Hoplocephalus bungaroides, is a small venomous species restricted to a handful of disjunct reserves near Sydney, Australia. Mark-recapture studies have indicated low vagility for this ambush predator, suggesting that gene flow also may be low. However, our analyses of 11 microsatellite loci from 163 snakes collected in Morton National Park, from six sites within a 10-km diameter, suggest relatively high rates of gene flow among sites. Most populations exchange genes with each other, with one large population serving as a source area and smaller populations apparently acting as sinks. About half of the juvenile snakes, for which we could reliably infer parentage, were collected from populations other than those in which we collected their putative parents. As expected from the snakes’ reliance on rocky outcrops during cooler months of the year, most gene flow appears to be along sandstone plateaux rather than across the densely forested valleys that separate plateaux. The unexpectedly high rates of gene flow on a landscape scale are encouraging for future conservation of this endangered taxon. For example, wildlife managers could conserve broad-headed snakes by restoring habitats near extant source populations in areas predicted to be least affected by future climate change.For endangered species that persist as apparently isolated populations within a previously more extensive range, the degree of genetic exchange between those populations is critical to conservation and management. A lack of gene flow can exacerbate impacts of threatening processes and delay or prevent colonization of sites after local extirpation. The broad-headed snake,  Many endangered species exist as small fragmented populations. The reasons for population fragmentation sometimes are obvious: for example, in habitat specialists that depend upon discontinuously distributed patches, or in formerly widespread taxa whose range has been fragmented by urbanization and habitat destruction e.g., . In otheHoplocephalus bungaroides, Elapidae; Information on the spatial extent and determinants of gene flow is crucial for conserving endangered species that persist as isolated populations. The broad-headed snake , we conducted parentage and landscape genetics analyses to ask the following questions: (1) do snakes move between plateaux , or strictly along plateaux? (2) How much gene flow is there between snake populations separated by long distances of apparently suitable but unoccupied habitat (determined by a lack of samples during surveys) ? and (3) What landscape features affect gene flow among populations of this endangered reptile species?H. bungaroides during ecological research conducted from 1998 to 2009, from six locations in Morton National Park in southeastern Australia , 0.1-mM dNTPs, 0.4 mM of each primer, 20-mM Tris-HCl, pH 8. 5, 50-mM KCl, 1.25-mM MgCl2, and 0.8 µL of DNA extraction. Cycling conditions included a hot start denaturation of 95°C for 3 min, followed by 40 cycles of 95°C for 30 sec, 55°C (58°C for Ns14) annealing temperature for 30 sec, 72°C for 30 sec, and a final extension of 72°C for 7 min. Amplified products were genotyped with a 3130 xl genetic analyzer (Applied Biosystems) using Genemapper software V3.7 (Applied Biosystems).PCR amplifications were performed in a 9800 Fast thermal cycler  as 5 µl reactions containing 0.075 U ISF) were estimated following ISF) per locus and sample were computed with a bootstrap procedure . We estimated expected (SH) and observed (OH) heterozygosities following the methods of To check for linked loci, we calculated the genotypic disequilibrium between loci in each sample based on 10,000 randomizations. Deviations from Hardy–Weinberg equilibrium (HWE) within samples were tested based on 10,000 randomizations. Wright's fixation indices for within-population deviation from random mating , we converted AIC values into AICc  using BAYESASS 1.3  iterations; for the final analysis, we used 3 × 107 MCMC iterations of which 1 × 107 were for the burn-in.We estimated migration rate . This software is based on a coalescence model with mutation and migration, and estimates a measure of effective population size, θ, defined as 4Neµ, where l denotes the mutation rate and Ne the effective population size, and migration M, defined as m?µ, where m denotes migration rate. Estimates were based on five long (105 MCMC steps) and 15 short chains (104 MCMC steps). We used, the “adaptive heating" option with one “cold" and three “hot" chains to ensure convergence.We used the software Migrate 3.2.15  to estim5 iterations (the first 2 × 104 considered as burn-in) for K= 1–6, including all the populations were performed. We defined the number of populations best fitting our dataset as described in K and the corresponding variance of log probabilities.The software Structure 2.1  was usedWe determined paternity of 101 juvenile snakes on the basis of a maximum likelihood method via the program CERVUS 3.0 , in ordeHO) varied from 0.43 to 0.68, and expected heterozygosity (HS from 0.44 to 0.57 (FST) ranged from –0.0004 to 0.1379 (FST of 0.044 (P < 0.001).We did not detect any significant linkage disequilibrium or null alleles, or deviation from HWE within our six populations. For the 11 microsatellite loci, the number of alleles per locus ranged from 2 to 22, with a total of 73 alleles across 11 loci. Observed heterozygosity within populations  in H. bungaroides , and the third one, the straight-line distance and the number of rivers between sites . There was little difference between the two highest-ranking models (ΔAICc= 0.58). Importantly, all three of the best models include the number of rivers as a significant variable. Because the true distance between sites was no more informative than the straight-line distance, only this latter variable was used in our analyses  from Site 2 into Site 1 . Site 2 appeared to be involved in most of the recent gene flow between populations. Similarly, the Migrate analysis suggested that migration occurs among the populations  and 48 offspring (47.5%) were assigned at ≥ 95% and ≥80% certainty, respectively, to at least one parent . Four offspring had both parents assigned at ≥80%, and another five had one of the parents assigned at ≥95% and the other at ≥80%. Only five trios (parents plus offspring) were assigned at ≥80%, and none at ≥95%. Seven of 13 offspring assigned at ≥ 95% to a parent were collected in the same population as their putative parents, whereas the other six were allocated to parents collected in a different population from their own . The maxFST= 0.04), with a maximum distance between collection sites of an offspring and its putative parent of about 10,700 m . Strikingly, almost half (46%) of the juvenile snakes assigned at ≥95% appear to be the progeny of parents that were collected from sites other than those in which the juveniles were collected, consistent with the overall low structure found between populations. As predicted from the snakes’ reliance upon rocky habitats in winter, most dispersal occurred between outcrops on the same plateaux rather than across valleys separating adjacent plateaux  between populations of more than 0.20 as important. Interestingly, the gene flow is much higher than in endangered species, like the Orsini's viper Vipera u. ursinii in France  are more specific, and more fragmented in their distribution, than are the forests used by H. stephensii. That difference in spatial heterogeneity of critical habitat features over long (evolutionary) time periods may explain why we see more population structure within the saxicolous H. bungaroides than the forest-dwelling H. stephensii.A previous analysis of genetic structure at a larger spatial scale in this species, based on molecular phylogeny , revealent times . The habH. bungaroides inferred from genetic data are higher than earlier estimates based on intensive mark-recapture studies. Genetic data can reveal even low rates of long-distance migration among populations, whereas mark-recapture studies are unlikely to reveal such occasional events , suggesting that managers should give high priority to protecting the source populations, as well as to improving the habitat quality of sink populations. Encouragingly, the high mobility of this snake species means that local extirpation will not necessarily result in a long-term absence of the species from the affected site, because such areas are likely to be subsequently recolonized from nearby areas (as long as populations in those source areas remain viable). That ability to recolonize may well prove to be crucial for the continued persistence of broad-headed snakes, because climate-change models have predicted range reductions of up to 86% by 2070, and an increasingly patchy distribution of the remaining suitable habitat . The loc


Naja atra) has a distribution from the mouth of the Yangtze River down to northern Vietnam and Laos, within which several large mountain ranges and water bodies may influence population structure. We combined 12 microsatellite loci and 1117 bp of the mitochondrial cytochrome b gene to explore genetic structure and demographic history in this species, using 269 individuals from various localities in Mainland China and Vietnam. High levels of genetic variation were identified for both mtDNA and microsatellites. mtDNA data revealed two main  and one minor  clades. Microsatellite data divided the eastern + southeastern China clade further into two genetic clusters, which include individuals from the eastern and southeastern regions, respectively. The Luoxiao and Nanling Mountains may be important barriers affecting the diversification of lineages. In the haplotype network of cytchrome b, many haplotypes were represented within a “star” cluster and this and other tests suggest recent expansion. However, microsatellite analyses did not yield strong evidence for a recent bottleneck for any population or genetic cluster. The three main clusters identified here should be considered as independent management units for conservation purposes. The release of Chinese cobras into the wild should cease unless their origin can be determined, and this will avoid problems arising from unnatural homogenization.An understanding of population structure and genetic diversity is crucial for wildlife conservation and for determining the integrity of wildlife populations. The vulnerable Chinese cobra ( Naja comprises 28 species of non-spitting and spitting cobras and was recently split into four subgenera. Six African and Arabian non-spitting cobras inhabiting savanna and open habitats are included in the subgenus Uraeus, four African non-spitting cobras using forest habitats in the subgenus Boulengerina, seven African spitting cobras in the subgenus Afronaja, and 11 Asian cobras in the subgenus NajaNajaN. naja and N. oxiana have some degree of adaptation to spitting b) supports monophyly of the Asian cobras The elapid genus Naja cobras in China, the Chinese cobra (N. atra) and the monocled cobra (N. kaouthia). The Chinese cobra is found from the mouth of the Yangtze River south to northern Vietnam and Laos N. atra have received little conservation attention. Until now, all local populations were treated as homogenous, so that if populations were under severe threat of extinction in a part of the cobra's range, this would not necessarily ring alarm bells, as there are individuals left in other places or in farms. Within the cobra's range large mountain ranges and water bodies such as the Wuyi Mountains, Luoxiao Mountains and Nanling Mountains, Taiwan Strait and Qiongzhou Strait can be found  Has genetic diversity been retained or lost following the recent and rapid decline of Chinese cobras across their range? (2) Does the cobra show genetic and population structuring across its range? (3) Are genes flowing between and within populations and are large mountain ranges and water bodies acting as geographic barriers? Answering these questions will allow us to assess the integrity, viability and population history of this taxon, thereby informing conservation efforts towards this vulnerable snake.b gene sequences (1117 bp) from 269 individuals yielded 39 distinct haplotypes . Eighty-six variable nucleotide sites were found, comprising 77 transitions, six transversions and three transition/transversion. While haplotypes C1 and C15 occupied a wide geographic distribution, 33 of 39 haplotypes were restricted to one population (Cytochrome pulation . High hapulation . Individpulation .NA) was 3.33–8.50; average HE and HO values were 0.440–0.753 and 0.347–0.631, respectively. Individuals from ZW had the lowest NA, HE and HO, and individuals from VS had the highest  and of ZW-QX (13.836), respectively; whereas the minimum and maximum genetic distance based on microsatellite data were from the comparisons of M-GY (0.056) and of ZW-M (0.672), respectively . The genK = 3, indicating probable substructure with three genetic groups . Therefore, we reanalyzed both clusters individually as suggested by the authors of STRUCTURE n = 212, omitting snakes from M and GY) suggested the existence of two genetic subgroups  and nucleotide (π = 0.827%) diversities are higher than values reported for the Jamaican boa (Epicrates subflavus), a similarly vulnerable snake with fragmented distributions, using the same marker (h = 0.79 and π = 0.76%) A. laevis where values were not calculated for all populations sampled as a whole but for each population h = 0.55–0.63) and nucleotide (π = 0.12–0.52%) diversities were lower for most populations of N. atra (π = 1.41%) detected across 86 individual D. acutus sampled across their range in China using the entire mitochondrial ND2 gene Our assessment of genetic variation based on mtDNA and microsatellites indicates high levels of genetic diversity in  N. atra . NucleotN. atra, with a NA of 11.42 and HE of 0.768  A. laevis (NA = 8.4 and HE = 0.263–0.881) S. c. catenatus (NA = 13.8 and HE = 0.49–0.77) N. atra, perhaps because of a large effective population size, or because bottlenecks due to very recent ecological disturbance are unlikely to dramatically reduce genetic diversity within a handful of generations unless they lead to catastrophically low population sizes.High microsatellite diversity was also detected in of 0.768 . At the N. atraD. acutus distributed almost in the same region, whereby an east-west division of the whole D. acutus population was found Our study utilized more and wider sampling locations compared to an earlier study of Generally, the degree of genetic structure in a continuously distributed species is expected to fall somewhere between (1) a mosaic of subpopulations delimited by effective physical  or ecological  Leiolepis reevesiiLineage sorting between island and mainland populations has not yet been completed in the most northeastern part of the cobra's range FST estimates based on microsatellite data also support the Bayesian clustering results (FST/1–FST) between ZW and M (0.672) and between ZW and GY (0.597) are the largest when compared with values between other populations, presumably suggesting that high latitude and cool climates play a significant role in the separation of northern and southern populations of the cobra. ZW is the northern limit of the species' range, and the cobra can be very abundant in the southern region (including islands) but absent in the northern part of the region  results . SlatkinN. atra. In fact, no distinct Quaternary glacier landform or deposits have been found within the distribution range of N. atra thus far N. atra. This differs partly from conclusions drawn using the Asiatic toad Bufo gargarizans, an important prey item of N. atra. Phylogenetic analysis suggests that dispersal, instead of vicariance, dominated the history of the B. gargarizans species group N. atra is somewhat similar to that of D. acutus where isolation, dispersal, bottlenecks and expansion jointly constitute its history There were many unobserved intermediate haplotypes missing along the long branches between the two central haplotypes (C17 and C14), whereas many haplotypes were represented within a “star” cluster in the haplotype network . Is thisN. atra, but great care should be taken to ensure that individuals are re-introduced to or translocated between appropriate populations within the same MU. Furthermore, as how much adaptive differentiation is present between these MUs or within them is unknown, we caution against long-distance transfers within a group, especially when environmental differences are apparent. In China current practices regarding the translocated release and artificial breeding of Chinese cobras in commercial farms will likely cause serious problems due to unnatural homogenization Phylogeography and population genetic studies can aid the identification of evolutionarily significant units and management units We employed local people to collect adult cobras larger than 900 mm snout-vent length between May and September of 2005–2010 from various localities  in the eastern , southeastern  and western  regions of the cobra's range in mainland China and northern Vietnam , Table 1b gene was amplified and sequenced according to the protocols described in Lin et al. (2008c) Preserved tissue samples were used to extract total genomic DNA using EasyPure Genomic DNA Extraction Kit (TransGen Biotech). DNA was resuspended in TE buffer  and stored at −80°C until ready for use. The cytochrome h) and nucleotide diversities (π) b (whole gene and each codon position separately). Within N. atra, signs of saturation were not present at any codon position; therefore, saturation was not considered to be a significant factor and all nucleotide positions were used in subsequent analyses.Sequences were translated to amino acids with the program SQUINT N. kaouthia (GenBank Accession No. AF217835) as the outgroup. ML analysis was carried out by a heuristic search of 10 random addition analyses with tree-bisection-reconnection (TBR) branch swapping using PAUP 4.0 beta We reconstructed a phylogenetic tree based on the maximum likelihood (ML), using SSD) and the Harpending's raggedness index D test Fs test τ = 2utt), where τ is the date of the growth or decline measured in units of mutational time and u is the mutation rate per sequence and per generation. The approximate time of expansion in years was calculated by multiplying t by the generation time of N. atra. The generation time for large snakes was estimated as four years based on the approximate time at which animals mature −1 myr−1  for cytochrome b within the Viperidae. We used the upper and lower values (0.005–0.009) to estimate the overall range of potential dates. Although this dating must be taken with extreme caution due to the lack of calibration of the substitution rate in N. atra and to the sensible overestimation of timing recent events induced by the time-dependency of molecular rates We used mismatch distributions to test demographic signatures of population expansions within mtDNA lineages NA) per locus and observed (HO) and expected heterozygosities (HE) were calculated using ARLEQUIN 3.5. FSTAT 2.9.3.2 AR) on a minimum of 17 individuals. Deviations from Hardy-Weinberg equilibrium across all loci for each population were assessed using the exact probability test in GENEPOP 4.0 All microsatellite loci were screened for null alleles and large allele dropouts using MICRO-CHECKER 2.2.3 FST/1–FST) derived from pairwise FST, which were estimated for 15 comparisons between six populations Genetic distances were computed as Slatkin's (1995) genetic distance (K) tested was set from 1 to 10, and 10 independent runs were carried out for each using no prior information, assumed admixture and correlated allele frequencies. The lengths of MCMC iteration and burn-in were set at 300 000 and 50 000, respectively. The true K is selected using the maximal value of the log likelihood [Ln Pr(X/K)] of the posterior probability of the data for a given ΔKK statistic, the second-order rate of change in the log probability of the data between successive values of K, was also estimated A Bayesian clustering method, STRUCTURE P-values from the Wilcoxon's test were used as evidence for bottlenecks and were assessed for significance at the 0.05 level.Demographic history based on microsatellites was assessed using two different and complementary methods. First, the Wilcoxon's sign rank test was used to examine whether populations exhibit a greater level of heterozygosity than predicted in a population at mutation-drift equilibrium. This test is most sensitive to detecting bottlenecks occurring over approximately the last 2–4 Ne generations and, for most parameters, has more power to detect more recent bottlenecks (e.g. 0.2–1Ne generations ago). Second, a mode-shift test


Suberites diversicolor (Porifera: Demospongiae: Suberitidae) is typical of marine lake habitats in the Indo-Australian Archipelago. Four molecular markers  were employed to study genetic structure of populations within and between marine lakes in Indonesia and three coastal locations in Indonesia, Singapore and Australia. Within populations of S. diversicolor two strongly divergent lineages (A & B) (COI: p = 0.4% and ITS: p = 7.3%) were found, that may constitute cryptic species. Lineage A only occurred in Kakaban lake , while lineage B was present in all sampled populations. Within lineage B, we found low levels of genetic diversity in lakes, though there was spatial genetic population structuring. The Australian population is genetically differentiated from the Indonesian populations. Within Indonesia we did not record an East-West barrier, which has frequently been reported for other marine invertebrates. Kakaban lake is the largest and most isolated marine lake in Indonesia and contains the highest genetic diversity with genetic variants not observed elsewhere. Kakaban lake may be an area where multiple putative refugia populations have come into secondary contact, resulting in high levels of genetic diversity and a high number of endemic species.The existence of multiple independently derived populations in landlocked marine lakes provides an opportunity for fundamental research into the role of isolation in population divergence and speciation in marine taxa. Marine lakes are landlocked water bodies that maintain a marine character through narrow submarine connections to the sea and could be regarded as the marine equivalents of terrestrial islands. The sponge  It has long been hypothesized that marine species have large geographic ranges with large population sizes, and are faced with weaker barriers to dispersal than terrestrial organisms, thus resulting in relatively slow rates of speciation e.g. . The assPhylogeographic studies of anchialine systems across the world typically show high levels of genetic differentiation between marine lake populations, suggesting little to no gene flow at small spatial scales ranging from 10 to 100 km . FurtherSuberites diversicolor [Porifera: Demospongiae: Suberitidae] is an ideal taxon to pursue this study as it allows comparison of multiple lakes at various scales and with varying degrees of connection to the sea  to investigate possible relationships between genetic diversity and the level of isolation of the lakes.Here we have conducted the first phylogeographic study of Indonesian marine lake populations. The sponge species  the sea . There aIndonesia: to L. Becking East Kalimantan IN 2008&2009: 0094/frp/sm/v/2009 and 1810/FRP/SM/VIII/2008; West Papua in 2011: 098/SIP/FRP/SM/V/2011.Singapore: Singapore National Biodiversity Center collection permit to S.C. Lim,Australia: Museum and Art Gallery of the Northern Territory permit to B. Alvarez.Suberites diversicolor. Populations of Suberites diversicolor were located in seven marine lakes  in the region of Berau, East Kalimantan province  and the regions of Northern Raja Ampat  and Southern Raja Ampat (Misool Jellyfish lake) in West Papua province, and in mangroves along the coast of the island of Maratua in the region of Berau, East Kalimantan province  as voucher specimens. The investigated specimens are listed in the Appendix A.Collections were made randomly along the entire coastline of each of the lakes and specimens were collected at least 25 m distance from each other to avoid collecting clone siblings. Our aim was to collect 20 individuals per location, but in most locations the resident population size was too small to attain this target see . Hence, Total DNA was extracted from 105 specimens using DNeasy tissue kit (Qiagen), following the instructions of the manufacturer. Partitions of four markers were amplified: two mitochondrial genes, cytochrome oxidase subunit 1 (COI) and subunit 2 (COII), and two nuclear markers, the nuclear ribosomal operons consisting of partial 18S rDNA, full-length internal transcribed spacer 1 and 2, 5.8S, and partial 28S rDNA fragments (ITS) and the D3–D5 region of the nuclear ribosomal 28S gene (28S). The nuclear markers are independent from the mitochondrial markers and therefore provide extra support in case of congruent results.Suberites SUB-COI-F: GGAATGATCGGGACAGCTTTTAGCATG and a degenerated reverse primer from Folmer et al. TTTTTCACGATCAGATTATGTTTA and CO2R: ATACTCGCACTGAGTTTGAATAGG. ITS amplified with primers from Wörheide (1998) RA2: GTCCCTGCCCTTTGTACACA and ITS2.2: CCTGGTTAGTTTCTTTTCCTCCGC. 28S was amplified in a subset of samples with primers from McCormack and Kelly (2002) RD3A: GACCCGTCTTGAAACACGA and RD5B2: ACACACTCCTTAGCGGA. Amplifications were carried out in 25 µl reaction volumes containing 5 µl Phire® Reaction Buffer,3 µl dNTPs (1 mM), 0.625 µl of each primer (10 µM), 0.25 µl Phire® Hotstart-Taq polymerase DNA , and 1 µl of DNA (10–20 ng/µl). The temperature regime for amplification: 94°C for 30 s; followed by 35 cycles of 94°C for 5 s; 50°C for 5 s; 72°C for 12 s; followed by 72°C for 1 min. PCR products were purified and sequenced by Macrogen Inc (Korea and The Netherlands).The standard DNA-barcoding fragment of COI was amplified by using a specific forward primer designed for http://blast.ncbi.nlm.nih.gov/Blast.cgi). Sequences were handled in SEQUENCHER 4.10.1 (Gene Codes Corporation) and aligned with CLUSTALW and MUSCLE as implemented in DAMBE The poriferan origin of the obtained sequences was verified through BLAST searches against Genbank (th generation at the default temperature (0.2). Analyses were terminated after the chains converged significantly as indicated by an average standard deviation of split frequencies <0.001. Convergence was also checked in Tracer v. 1.5.0 p-distance (uncorrected), as well as net nucleotide divergence between groups were calculated in MEGA. A Kruskall-Wallis test was performed to test whether color or substrate preference significantly differed between lineages. To test for spatial structuring of samples we performed an analysis of molecular variance (AMOVA) and calculated pairwise Φst values between separate populations using Arlequin 3.5.1.2 Φst values (based on p-distances) was determined by 10,000 permutations and exact tests of population differentiation in Arlequin.Phylogeographic analyses were carried out for COI and ITS. We used ITS outgroup sequences obtained from Genbank from the family Halichondriidae , as the S. diversicolor of 519 bp for COI with four haplotypes , 331 bp for COII with one haplotype , 689 bp of ITS with nine genetic variants  (All sequences were submitted to GenBank with accession numbers KF568951-KF568965 . We obtaF568959) . For a sp-distance within lineage B was 0.25% (n = 91). The net nucleotide divergence between lineage A & B for COI was 0.38%. Haplotype C1 (lineage A) differed by two basepairs from C2 (the dominant haplotype from lineage B) of which one resulted in a non-synonymous substitution between two unpolar amino acids, from isoleucine to valine. For ITS the average p-distance within lineage A was 0.44% (n = 13), while the average p-distance within lineage B was 0.29% (n = 91). The net nucleotide divergence between lineages A & B for ITS was 7.26%. Several indels of 1–3 bp length were observed and were consistent within lineages and differed between lineages. There were insertions in lineage A with respect to lineage B from 102–103 bp (either CT or TT), 380–381 bp (CA), 470–473 bp (GGA or GAA). There were gaps in lineage A with respect to lineage B from 139, 178–180, 549–555 bp. No double peaks were observed, and it we therefore assumed that no intragenomic polymorphisms occur within this species. The level of intragenomic polymorphisms differs per species COI and ITS sequences were obtained from the same specimens and fall apart into two major lineages, termed A and B . These lLineage A was only present in Kakaban lake while lineage B was present in all populations. The geographical distribution of COI haplotypes is shown in Within lineage A in Kakaban lake there was only a single haplotype of COI while the ITS gene diversity was 0.8242+/- 0.0567, and ITS nucleotide diversity was 0.005656+/- 0.003392. Within lineage B all populations contained a single COI haplotype except Urani lake, which had two haplotypes with a haplotype diversity of 0.3333+/− 0.2152 and nucleotide diversity of 0.000624+/− 0.000822. For ITS, the majority of the populations contained only a single genotype, except for Kakaban lake, Darwin and Misool Jellyfish lake. The population in Kakaban lake had the highest gene and nucleotide diversity in lineage B, followed by Darwin and Misool Jellyfish lake . Tajima'Φst between 0.53–1) and significantly differentiated from all marine lakes populations  are indicative of reproductive isolation, and thus we suggest that the two lineages (A and B) constitute different species. We searched for morphological and ecological characters to distinguish the two lineages, but did not find any. Genetic divergence can preclude morphological or ecological distinction. The skeletal structure and spicule lengths do not differ between lineages and fall within the natural variation of this species  displays different colors at different depths as a result of a symbiosis with the unicellular green algae Chlorella and symbiotic bacteria Two major lineages were uncovered in the populations of the sponge see also . The colTedania spp. in mangroves Scopalina lophyropodaCliona spp. Hexadella spp. 2 it is tenfold larger than any of the other marine lakes in Indonesia and the majority of Palau . Within sponges there are several reports of sympatric cryptic species: of Palau . AlternaS. diversicolor are unknown, but this species does produce asexual buds [Becking pers. obs.] which may survive a considerable amount of time in the plankton or by rafting before colonizing distant locations as proposed by Wörheide et al. Leucetta chagosensis.Lineage A is only present in Kakaban lake, while lineage B is present in all populations. Within lineage B the spatial genetic structure shows three groups: 1. the three Berau lakes and southern Raja Ampat lake, 2. Berau coast, Singapore coast and the three northern Raja Ampat lakes, 3. Darwin, Australia. At present there is no comprehensive phylogeographic study of sponges spanning the Indonesian archipelago, yet pronounced genetic differences in populations of other marine invertebrates and vertebrates are present between the Java Sea, the Indonesian Through Flow, and the seas of East Sulawesi Mastigias papuaSphaeramia orbicularisBrachidontes sp. Mastigias papua and Brachidontes sp. rapid morphological evolution in the marine lakes p-distances)  e.g. . HoweverS. diversicolor. The D3–D5 region of the 28S fragment has been used to distinguish genera and species of a wide range of demosponge taxa including halichondrids Hymeniacidon heliophila but attributed their results to the collection of clone-mates. In the present study COII showed no variation between any of the samples spanning a wide geographic range. We conclude that COII is not a suitable marker for intraspecific variation or distinction between closely related species of the genus Suberites in particular, and probably more generally for the families Suberitidae and Halichondriidae.We found no sequence variation in 28S and COII markers between any of the populations or between the two lineages of Suberites diversicolor displayed the highest genetic diversity with unique genetic variants that were not shared with two marine lakes at just 6 km distance Click here for additional data file.Table S2Φst values between all populations of the sponge Suberites diversicolor lineage B based COI of Suberites diversicolor (location codes indicated in Pairwise (XLSX)Click here for additional data file.


The Altiplano region of the South American Andes is marked by an inhospitable climate to which the autochthonous human populations adapted and then developed great ancient civilizations, such as the Tiwanaku culture and the Inca Empire. Since pre-Columbian times, different rulers established themselves around the Titicaca and Poopo Lakes. By the time of the arrival of Spaniards, Aymara and Quechua languages were predominant on the Altiplano under the rule of the Incas, although the occurrence of other spoken languages, such as Puquina and Uruquilla, suggests the existence of different ethnic groups in this region. In this study, we focused on the pre-Columbian history of the autochthonous Altiplano populations, particularly the Uros ethnic group, which claims to directly descend from the first settlers of the Andes, and some linguists suggest they might otherwise be related to Arawak speaking groups from the Amazon. Using phylogeographic, population structure and spatial genetic analyses of Y-chromosome and mtDNA data, we inferred the genetic relationships among Uros populations , and compared their haplotype profiles with eight Aymara, nine Quechua and two Arawak (Machiguenga and Yanesha) speaking populations from Peru and Bolivia. Our results indicated that Uros populations stand out among the Altiplano populations, while appearing more closely related to the Aymara and Quechua from Lake Titicaca and surrounding regions than to the Amazon Arawaks. Moreover, the Uros populations from Peru and Bolivia are genetically differentiated from each other, indicating a high heterogeneity in this ethnic group. Finally, our results support the distinctive ancestry for the Uros populations of Peru and Bolivia, which are likely derived from ancient Andean lineages that were partially replaced during more recent farming expansion events and the establishment of complex civilizations in the Andes. Since pre-Columbian times, the inhabitants of the Altiplano region in the South American Andes  have been engaged in agriculture, raising livestock and fishing. The Altiplano region or Collao Plateau lies in the central Andes, and presents an average height of about 3,750 meters above sea level, mean annual temperatures below 10°C, and total annual rainfall less than 1000 mm. Despite its harsh conditions, radiocarbon dating of artifacts suggested that the Altiplano was initially occupied by humans at approximately 3,700 years before the present th century, most of this region was inhabited by Kollas, Lupakas, Pakaxes, and Carangas (mostly Aymara speaking clans or “señorios”) who were subjugated by the Inca Empire At the time of arrival of the Spaniards in the 16th century, the Uruquilla speakers who were named Uros  were distributed along the aquatic axis comprising Lake Titicaca (Peru/Bolivia), the Azangaro and Desaguadero Rivers, Lake Poopo, the Lacajahuira and Lauca Rivers, and Lake Coipasa in Bolivia Until the 16http://www.inei.gob.pe) speaks only Aymara and Spanish. Bolivian Uros  are composed by about 2600 individuals (Bolivian census at http://www.ine.gob.bo), with the majority speaking only Aymara or Spanish The reduction of the Uros population has been suggested by linguists to have resulted from a gradual “transculturation” during pre-Inca, Inca and Spanish dominance, when many Uros were “puquinized”, “aymarized”, “quechuized”, and “castellanized” According to some researchers, the Uros were the first settlers of the Andean Altiplano, yet their origin has been subjected to considerable academic debate Because of the complexity of their reported history and subjugation to other dominant societies since pre-Columbian times, the Uros have been the target of many research studies, particularly in the fields of anthropology and linguistics Here, we generated and analyzed Y-chromosome and mtDNA diversity in 388 indigenous participants from Peru and Bolivia, focusing mainly on the pre-Columbian settlement of the Altiplano region and the ancestry of the Uros. Our results clarify the genetic relationships and population structure of the Peruvian and Bolivian Altiplano communities, as well as unveil the degree of kinship and shared ancestry between currently known Uros from Peru (Los Uros) and Bolivia (Uru-Chipaya and Uru-Poopo), and the major ethnic groups from the Andes, the Aymaras and Quechuas, and Arawaks from the Peruvian Amazon. With these data, we investigated three major questions: (i) Do the Uros from Bolivia and Peru share a recent common ancestry, or do they descend from distinct ancestors? (ii) How do the Uros relate to the neighboring Aymara and Quechua, and other populations from the Altiplano? and (iii) Do the Uros present any evidence of having close genetic kinship to Amazonian native communities of the Arawak family?rd degree were avoided, and men representing unique families were preferentially sampled to allow analyses with both Y-chromosome and mtDNA markers using a lower number of individuals, as women would only contribute to mtDNA analysis.Ethical approval for the present study (The South American Genographic Project) was provided by the Brazilian National Ethics Commission , and by local ethical committees from Peru and Bolivia . The project was explained to the volunteers after previous contact with indigenous confederations, authorities and/or community leaders, and in some cases in their indigenous languages. Signed informed consents for all subjects were obtained before collection of mouth swab samples. In most cases, collection expeditions occurred in the relatively isolated villages of the native participants, who were interviewed in order to assess the birthplace and ethnicity of their parents and grandparents, and to certify that at least three preceding generations of their ancestors had been living in the same locality. Relatives to the 3For the present study, we analyzed DNAs from 388 individuals residing in 22 sampling localities or communities  that werDNA samples were extracted from buccal swabs using standard procedures. Samples were initially genotyped for the four more prevalent Y-SNPs autochthonous from South America, M130, M242, M346, and M3 Although the assignment of the two-repeat blocks of DYS385 could not be accurately made without further genotyping, it was suggested the shorter repeat allele was associated with DYS385a For all samples, the complete mtDNA control region  The sequence alignments were performed in relation to rCRS through SeqScape 2.6 (Applied Biosystems). Variable positions were determined and major haplogroup assignment was obtained by MitoTool www.fluxus-engineering.com), with and without weighting the Y-STRs or mtDNA haplotypes www.fluxus-engineering.com).To reveal the genetic relationships among individuals we used the Median Joining algorithm through the program Network  were obtained to evaluate the genetic differentiation of the 22 communities. For estimation of a substitution model on mtDNA analyses, we used jModelTest 0.1.1 stR and Φst distances into Reynolds’ coancestry coefficients in Arlequin, which were used in metric MDS analyses with GenAlEx http://folk.uio.no/ohammer/past) to visualize population relationships in a bidimensional space. The goodness of fit stress values were estimated for nmMDS. The linearized Reynolds’ distances were also used in the Mantel test, comparing matrices of genetic and geographic distances , and also in the spatial analysis using the Monmonier’s algorithm and Delaunay triangulation performed with the program Barrier Analysis of Molecular Variance (AMOVA) was performed with Arlequin 3.5 After SNP genotyping samples from all 22 populations, we obtained Y chromosomal haplotypes from only two related Q lineages, Q-M346* (n = 24) and Q-M3 (n = 363). Because our main focus was investigating the origin of the Uros and all displayed only Q-M3 haplogroup, we selected 363 Q-M3 samples for individual level genetic analyses in phylogenetic networks, using 15 or 17 Y-STRs, with and without weighting. A complete list of Y-STR haplotypes is available in Following the stepwise mutation model and parsimony criteria, we obtained a median-joining (MJ) phylogeographic network  of Y-chrHowever, other Peruvian Uros’ haplotypes appeared in different clusters, all of which being more closely related to those found in communities around Lake Titicaca . For exaAmong Bolivian Uros, the Uru-Chipaya (Chp) haplotypes also formed a cluster bearing haplotypes H12 and H13, which was closely related to individuals bearing haplotype H15 from Pampa Aullagas (Pam) and H14 from Chimu (Chi), both neighboring Aymara communities. However, another haplotype H16 (Uru-Chipaya) did not belong to this cluster, but appeared genetically related to an Uru-Poopo (Ppo) haplotype H17. Both haplotypes (H16 and H17) were related to H18, which appeared in one Aymara from Pajchiri (Paj) on the border of Lake Titicaca. Moreover, haplotype H24 was shared between individuals from Uru-Chipaya and Santa Rosa de Yanaque (SRY), also at the shores of Lake Titicaca. In another cluster, H19 from one individual Uru-Poopo was closely related to H20 (one repeat unit difference) from Quechua of Potosi, very close to the Uro-Poopo locality. For two Uru-Poopo individuals, there was one difference between haplotypes H21 and H22, and those haplotypes were close to haplotype H23 (a Quechua from HVC). The major results of clustering and sharing of Peruvian and Bolivian Uros haplotypes can be also observed using all 17 Y-STRs and without weighting . Anyway,In the nmMDS plot  of 17 Y-stR = 0.088), a moderate differentiation among Quechua groups (stR = 0.136), and high differentiation among Arawaks (stR = 0.245) and Uros (stR = 0.478). The stR comparisons in the three levels AMOVA also showed higher values when Uros were analyzed together Aymaras, Quechuas or Arawaks than analyses without them. However, there was little intergroup differentiation when subpopulations were clustered in four linguistic groups (4.41%). Besides a negative (ctF = −0.13) and non-significant differentiation (p = 0.9) was observed in the tree levels AMOVA between Uros and Arawaks . We also performed two levels AMOVAs to compare linguistic groupings with artificially merged subpopulations (stR = 0.111), the latter appeared much closer to Aymara and Quechua . The population pairwise stR ´s between Peruvian and Bolivian Uros and other communities were all significant (p<0.05), except for Uro-Poopó that presented non-significant differentiation to Qui, Apu, Amt, ViM, Chi, Pam, and Pun likely due to its very small sample size (n = 4). Furthermore, the AMOVA between different population pairs  and geographic distances (Km) in the Mantel test among the 22 communities , suggesting gene flow discontinuities. Moreover, the test considering geographic location and genetic distances through Barrier software showed that the Peruvian Uros, Taquile, Uru-Chipaya, Uru-Poopo, and Machiguenga communities were relatively isolated by gene flow barriers , 133 haplotypes to haplogroup B2 (n = 285), 20 haplotypes to haplogroup C1 (n = 23), and 25 haplotypes to haplogroup D1 (n = 40). The haplotypes found in the Uros populations are listed in The distribution of mtDNA haplogroup frequencies in the 22 communities is shown in the In other B2 clusters within the MJ network , we obseAmong Bolivian Uros, three Uru-Poopo individuals appeared in an exclusive and highly differentiated B2 cluster  of relatAmong sequences of haplogroup D1 , we obsehttp://www.atgc-montpellier.fr/phyml) supports the close relationship of Uroś haplotypes discussed above, although the tree presents a branching topology that cannot be directly observed in the networks.A maximum likelihood phylogenetic reconstruction  built wiThe nmMDS plot generated from Reynolds’ linearized distances  showed tTo characterize population structure and genetic diversity within and between the 22 Peruvian and Bolivian autochthonous communities under study, we carried out AMOVA tests following different population comparisons . We perfst = 0.310), followed by the Arawaks (Φst = 0.252), Quechua (Φst = 0.137), and Aymara (Φst = 0.024). Thus, there was high heterogeneity among populations of Uros and Arawaks in comparison to Aymara and Quechua, a pattern similar to that produced by the AMOVA using Y-STR haplotypes. In the three level AMOVA results, we observed a lower interpopulation differentiation between Uros and Aymara (Φst = 0.090) when compared to Uros and Quechua (Φst = 0.169), but a particularly high difference between Uros and Arawaks (Φst = 0.352). On the other hand, Aymara and Quechua populations were more closely related through their maternal lineages (Φst = 0.090) than either was to Arawaks (Φst>0.23). The population pairwise Φst’s between Peruvian and Bolivian Uros, and other communities were all significant (p<0.05), except between Uru-Chipaya and Pajchiri (p = 0.06), We also conducted several two level AMOVAs between specific population pairs, or joining populations in linguistic groups  among Potosi and Villa Molino populations, suggesting they have gone through demographic expansions in the past , Uros-Puno (h = 0.521), and Uru-Chipaya (h = 0.643), and this finding partially explains the relationships observed in the MDS plot . Tajima’the past .2 = 0.010; p = 0.248). However, the spatial analysis using genetic distances and geographic coordinates through Monmonier’s algorithm and Delaunay triangulation showed that the Machiguenga, Peruvian Uros, Uru-Poopo, Uru-Chipaya, and Taquile communities were isolated by geographic barriers . It revealed some exclusive clusters of Uros, particularly among Peruvian Uros from Puno Bay. The few related haplotypes in this exclusive cluster  are found in two Aymara individuals from Santa Ana, at the shore of Lake Titicaca, and this finding can be explained by unidirectional gene flow from Uros to Aymara or “Aymarization” of Uros, as suggested by linguists The AMOVAs of both Y-chromosome and mtDNA haplotypes  and 2 inThe mtDNA results indicate that the Machiguenga (Mac) and Uru-Chipaya (Chp) are the most differentiated populations, followed by Peruvian Uros (Pun) and Uru-Poopo (Ppo). Among the Uros from Puno, the sharing of a mtDNA haplotype with an individual from Chimu community suggests that these individuals could be descendents of Aymarized “Uros”, as suggested by linguists Likewise, the cluster depicting the distribution of A2 haplotypes  suggestsIn conclusion, the genetic evidence obtained through analyses of autochthonous paternal and maternal lineages from the Andean Altiplano showed that Uros from Puno Bay in Peru and Bolivian Uros communities of Uru-Chipaya and Uru-Poopo are heterogeneous groups, bearing genetic lineages derived from divergent ancestors when compared to most of the current Andean populations. Our results also indicate more gene flow with neighboring Andeans (Aymara and Quechua) than with more distant Andean communities or Arawakan speaking populations. Moreover, the results suggest the Uros could be derived from ancestral Andean stocks that were intermingled and partially replaced by lineages arriving with populations expanding due to a more recent farming expansion and posterior establishment of complex civilizations on the Andes.Figure S1Median Joining network for 17-YSTR Q-M3 haplotypes among 22 Peruvian and Bolivian populations without weighting. Different population groups are defined by distinct colors, where Aymara and Quechua communities from the border of the lakes (Titicaca and Poopo) are discriminated. The Y-STR haplotypes (H#) named according to (TIF)Click here for additional data file.Figure S2Detailed map of the Altiplano region with four gene flow barriers detected among 22 Bolivian and Peruvian populations using the Barrier software. The ranking order of barriers isolating populations is as follows: Peruvian Uros (Pun), Taquile (Taq), Uru-Chipaya (Chp), Uru-Poopo (Ppo), and Machiguenga  for Y-STR data analysis; and Machiguenga (not shown), Peruvian Uros, Uru-Poopo, Uru-Chipaya, and Taquile for mtDNA data analysis.(TIF)Click here for additional data file.Figure S3A Maximum likelihood phylogenetic tree of all mtDNA control region haplotypes among Peruvian and Bolivian individuals (only topology is shown). The haplogroups are indicated at the end of haplotype names. Red sequence names appear on the Uroś communities (munities  and are (TIF)Click here for additional data file.Table S1Description of the 22 Peruvian and Bolivian populations analyzed.(XLSX)Click here for additional data file.Table S2Y-STR haplotypes found among the 22 Peruvian and Bolivian populations. a) individual 17 Y-STR haplotype data for Q-M3 and Q-M346* lineages; b) population data for Q-M3 Y-STR haplotypes; c) 15 Y-STR haplotypes.(XLSX)Click here for additional data file.Table S3Two levels AMOVA results using 17 Y-STRs of Q-M3 and Q-M346 lineages (a) and four mtDNA haplogroups (b) involving comparisons of four linguistic groups without subpopulation division, and specific population pairs.(DOCX)Click here for additional data file.Table S4Control region mtDNA haplotypes and SNP variant positions found among Uros and related individuals from Peruvian and Bolivian populations.(DOCX)Click here for additional data file.Table S5Polymorphic sites in mtDNA CR sequences among Peruvian and Bolivian autochthonous individuals, determined by MitoTool program.(XLSX)Click here for additional data file.Table S6Distribution of mtDNA haplogroup frequencies  among the 22 Peruvian and Bolivian populations.(DOCX)Click here for additional data file.Table S7Diversity indices and neutrality tests per population on 388 mtDNA CR sequences of A2, B2, C1 and D1 haplogroups.(DOCX)Click here for additional data file.


Daphnia . D. galeata and D. longispina both occur in lakes in the Tatra Mountains , but their populations show distinct life history strategies in that region. In three studied lakes inhabited by D. galeata, daphnids overwinter under the ice as adult females. In contrast, in lakes inhabited by D. longispina, populations apparently disappear from the water column and overwinter as dormant eggs in lake sediments. We investigated to what extent these different strategies lead to differences in the clonal composition of late summer populations.The population structure of cyclical parthenogens such as water fleas is strongly influenced by the frequency of alternations between sexual and asexual (parthenogenetic) reproduction, which may differ among populations and species. We studied genetic variation within six populations of two closely related species of water fleas of the genus D. longispina populations, very high clonal richness was found (MLG/N ranging from 0.97 to 1.00), whereas in D. galeata it was much lower (0.05 to 0.50). The dominant MLGs in all D. galeata populations were heterozygous at five or more loci, suggesting that such individuals all represented the same clonal lineages rather than insufficiently resolved groups of different clones.Analysis of genetic variation at nine microsatellite loci revealed that clonal richness  consistently differed between the two studied species. In the three D. galeata populations were likely a consequence of strong clonal erosion over extended periods of time  and the limited influence of sexual reproduction. Our data reveal that populations of closely related Daphnia species living in relatively similar habitats  within the same region may show strikingly different genetic structures, which most likely depend on their reproductive strategy during unfavourable periods. We assume that similar impacts of life history on population structures are also relevant for other cyclical parthenogen groups. In extreme cases, prolonged clonal erosion may result in the dominance of a single clone within a population, which might limit its microevolutionary potential if selection pressures suddenly change.The low clonal diversities and significant deviations from Hardy-Weinberg equilibrium in  Many organisms are capable of alternating sexual and asexual reproduction throughout their life cycle, including protists, cnidarians, rotifers, crustaceans, insects, mosses, vascular plants and macro-algae . The relIn aquatic environments that undergo regular seasonal changes, including cold temperate lakes or temporary ponds, many zooplankton groups such as water fleas (Cladocera) usually survive unfavourable periods of winter or drought as dormant stages, and hatch at the onset of more favourable conditions. The typical cladoceran life cycle (reviewed in ) is thusFertilized dormant eggs, usually encased in an ephippium, enter dormancy at an early embryonic stage, endure unfavourable periods, and can hatch when the conditions improve again. Each hatchling from a dormant egg of a cyclically parthenogenetic cladoceran has a unique genotype ,5. UnderDuring parthenogenetic reproduction in the growing season, genotypes best adapted to their local environment are favoured. This process often results in the coexistence of a limited number of genotypes ,8. ClonaThe above-mentioned processes are of course not limited to cladocerans. The genetic structure of cyclical parthenogen populations in general is affected by the strength of the clonal selection, length of the growing season, and frequency of sexual reproduction ,5. A redDaphnia. These small crustaceans are key grazers of phytoplankton in temperate lakes. In such habitats, Daphnia have two options to survive unfavourable winter conditions [Daphnia female may produce dormant eggs sexually and subsequently switch back to parthenogenetic reproduction [Daphnia species inhabiting the same lake  [Daphnia life cycle may be crucial for the resulting clonal diversity. Therefore, different overwintering strategies are expected to have very different impacts on the clonal structure of Daphnia populations, their effective population size, and in the long term on their microevolutionary potential [An important group in which these phenomena have been studied in detail is the cladoceran genus nditions : 1) as doduction . Variatie  and wite . It haswinters) . In the winters) ,5. Indivwinters)  predictsotential .Daphnia clonal diversity, however, have long been hindered by a lack of suitable genetic markers. Most studies in the past have used allozyme electrophoresis with limited genetic resolution to detect multilocus genotypes . ,20. Daphs .,22.DaphnDaphnia species inhabiting mountain lakes in relation to their presumed overwintering strategy. We studied six Daphnia populations from lakes in the Tatra Mountains  are reflected in clonal richness and diversity.The survival of parthenogenetic females, even at low densities, may have profound consequences for population structure, as such females have a short-term advantage at the beginning of the growing season compared to dormant eggs . This adD. longispina populations showed very high clonal richness as well as diversity in the analysed samples. Clonal richness, calculated as the number of multilocus genotypes (MLGs) detected in the sample divided by the total number of studied individuals in each population (MLG/N), ranged from 0.97 to 1.00; clonal diversity, calculated as the complement of the maximum likelihood estimator of the Simpson's index, (1-D), reached the maximum possible value of 1.00 in all three populations. As these values indicate, almost every single analysed D. longispina individual had a different multilocus genotype (MLG), with the exception of two individuals with an identical MLG from Vyšné Furkotské Lake. None of the D. longispina populations significantly deviated from expectations of the Hardy-Weinberg equilibrium (HWE). The observed among-population differentiation within this species was high (Dest = 0.33). These patterns are apparent in the factorial correspondence analysis (FCA) of multilocus genotypes  were each dominated by a single local genotype. Only one individual differed from the dominant MLG in each of these two populations  and diversity (0.97-1.00) than in the original samples, approaching rather those observed in D. longispina populations. Furthermore, only 2.7 to 3.8% out of 1000 simulated "artificial hybrids" had MLGs identical to the respective dominant clones.The dominant MLG in these populations was heterozygous at five out of nine loci, making it highly unlikely that it harbours a substantial number of genotypes produced by sexual reproduction within the clone . Moreover, the percentage of polymorphic loci and allele richness in those populations was sufficient to detect possible sexually-produced MLGs. This was confirmed by the analysis of MLG richness and diversity of the randomised datasets simulating sexual reproduction within populations with the same MLG structure as recorded in the field Table . AverageThe individuals that exhibited different MLGs in these two lakes differed from the dominant ones at one locus in Nižné Žabie Bielovodské and at two loci in Morskie Oko, respectively; one allele at those variable loci was always shared with the dominant MLG. Five and four loci that were heterozygous in the dominant clones were also identical in the rare MLGs. Such differences correspond to distance classes 2 and 8, respectively, between dominant and rare MLGs, as calculated in the software GenoType. These are relatively small values; identical or lower distances between pairs of 1000 randomised MLGs were observed in only 1.8 and 5.3% of pairwise comparisons, respectively.D. galeata population (from Štrbské Lake) was more variable than the other two, with observed values of clonal richness and diversity reaching 0.50 and 0.91, respectively. Several MLGs were detected multiple times in the sample. The two most common ones were detected seven and nine times, reaching respective proportions of 18 and 24% in the analysed sample of 38 individuals. These two locally dominant clones were heterozygous at nine and eight loci, respectively.The third D. galeata populations showed significant deviations from HWE, with a strong heterozygote excess as a result of the dominance of heterozygous clones. The observed among-population differentiation expressed as the Dest value was even higher than in D. longispina (0.42).All three Daphnia species from the same mountain range strongly differed in their degree of clonal diversity, consistently with assumptions of their overwintering strategy. The differences in population structure between the two groups agree well with predictions of a physiologically-structured model of Daphnia life-history [The populations of two related -history , which aD. galeata, which undergo a prolonged period of clonal selection, had much lower levels of clonal diversity, being almost completely dominated by a single clone in two cases. Moreover, the single individuals in these lakes that exhibited different multilocus genotypes differed only slightly from the dominant clones, being identical at a number of heterozygous loci. It is therefore likely that these individuals did not represent other hatchlings from sexually-produced dormant eggs but the observed differences were rather due to PCR artefacts or possibly somatic mutations of the dominant clones. This is particularly likely for Nižné Žabie Bielovodské, in which the rare MLG differed from the dominant MLG by a single allele of one locus.Actively overwintering populations of D. galeata, intrapopulation variation of D. longispina was very high, similar to what could be expected in an obligately sexual species . Althougg banks .Daphnia longispina in an artificial German lake [D. galeata in Štrbské Lake.The observed patterns of clonal variation suggest that most populations we studied (apart from Štrbské Lake) represent extreme cases in a continuum of overwintering strategies. The contribution of individuals surviving the winter period to the next season's population may substantially differ, both among different localities (as observed by us) and among years within the same locality . The oveman lake ; two sucD. longispina complex from various European habitats analysed by Thielsch et al. [D. longispina populations were characterised by very high clonal richness and diversity, but two out of three populations of D. galeata included in the analysis showed reduced variation. One of these was from a Dutch lake, in which we assume Daphnia may indeed persist year-round due to the relatively mild oceanic climate. However, low MLG diversity does not always correspond to long-term or strong clonal erosion. For example, the lowest diversity among D. longispina populations was reported from a small Tatra Mountain lake, Vyšné Satanie [A wide range of clonal diversities were observed for multiple populations of the h et al. . The pat Satanie . In that Satanie ,24.D. longispina complex is not necessarily species-specific. Whether or not Daphnia successfully survive the winter period is rather influenced by the ability to adapt to local conditions in a particular lake [D. longispina  overwintered in Lake Constance and did not invest in sexual reproduction, while D. galeata produced dormant eggs and disappeared from the water column in winter [D. longispina than in D. galeata [Although we studied two different species, the overwintering strategy in the lar lake ,25,26. Fn winter . This wa galeata .Daphnia taxa in Lake Constance differed in their overwintering strategy within a single lake suggests that this life history trait has a genetic component. Although the conspecific Tatra Mountain populations analysed here showed a similar genetic structure, this is not necessarily a consequence of genetic relatedness. The patterns of mitochondrial variation suggest that each of these species colonised the Tatra Mountains multiple times, and in only two of the studied lakes from the West Tatras, Dolné Roháčské and Vyšné Jamnícke, did they likely originate from the same source [D. galeata tends to be similar despite the fact that some important selection factors, such as predation pressure, strongly differ among the lakes. In two of them (Morskie Oko and Štrbské Lake), fish predation on Daphnia is high [The fact that two coexisting e source ,27. This is high ,29, whilD. galeata inhabits several relatively large and deep lakes, while D. longispina is found in smaller ones [Daphnia individuals is higher, as they may survive in the deep refuge [D. galeata populations from the Tatra Mountains apparently regularly overwinter, as observed in previous studies , as alsD. galeata populations that survive winter as active animals, in contrast to D. longispina populations. The third consequence of clonal erosion may be among-population differentiation [D. galeata did show higher differentiation , which would conform to the prediction. In this case, however, factors other than clonal selection are probably more important, in particular persistent founder effects [Apart from reductions of clonal diversity, another predicted consequence of prolonged clonal erosion is deviation from the Hardy-Weinberg equilibrium. Simulation data  suggest ntiation . This wa effects . Pattern effects ,27, and Daphnia population may be strongly influenced by the choice of reproductive strategy during unfavourable periods, and can show greatly different patterns among populations living in close proximity to one another, depending on local environmental conditions. What are the benefits and costs of an active overwintering strategy? Females surviving under the ice are ready to start reproduction as soon as conditions improve in spring, which is a great advantage against genotypes hatching from dormant eggs [Our data show that the genetic diversity of a ant eggs . If seasant eggs ). Furtheant eggs , and eveD. galeata populations. However, if these clones invest little or nothing into the production of dormant eggs, this may be an evolutionary trap. Ephippia produced earlier in the population are effectively buried in the sediment, and re-establishment from the dormant egg bank may be difficult . A low Daphnia species living in mountain lakes differed in overwintering strategy, which influences the length of the period of clonal erosion and frequency of sexual reproduction. Consequently, strikingly different patterns of genetic diversity were observed in populations of the two species. The use of microsatellite markers, which are variable enough to reveal true clonal structures in cyclical parthenogen populations, revealed that populations surviving unfavourable conditions as active individuals may be dominated by a single clone. Such an extreme reduction of clonal diversity due to long-term clonal erosion may have profound impacts on the microevolutionary potential of such populations.Our study demonstrates how different life history adaptations to unfavourable periods may impact the population genetic diversity in cyclical parthenogens. Two closely related Daphnia populations. The two studied species are locally the commonest members of the D. longispina complex . We a Figure .D. longispina, no individuals were found [D. longispina lakes, Vyšné Jamnické and Dolné Roháčské , but given their similarity to Vyšné Furkotské Lake . Sampling during this period ensured that clonal structure that might have arisen by hatching early in the growing season  would be noticed genetically. Samples were collected by tows of a plankton net from the lake shore or from a rubber boat by vertical hauls from the depth, and preserved in 96% ethanol. DNA from individuals was extracted in 100 μl proteinase K solution, using the protocol from .D. galeata populations were carefully checked to rule out scoring errors.Genotypes of individuals were determined by analysis of nine microsatellite loci described previously , amplifiEach individual was characterized by its multilocus genotype (MLG), and the patterns of MLG similarity were visualised by factorial correspondence analysis (FCA) calculated in Genetix v4.03 . Clonal D. galeata population with those expected under the strong influence of recruitment from dormant eggs, we used the program HYBRIDLAB [D. galeata population, and compared the averaged values with those from field samples of both Daphnia species.To compare the observed clonal structures in each YBRIDLAB  to generD. galeata populations dominated by a single clone.Additionally, we evaluated whether rare genotypes observed in populations dominated by a single MLG more likely arose independently, or resulted from a somatic mutation or methodological artefact. The software GenoType  was usedest [The extent of deviations from the Hardy-Weinberg equilibrium, which are predicted under the scenario of strong clonal erosion , were teest , using test .AP and EH designed the study and did the sampling. EH and JM carried out the molecular work. All authors contributed to data analyses and preparation of the manuscript, read and approved the final version.


Juniperus thurifera L. is an important tree endemic to the western Mediterranean basin that it is able to grow in semi-arid climates. It nowadays exhibits a disjunct distribution pattern, occurring in North Africa, Spain, France and the Italian Alps. The Strait of Gibraltar has acted as an efficient barrier against gene flow between African and European populations, which are considered different subspecies by some authors. We aimed at describing the intraspecific genetic diversity of J. thurifera in populations from the Iberian Peninsula and Morocco and the phylogeographical relationships among these populations. The ploidy level of J. thurifera was examined and eleven nuclear microsatellites (nSSRs) developed for J. thurifera were assessed for genotyping this species. Six nSSRs were polymorphic and subsequently used to assess the genetic diversity and structure of the studied populations. Genotyping of the tetraploid J. thurifera using nuclear microsatellites supports the separation of Moroccan and Spanish populations into two genetically differentiated groups that correspond to the proposed subspecies africana and thurifera. High values of within population genetic diversity were found, that accounted for 90% of the total genetic variance, while population structure was weak. The estimators of genetic diversity were higher in populations of Spain than in populations of Morocco pointing for a possible loss of genetic diversity during the spread of this species to Africa from Europe. Juniperus thurifera L. (section Sabina) is a dioecious tree endemic to the western Mediterranean basin Juniperus thurifera was likely widespread during cold stages of the Pleistocene but nowadays it exhibits a disjunct distribution pattern, occurring in North Africa (Morocco and Algeria), Spain, France  and in the Italian Alps J. thurifera populations was initially driven by increased aridity and warmer climates, but it is currently caused by human activities such as intense wood removal and grazing in North Africa and land use changes in the Iberian Peninsula that lead to the colonization of Juniperus stands by pines or oaks Juniperus thurifera is most often found in calcareous soils in the Iberian Peninsula and acidic soils in North Africa Juniperus thurifera is a morphologically variable species due to the long-term isolation of disjunct populations. The Strait of Gibraltar has acted as an efficient barrier against gene flow between Morocco and European populations for a long time thurifera in Europe and subsp. africana in Africa. Juniperus thurifera subsp. africana (Maire) Romo et Boratyński differs from J. thurifera subsp. thurifera by its smaller cone diameter (5–6 vs. 7–12 mm), lower number of seeds per cone (only 1–2 vs. 2–5) and shorter leaf scales (1–1.5 vs. 2–2.5 mm) africana. Farjon A number of PCR-based techniques can be used to detect polymorphisms for studies of genetic diversity, being AFLP (Amplified Fragment Length Polymorphism) and SSR (Simple Sequence Repeat or microsatellites) the most popular molecular markers used J. thurifera populations from the Iberian Peninsula and Morocco. Specifically, our aims were (1) to analyse the ploidy level of J. thurifera in both sides of the Strait of Gibraltar, (2) to describe the intraspecific genetic diversity of J. thurifera in populations from the Iberian Peninsula and Morocco, and (3) to study the phylogeographical relationships among these populations.In the present study, we used nuclear microsatellites (nSSRs) to assess the genetic structure of Juniperus thurifera. Five populations were sampled in Morocco and six in Spain. A total of 261 individuals were included in the study. Fresh leaves were collected from 20–30 individuals selected randomly in 11 natural populations of The leaves collected from each individual were dried at 30°C overnight before DNA extraction. Genomic DNA was extracted from 20 mg of leaves using the DNeasy Plant Mini Kit (Qiagen). DNA extraction followed the manufacturer's protocol with a few modifications: instead of 200 µl of buffer AE we only added 150 µl and the time of incubation was extended to 10–15 min. The quality of the extracted DNA was checked on 1% TBE agarose gels stained with RedSafe. DNA concentration was estimated using NanoDrop 2000c  and samples were stored at −20°C.J. thurifera was inferred using flow cytometry following Castro et al. J. thurifera, J. phoenicea and Vicia faba with a razor blade in a Petri dish containing 1 ml of WPB buffer . Vicia faba was used as the internal reference standard (2C = 26.90 pg DNA). The nuclear suspension obtained was filtered with a nylon filter and 50 µg/ml of propidium iodide was added to stain the DNA. 50 µg/ml of RNAse was added to avoid staining of double stranded RNA. All samples were analyzed in a Partec CyFlow Space flow cytometer and the results were checked using Partec FloMax software v2.4d . At least 5000 particles were analysed per sample. For each sample, the DNA index was calculated and the nuclear DNA content of J. thurifera was estimated by multiplying the DNA index by the known genome size of the V. faba, using the formula 1 pg = 978 Mbp.The genome size of J. thurifera by Genoscreen  was tested  was performed in a total volume of 20 µl, containing 20 ng of DNA template, 2× QIAGEN Multiplex PCR Master Mix and 1 µM of each forward (F) and reverse (R) primers and RNase-free water. All PCR were run in a Gene Amp 9700 (Applied Biosystems) using the following program: Initial denaturation step for 15 min at 95°C, followed by 40 cycles of 30 s at 94°C, 90 s at locus-specific annealing temperature see  and 60 se), cannot be used to study genetic diversity in polyploids such as J. thuriferaJ. thurifera is an autopolyploid or allopolyploid species. The effective number of alleles per locus (Ae′) and heterozygosity within each population (Hs) were calculated using GenoDive, a software that allows analysing polyploids with unknown dosage of alleles The quantification of genetic diversity in polyploids can be problematic because of the difficulties in assigning the correct allele dosage for each loci and individual r) was computed using the geographic distance and the multilocus genetic distance between individuals. Tests for statistical significance were conducted by random shuffling (999 times) of individual geographic locations to define the upper and lower bounds of the 95% confidence interval for each distance class, and estimating 95% confidence intervals around mean r values by bootstrapping pair-wise comparisons within each distance class (1000 repeats). Analyses were performed using the variable distance classes option based on the geographical distances estimated in each population.The multilocus data was transformed to a binary matrix of presence/absence of each allele for each individual The original genotypic data matrix was used to perform a PCA using the Bruvo distance to explore the genetic similarity between samples. This analysis was performed with PolySat The genetic structure of each population was inferred with the BAPS software Juniperus species resulted in clear fluorescence peaks. Juniperus phoenicea, a known diploid species, had 2C of ≈20 pg, while J. thurifera had 2C of ≈40 pg, revealing that the genomic size of J. thurifera is double than that in J. phoenicea. Knowing that J. phoenicea is a diploid species, the value observed for J. thurifera would correspond to a tetraploid species. The 2C values obtained were the same for all J. thurifera samples, meaning that ploidy levels did not experience variation among regions or populations. The values obtained were similar to those published by Romo et al. J. thurifera and correspond to a tetraploid species.Flow cytometry from individuals of the two P = 0.12). All populations showed also similar high values of heterozygosity (P = 0.01). Significantly higher values were also found in the Iberian Peninsula for the Shannon's information index (I) and the estimator of genetic diversity (H) (P = 0.05 and P = 0.04 respectively).A total of 153 alleles were amplified from the 6 microsatellite loci. All 6 loci were found to be highly polymorphic and displayed up to 4 alleles per sample, in agreement with the ploidy level detected by flow cytometry. The number of different alleles found within each population (An) was generally higher in the populations from Spain than in Morocco, while the average number of effective alleles per locus was similar in all populations . No signzygosity . PrivateFrom the 153 alleles observed, there were 105 alleles (69%) shared by all populations. The remaining alleles were exclusively found in populations of Spain or in populations of Morocco. The number of private alleles found in Spain was higher than in Morocco . 40 privPairwise PhiPT genetic distances ranged between 0.004 (Azzaden Oussem/Matat) and 0.127 (Azzaden Oussem/Luna B). The PhiPT distances between Azzaden Oussem and Matat (0.004) and between Tizi Techt and Matat (0.012) were not statistically significant . The Ana2 = 0.048, P = 0.01). Spatial autocorrelation was detected only for the smallest distance class  separated samples from Spain and Morocco into two groups along the first axis, although there was some overlap between them . The Mance class .Bayesian analysis using BAPS showed the existence of 2 ancestral groups (K = 2) that were dominant in each of the two regions, Morocco and Spain . The twoJ. thurifera using 6 polymorphic nSSR revealed high values of genetic diversity within populations and a clear separation between the populations of Morocco and Spain. This is the first study of the polyploid J. thurifera using nuclear microsatellites. Six primer pairs were developed that are useful for genotyping both J. thurifera subspecies. Our results show some geographical differentiation following the current division of this species into two different subspecies separated by the Strait of Gibraltar: J. thurifera subsp. africana in Africa and J. thurifera subsp. thurifera in Europe The analysis of 11 populations of Juniperus is the gymnosperm genus with most number of cases of natural polyploidy. There are three known cases of natural tetraploidy  and others cases of non-natural triploidy and tetraploidy . But J. thurifera is the only species exclusively polyploidy in the genus J. thurifera individuals had the same genome size and no differences in genome size were found between J. thurifera subsp. thurifera and J. thurifera subsp. africana. These results are in accordance with the survey published by Romo et al. J. thurifera happened early in its evolutionary history.Polyploidy is a rare event in gymnosperm species. The genus J. thurifera are in accordance with other studies in conifers Turdus spp.), rabbits (Oryctolagus cuniculus), red foxes (Vulpes vulpes), stone martens (Martes foina) and even domestic sheeps (Ovis aries) consume J. thurifera fleshy fruits and can be effective seed dispersers for this species at medium and long distances J. thurifera during cold periods of the Pleistocene that allowed gene flow between the currently fragmented populations might also have contributed to the low genetic structure detected within regions The high values of within population genetic diversity found in africana) and Spanish (subsp. thurifera) populations of J. thurifera, likely due to the geographical barrier imposed by the Strait of Gibraltar J. thuriferaWe did find evidences of genetic differentiation between the Moroccan , and variable distance classes. 95% confidence error bars for r were estimated by bootstrapping over pairs of samples; dashed lines  represent upper and lower bounds of a 95% CI for r generated under the null hypothesis of random geographic distribution.(DOCX)Click here for additional data file.Table S1Geographic locations of the 11 populations of J. thurifera in study.(DOCX)Click here for additional data file.Table S2Number of alleles per locus: total number of alleles, alleles shared between both regions and private alleles found for each region.(DOCX)Click here for additional data file.


Alosa pseudoharengus) and blueback herring , species for which long-term demographic data are limited. Recent decades have seen dramatic declines in these species, which are an important ecological component of coastal ecosystems and once represented an important fishery resource. Results show that most populations comprise genetically distinguishable units, which are nested geographically within genetically distinct clusters or stocks. We identified three distinct stocks in alewife and four stocks in blueback herring. Analysis of available time series data for spawning adult abundance and body size indicate declines across the US ranges of both species, with the most severe declines having occurred for populations belonging to the Southern New England and the Mid-Atlantic Stocks. While all alewife and blueback herring populations deserve conservation attention, those belonging to these genetic stocks warrant the highest conservation prioritization.A major challenge in conservation biology is the need to broadly prioritize conservation efforts when demographic data are limited. One method to address this challenge is to use population genetic data to define groups of populations linked by migration and then use demographic information from monitored populations to draw inferences about the status of unmonitored populations within those groups. We applied this method to anadromous alewife ( The inherent value of integrating genetic and demographic data in the design of conservation and recovery plans has been recognized for some time, particularly in the context of evaluating extinction risk in small, isolated populations Lande . A somewAlosa pseudoharengus) and blueback herring  – species for which demographic information is limited to a handful of rivers Atlantic States Marine Fisheries Commission  . Amplification, size-fragment analysis, and scoring were conducted following A'Hara et al. . Genomic DNA was stored at −20° C. Specimens were genotyped at 15 polymorphic microsatellite loci , observed heterozygosity (HO), an unbiased estimate of expected heterozygosity (HE) (Nei FIS) (Weir and Cockerham R) per locus was calculated for each river using FSTAT 2.9.3.2  Nei , and inb2 Goudet  standard2 Goudet .amova was conducted to partition components of genetic variation among rivers, among collections, and among individuals within collections, using a permutation procedure (10 000 iterations) in Arlequin 3.1  (Weir and Cockerham FST(max) for each pairwise comparison. Standardized estimates of differentiation were then calculated as FST/FST(max)  of the pairwise genetic distance matrix for K (number of clusters) from 1 to 13, allowing an estimation of the most likely number of clusters. Both the plateau of likelihood values   were conducted among populations to determine the number of genetically homogeneous groups. Admixture analysis was then conducted to estimate individual admixture proportions with regards to the most likely number of K clusters identified  was conducted among rivers to test for correlations between geographic distance and genetic differentiation using 10 000 permutations of the Mantel test implemented in IBDWS v.3.15  and tested for significant trends over time using Mann–Kendall tests. Both procedures were conducted using Package ‘rkt’ .We used general linear models to test for differences in demographic trends between species and among stocks within each species. Many populations for which we had time series information were also included in our genetic analysis, making stock assignments unambiguous . PopulatWe combined genetic and demographic data to develop a quantitative conservation prioritization for river herring populations that the Stock Assessment identified as being of current or historical importance. We examined the distribution of slope values for mean length and run size time series (both species examined together). We considered demographically increasing populations (slope > 0) to be low priority , stable or slightly declining populations as medium priority, and steeply declining populations as high priority. We set the thresholds between medium and high priority populations at slope = −0.75 for mean length and slope = −0.05 for run size. These values resulted in approximately equal numbers of cases being categorized as medium and high priority. In cases where mean length and run size data were both available but designations did not agree , we applied the more conservative designation  due to the precautionary principle. We used genetic information to extend conservation prioritization to demographically unmonitored populations. We assigned all populations to genetic stocks as described above and calculated the average slope values for each genetic stock. These average slope values were used to designate stock-level prioritizations, which were then applied to any unmonitored rivers within a given stock.Aa082, Ap037, Ap047, Ap070) and blueback herring  prior to further analyses. Remaining loci were retained as evidence for null alleles was sporadically distributed among loci and rivers. Exact tests revealed that genotypic frequencies were largely in accordance with HWE for both species . HWE departures for alewife and blueback herring remained for 11 and 20 locus river comparisons, respectively, and were due to heterozygote deficiencies from sporadic null alleles. Exact tests of LD revealed that loci were physically unlinked and statistically independent . Relative variance in repeat number (lnRV) and heterozygosity (lnRH) failed to detect outlier loci for either species, and provided no evidence of non-neutrality.Evidence for null alleles resulted in the exclusion of loci for both alewife (Aa046) to 19 (Ap010). Ho varied from 0.50 (Town Brook) to 0.67 (Delaware), and R from 4.00 (Lamprey) to 5.49 (Delaware) (Table S1). For blueback herring, the number of alleles per locus ranged from 7  to 28 (Ap037). HO varied from 0.50 (Gilbert Stuart) to 0.57 (Nanticoke), and R from 4.59 (Monument) to 6.81 (Delaware) (Table S2).Genetic polymorphism varied for both alewife and blueback herring depending on the locus and river considered (Tables S1 and S2). For alewife, the number of alleles per locus ranged from 5 (P < 0.05) result in contingency tests among populations with an FST of 0.001 was 0.86 and 0.98 (χ2) for alewife and blueback herring, respectively, while maintaining the realized α-error at the intended level (0.05) for tests of genetic homogeneity.An assessment of statistical power indicated that our microsatellite loci provided sufficient resolution to detect weak differentiation among alewife and blueback herring populations. The probability of obtaining a significant (P < 0.05) genic differentiation between populations was observed for 179/190 pairwise comparisons, with nonsignificant comparisons occurring among neighboring and geographically proximal populations (P < 0.05) genic differentiation between populations was observed for 178/190 pairwise comparisons, with nonsignificant comparisons occurring predominately among neighboring and geographically proximal rivers in the center of the species range  (Table S3); multilocus global FST = 0.049). Nonsignificant (P > 0.05) genetic differentiation was observed primarily among pairwise comparisons of neighboring and geographically proximal alewife populations (Table S3). For blueback herring, FST = −0.003 to 0.106) (Table S4); multilocus global FST = 0.030). Nonsignificant (P > 0.05) genetic differentiation was observed predominately (27/28) among pairwise comparisons of neighboring and geographically proximal blueback herring populations in the center of the species’ range (Table S4).For alewife, standardized pairwise estimates of genetic differentiation  proportion of genetic variance partitioned among populations, and among individuals within populations (Table S5). Nonsignificant variation among temporal replicates for both alewife and blueback herring suggested stable population structure over at least short  temporal scales.For both species, hierarchical DA) among populations  relationship with latitude (DA) among populations  relationship with latitude  using STRUCTURE was observed at K = 4 (−24465.20). However, this estimate was only slightly greater than at K = 3 (−24470.13) but had considerably more variation, suggesting that K = 3 was more accurate . BAPS corroborated this result with significant (P < 0.001) support for three genetically distinguishable clusters. Both methods identified the same three clusters (hereafter referred to as stocks): Northern New England, Southern New England, and Mid-Atlantic . amova revealed more variation among these three stocks  than among rivers within stock  (Table S5). The detection of significant variation among rivers within stocks is consistent with the significant genic differentiation detected among most populations  using STRUCTURE was observed at K = 6 (−35108.260). However, this estimate was only slightly greater than when K = 4 (−35189.77), or K = 5 (−35163.20) . BAPS had some difficulty resolving population structure and provided nearly equivalent support for either K = 4 (P = 0.503) or K = 5 (P = 0.497). However, the greater variation in estimates for K = 5  suggests four clusters across the US range for blueback herring. Both STRUCTURE and BAPS identified the same four clusters (hereafter referred to as stocks): Northern New England, Southern New England, Mid-Atlantic, and South Atlantic  and suggested ‘deep-rooted’ structure among the populations surveyed. amova revealed more variation among the four stocks  than among rivers within stocks  and was comparable with the among river component of variation  when populations were not grouped into stocks (Table S5). That amova detected significant variation among rivers within stocks was consistent with the significant genic differentiation observed among most populations sampled  pattern of IBD for both alewife (r = 0.73) and blueback herring (r = 0.71) across their US range. The slope of the IBD relationship was steeper in alewife (slope = 2.3 e-4) compared with blueback herring (slope = 8.9 e-5), suggesting greater genetic isolation among alewife populations or, conversely, more gene flow among blueback herring populations . Mann–Kendall tests revealed that mean length for spawning adult alewives has declined significantly in 4 of 10 rivers examined , and results were similar for males and females (Table S6). Alewife run size declined significantly in 3 of 20 rivers examined  and increased significantly in one river .Of a total of 29 time series analyzed for blueback herring, 18 showed significant declines, six showed nonsignificant declines, one showed no change, three showed nonsignificant increases, and none showed significant increases (Table S7). Mann–Kendall tests revealed that mean length for spawning adult blueback herring has declined significantly in seven of nine rivers examined . Results were similar for males and females with the exception of the St. Johns, for which declines were significant for females only (Table S7). Blueback herring run size declined significantly in four of nine rivers examined .Time series clearly show declines over time and general linear models revealed significant differences in the magnitude of declines between species and among stocks. For both species, all stocks showed average declines in mean length and run size over time . Overall, declines have been most dramatic in the central portions of each species range, especially for mean length of spawning adults .F1, 35 = 4.159, P = 0.049; F1, 30 = 1.158, P = 0.290; When comparing between species, the mean length of spawning adults has declined significantly more in blueback herring compared with alewife , with the Southern New England Stock showing more dramatic declines than either the Northern New England Stock (Tukey's HSD: P = 0.001) or the Mid-Atlantic Stock (Tukey's HSD: P = 0.011) . Declines in mean alewife run size were evident across all stocks but did not differ among stocks  , with the Southern New England and Mid-Atlantic Stocks declining more steeply than the Northern New England and Southern Atlantic Stocks  . Declines in blueback herring run size were observed across all stocks but did not differ among stocks  , six populations were designated as low priority, 23 as medium priority, and 15 as high priority . High-prWe analyzed population genetic structure and recent demographic trends in anadromous alewife and blueback herring to designate management units and prioritize populations within those units for conservation efforts. Our results show that the majority of rivers examined comprise genetically distinguishable groups Tables  and 3. TFST-based methods  (Hasselman and Limburg Our findings have important implications for managing interbasin transfers of gravid adults, a strategy that is being increasingly implemented in the name of alewife and blueback herring restoration (Hasselman and Limburg We combined genetic and demographic information to define management units and prioritize populations within those units for conservation action. The rationale for this approach is based on the fact that population genetic structure is the legacy of demographic nonindependence caused by migration. Specifically, linking ‘evolutionary measures’ of population genetic structure and ‘ecological measures’ of demographic nonindependence remain challenging because the power to detect population structure using genetic data varies between methods and marker types (Waples and Gaggiotti