) Wright .A and h of rDNA and ORF, and with He and A of allozymes. Of the three PCs, only PC2 significantly correlated with A and h of rDNA (a concentration (FL = −0.84), PAR (FL = 0.80), and light attenuation coefficient (FL = −0.87).The PCA applied to the 19 environmental variables produced three PCs that captured most (85.92%) of the environmental variance . To test the influence of environmental characteristics on standardized genetic diversity, we correlated the three principal components with  of rDNA  and noneΦst, geographic and environmental distances that revealed significant correlations between genetic differentiation and the geographical distance with rDNA (P = 0.03), but not between the former and environmental distance (P = 0.02) revealed the presence of an environmental gradient. However, a detailed analysis of the scatter plot of Φst versus geographic distance shows that there is no significant correlation among peninsular  in several regions, with notable exceptions. Two of the sampled continental localities stand out as less suitable for P. panamensis, as per the MaxEnt model predictions. One is Mazatlan, Sinaloa, representing the northernmost limit of distribution of the species on the Pacific continental margin. The coast of the state of Sinaloa is mostly sedimentary and is subject to strong fluctuations in sediment deposition and resuspension due to wind-wave action and considerable runoff. Near Mazatlan, these unstable and unfavorable conditions for corals are mainly responsible for their rarity. Coral forms are mainly incrusting (such as Porites) and coral reef structures are conspicuously absent. The second is Carrizales, Colima, which has the lowest probability of occurrence of all sampled locations yet has a coral cover comparable to the much more environmentally friendly Islas Marietas in Bahía de Banderas, Jalisco  to the abundant central region to the north, where this species becomes increasingly dominant  expected in central populations. This holds true in the case of P. panamensis, as peninsular populations manifested both large model-based estimations of θ  and high values of genetic diversity  or high levels of inbreeding (CA), as discussed earlier.The patterns of genetic diversity of ar coast , which iiversity . Howeveriversity ; Fig. S7Acropora palmata tended to have lower levels of genotypic diversity mostly due to asexual reproduction  away from their center of abundance (Ayre and Hughes a concentration (negative covariation), PAR (positive covariation), and light attenuation coefficient (negative covariation). This suggests that increased water clarity favoring photosynthetic output by the symbionts is playing a role in favoring coral survivorship and reproduction leading to increased genetic diversification. Admittedly, determining the taxonomic/evolutionary identity and ecological attributes of P. panamensis endosymbionts across the Mexican Pacific is essential to understanding the mechanisms behind the patterns unveiled in the genetic architecture of the cnidarian host of this study. Collaborative efforts are currently being directed toward that goal.A significant association of environmental factors with levels of genetic diversity suggests an indirect effect through abundance and genetic drift, assuming neutral dynamics, or a direct adaptive effect, under selective conditions. In our study, genetic diversity using rDNA of the host correlates with environmental variables not only favoring the survival of the holobiont, such as temperature and substrate availability, but also impinging on water clarity: chlorophyll P. panamensis partially agree with predictions of the rel-ACH. Genetic differentiation was pervasive throughout the studied area and most patterns were consistent not only among the three molecular markers but also between two temporal samples , suggesting that they are not marker dependent and stable in recent time. Genetic architecture among abundant peninsular populations contrasted with those at the periphery (Acropora palmata and Montastraea cavernosa, which showed significant geographical subdivision due to the presence of peripheral populations with different genetic architecture (Baums et al. Patterns of genetic subdivision within eriphery ; Table 3eriphery ; Table 4P. panamensis produces short-lived lecithotrophic planulae that settle near the parental colony, limiting the long-distance dispersal potential (Glynn et al. Our results suggest that continental populations are not only isolated from peninsular populations, but also among themselves. Although geographic distance seemed to be responsible for population divergence at a large spatial scale , there wNe, this polymorphism has endured at much lower frequencies. This hypothetical scenario is also supported by the locus LGG-1, in which the allele D present throughout the gulf, is absent from IM to be found again in BH (Pavona gigantea (Saavedra-Sotelo et al. Although the pattern of genetic structure matches with the overall predictions of the rel-ACH, the high levels of inferred gene flow from BH to peninsular CP and LO is not only contradictory but also improbable, given the limited dispersal of this species. Reconciliation of this result requires considering the ghosts of dispersal past Hellberg . Given tin in BH . SimilarFst values. This could be the result of inbreeding, clonality or null alleles. However, we think that this does not interfere with their power to corroborate the genetic structure patterns observed in the other loci. Indeed, in the worst-case scenario of the presence of null alleles, it is extremely unlikely that their random effect across loci and populations would be responsible for the geographic patterns observed, which largely coincides with an independent sample and loci unaffected by this problem.Finally, the large heterozygote deficit found in allozyme loci warrants caution in the interpretation of the numerical P. panamensis conforms to a large extent to the expectations of the relaxed biogeographical model. In addition, our results clearly revealed added complexity stemming from significant habitat discontinuities in the face of a limited dispersal and outlier localities, but also, and perhaps more importantly, they allowed us to identify the key habitat covariates, such as temperature and photosynthetically relevant variables, that are influencing the genetic patterns of diversification and structure, and may hold the key to the survival of the species in the face of widespread environmental degradation.We set out to test the predictions of the rel-ACH in a dominant hermatypic coral inhabiting the reef communities of the ETP. Our genetic seascape analyses, integrating niche modeling, environmental, and genetic patterns from three molecular markers with complementary evolutionary rates, allowed us to unveil that the genetic architecture of


CYP1A1, CYP2D6, CYP2E1, GSTM1, GSTT1, and GSTP1) and estimate population parameters in 115 Euro-descendants and 196 Afro-descendants from Curitiba, South of Brazil. PCR-based methods were used for genotyping, and statistical analysis were performed by AMOVA with ARLEQUIN software. The mutant allele frequencies in the Afro-descendants and Euro-descendants, respectively, were: CYP1A1*2A = 30.1% and 15.2%; CYP2D6*4 = 14.5% and 21.5%; CYP2E1*5B = 7.9% and 5%; GSTP1*B = 37.8% and 28.3%. The null genotype frequencies were: GSTM1*0 = 36.8% and 46.1%; GSTT1*0 = 24.2% and 17.4%.Individual variability in xenobiotic metabolism has been associated with susceptibility to developing complex diseases. Genes involved in xenobiotic metabolism have been evaluated in association studies; the difficulty of obtaining accurate gene frequencies in mixed populations makes interpretation of the results difficult. We sought to estimate population parameters for the cytochrome P450 and glutathione S-transferase gene families, thus contributing to studies using these genes as markers. We describe the frequencies of six genes ( Genetic marker studies assessing individual backgrounds from specific populations can provide information on gene flow, evolutionary history, and population dispersions, and can also help in the prediction of risks for particular diseases. Based on these studies, pharmacogenetic data have shown significant inter- and intra-population differences in the metabolism, efficiencies, and toxicities of several types of drugs. These findings have important implications for the management and treatment of human diseases .e.g., Europeans, Africans, Asians, and their mixed descendants) in case-control studies of complex diseases. With regard to cancers, these studies focus primarily on lung, breast, and head and neck tumors  in phase I, as well as glutathione S-transferases (GSTs) and N-acetyl-transferases (NATs) in phase II . Severalet al., 2004; Variants of GSTs enzymes have been extensively studied and were found to be associated with several types of neoplasias in different populations, such as Europeans and Euro-descendants . The group of Euro-descendants was comprised of 115 healthy individuals  with an average age of 42.6 ± 7.3 years. The group of Afro-descendants was comprised of 196 healthy individuals  with an average age of 33.4 ± 8.6 years. The ethnic differentiation from these groups was determined through a survey with self-declared information from the individuals that was attached to the Informed Consent agreement. The blood samples were collected in the Hematology and Hemotherapy Center of Paraná State (HEMEPAR), a center for blood donation, by the staff of the Immunogenetics and Histocompatibility Laboratory (LIGH). Genomic DNA was isolated from peripheral white blood cells from all individuals and sampled by a salting out procedure . Polymerase chain reaction (PCR) primers were designed according to the Genome Data Bank. The genotyping of CYP1A1*2A, CYP2D6*4, CYP2E1*5B, and GSTP1*B was performed by PCR RFLP according to the following protocols, respectively: GSTM1*0 and GSTT1*0 genotyping was performed by PCR multiplex according to the protocol described by In the present report, two distinct groups (Euro-descendants and Afro-descendants) from Curitiba in the South of Brazil were analyzed in order to describe the frequency of six metabolic genes  compare the frequencies of dominant and recessive genotypes of the genes GSTM1*0 and GSTT1*0 in individuals of the Euro and Afro-descendant groups, 2) verify whether the genes CYP1A1*2A, CYP2D6*4, CYP2E*5B, and GSTP1*B were in Hardy-Weinberg equilibrium (HWE), and 3) compare the frequencies of the mutant allele of these genes and the genotypes GSTM1*0 and GSTT1*0 with published data. The frequencies of CYP1A1*2A, CYP2D6*4, CYP2E*5B, and GSTP1*B, genotyped in 311 unrelated persons (622 chromosomes) in both samples, were compared via the analysis of the molecular variance (ARLEQUIN 3.1) according to The allele frequencies of the CYP1A1*2A, CYP2D6*4, CYP2E*5B and GSTP1*B. The mutant allele and null genotype frequencies found in the present study were compared with others described in the literature from both non-Brazilian and Brazilian populations  for the genes studied were homogeneously distributed between populations when the non-Brazilian Europeans and Euro-descendants were considered . The analysis of molecular variance (AMOVA) for the genes CYP1A1*2A, CYP2D6*4, CYP2E1*5B, and GSTP1*B showed that 97.47% of the component of genetic variance is present within the ethnic groups and 2.53% (p < 10-4) between them. This lower value justify the lower value of the fixation index or co-ancestry coefficient  observed in this study. Fst, is computed as a measure of the population division effect and values up to 0.05 indicate negligible genetic differentiation . Such studies must consider the impact of the population stratification and miscegenation degree of the control population (Biometabolism genes have been widely used in association studies, and they have contributed to the improvement in understanding the genetic basis of quantitative features (pulation .The present report provides data that can contribute to the general profile of frequency and population dynamics of biometabolizing genes in groups of the Southern Brazilian population. These data constitute a valuable resource for the planning of future association studies in complex diseases like cancers.