Although studies of ancient lake fauna have provided important insights about speciation patterns and processes of organisms in heterogeneous benthic environments, evolutionary forces responsible for speciation in the relatively homogenous planktonic environment remain largely unexplored. In this study, we investigate possible mechanisms of speciation in zooplankton using the freshwater diaptomids of the ancient lakes of Sulawesi, Indonesia, as a model system. We integrate phylogenetic and population genetic analyses of mitochondrial and nuclear genes with morphological and genome size data. Overall, our results support the conclusion that colonization order and local adaptation are dominant at the large, island scale, whereas at local and intralacustrine scales, speciation processes are regulated by gene flow among genetically differentiated and locally adapted populations. In the Malili lakes, the diaptomid populations are homogenous at nuclear loci, but show two highly divergent mitochondrial clades that are geographically restricted to single lakes despite the interconnectivity of the lake systems. Our study, based on coalescent simulations and population genetic analyses, indicates that unidirectional hybridization allows gene flow across the nuclear genome, but prevents the introgression of mitochondria into downstream populations. We suggest that hybridization and introgression between young lineages is a significant evolutionary force in freshwater plankton. A fundamental goal of evolutionary biology is to understand the forces that develop species and maintain biodiversity. For hundreds of years, evolutionists have studied species radiations across isolated island archipelagos to investigate and test predictions about speciation processes. Similarly, ancient, long-lived lakes  represent the aquatic equivalents of islands and have become the focus of many current empirical speciation researchers. In these unique habitats, species are often found to rapidly radiate ecologically and form many flocks of closely related lineages Martens  making tMost ancient lakes have simple food webs and planktonic communities consisting of a single endemic grazing calanoid and small number of predatory species Dumont . Dumont The large lakes of Sulawesi centered in the unique biogeographic region of Wallacea have peculiar ecological characteristics. In this study we compare three isolated lake systems  populations of Sulawesi using cytogenetic, phylogenetic, and population genetic analyses at the island, local , and intralacustrine scales. We contrast the drivers of speciation in the relatively homogenous planktonic environment with the speciation forces observed in the more heterogeneous benthic and littoral habitats. We hypothesize that at the island scale, each isolated lake system has been colonized independently. Thus, we predict that each major basin will harbor a different, possibly endemic, species. In contrast to littoral and benthic organisms, we hypothesize that planktonic organisms will have little genetic differentiation at local and intralacustrine scale due to homogenous pelagic environments that allow unrestricted gene flow. Therefore, we predict panmictic intralacustrine populations as well as genetically homogenous populations among the interconnected Malili lakes.Gallus gallus domesticus) blood as a standard. Means and standard errors were calculated from four individuals of each lake population. A one-way ANOVA and Tukey post hoc comparisons were performed with the STATISTICA v. 8.0 software package to test for differences between all lake populations.We surveyed five lakes on the island of Sulawesi, Indonesia: Lakes Tondano, Poso, and the three major lakes of the Malili lake system . Three to five sites were sampled per lake for a total of 19 sites ; Fig. 1AK method  gene. A subset of these individuals, chosen to represent each of the major COI clades, were analyzed for the nuclear internal transcribed spacer 1 (ITS1) region , and 18S and 28S ribosomal RNA genes . All 20 μL polymerase chain reactions contained 1× PCR buffer (Genscript), 1 mmol/L MgCl2, 1.0 μmol/L of each primer, 0.08 μmol/L dNTPs, 0.4 units of Taq DNA polymerase (Genscript), and 100 ng gDNA. Primers and reaction temperature profiles are given in Total genomic DNA (gDNA) was extracted from single individuals (adults or late copepodite stages) using a modified proteinase 3 bootstrap replicates. BI reconstructions were conducted with MrBayes v.3.1 . Neighbor-joining (NJ) and Bayesian (BI) phylogenetic reconstructions were conducted for three data sets: COI, ITS1, and concatenated 18S + 28S. NJ phylogenetic reconstructions were performed in MEGA v. 4  with coalescent simulations using the full COI alignment and the longest nonrecombining stretch of DNA from the ITS1 alignment in the program IMa2 Hey . We condNh), haplotype diversity (h), nucleotide diversity (π), Tajima's D, and Fu's FS was calculated for the COI data with DnaSP v.5  among the Malili lakes populations eliminating polyteny as a potential source of cryptic speciation  and 418 sequences and contained 175 synonymous and four nonsynonymous mutations comprising 150 unique haplotypes. The mitochondrial phylogeny was used to select 49 representative individuals for sequencing the nuclear genes. The ITS1 alignment consisted of 708 bp and 49 sequences and contained 154 variable sites and 10 unique haplotypes, whereas the 980 bp 18S (352 bp) and 28S (628 bp) alignment consisted of 21 sequences with 36 variable sites and four unique haplotypes.Phylogenetic tree topologies were consistent between NJ and BI inference methods. For each marker, BI analyses converged and produced low average standard deviation of split frequencies. The mitochondrial COI phylogeny reveals four well supported and highly divergent clades corresponding to populations in lakes Tondano TD1, TD2), Poso (PO), Matano/Mahalona (MA/MH), and Towuti (TO) , indicating unidirectional gene flow from Lake Mahalona into Lake Towuti.Coalescent simulations in IMa2 Hey  providedh = 0.53), indicative of a very recent and severe bottleneck or selective sweep. Populations in Poso and the Malili lakes had very high haplotype diversities (h = 0.91–1.0), suggesting high genetic diversity and large effective population sizes. Most notably, Lake Poso had a haplotype diversity of 1.0, where every sampled individual possessed a unique haplotype. Tajima's D  was colonized independently by a different genus of copepod, and each species is endemic to Sulawesi Island. Nearly all their congeners are inhabitants of Southeast Asia (Lai and Fernando h = 1) and sprawling haplotype network  in this population. Such pronounced genetic scarcity may be the result of either very recent colonization, drastic changes in environmental conditions  and sprawling haplotype networks  over regional geographic scales (Adamowicz et al.


Human pressures have put many top predator populations at risk of extinction. Recent years have seen alarming declines in sharks worldwide, while their resilience remains poorly understood. Studying the ecology of small populations of marine predators is a priority to better understand their ability to withstand anthropogenic and environmental stressors. In the present study, we monitored a naturally small island population of 40 adult sicklefin lemon sharks in Moorea, French Polynesia over 5 years. We reconstructed the genetic relationships among individuals and determined the population’s mating system. The genetic network illustrates that all individuals, except one, are interconnected at least through one first order genetic relationship. While this species developed a clear inbreeding avoidance strategy involving dispersal and migration, the small population size, low number of breeders, and the fragmented environment characterizing these tropical islands, limits its complete effectiveness. Human population growth has fragmented the range of many species , leadingGlobally, sharks are threatened by overfishing –12. WhilNegaprionacutidens) population around Moorea, French Polynesia [For 5 years, we monitored a small sicklefin lemon shark . Every shark species is protected under French Polynesian laws, however, DNA samples were taken by non-invasive methods approved and conducted under the French Polynesia Ministry of Research permitting authority (Permit # 653/MRM/SPE/DEV), and no lethal sampling was conducted. Juvenile sharks were captured with gillnets and released in the water alive under good conditions and adults were remotely sampled with a modified speargun with a biopsy tip.Negaprionacutidens) visiting a recreational diving site during a 5-year period in Moorea , which is expected to increase the effective isolation of reef-associated populations . We moniResident sharks of Moorea: composed of 7 males  and 7 females ;• Non-resident sharks visiting Moorea occasionally: composed of 6 males  and 9 females ;• Bora-Bora resident sharks: composed of 4 females only sighted in Bora Bora .• In addition to adults on Moorea and Bora-Bora sites we also captured 52 newborn and immature sharks from different cohorts between 2006 and 2009 in Moorea and three neighbouring islands: Tetiaora (40 km), Tahaa (200 km) and Rangiroa (330 km) . Annual During our underwater photo-identification surveys, we reported the presence of dermal bite wounds and scars on females’ body as a sign of mating activity ,28. CourNegaprionacutidens and selected 14 polymorphic microsatellite loci developed for other shark species . We isolated 2 species-specific polymorphic microsatellite loci from  species . All spe species . AmplifiENALEX 6 . The preENALEX 6 . Tests fENALEX 6  and signechnique , GENALEXechnique  was usedMaximum likelihood estimates of pairwise relatedness coefficients and genealogical relationships were calculated with the software ML-RELATE  computinInstead of trying to reconstruct an exact pedigree that is challenging from genetic data alone in wild populations, we built a genetic network using relatedness information . NetworkThe program CERVUS  was firsTo test for inbreeding, we used a Monte Carlo simulation implemented in STORM 1.0 . This anNegaprionacutidens collected for this work, polymorphism varied from 2 to 15 alleles per locus. Observed heterozygosity ranged from 0.365 to 0.871 per locus and expected heterozygosity from 0.395 to 0.871  even when excluding resident sharks of Bora Bora  and when we included individuals from Bora Bora , with a stable calendar each year. Several males can be involved in courtship behavior at the same time following the same female  of the 20 fathers sired more than one litter . Five maP = 0.142). IR values calculated from loci with over 5 alleles were not different from those calculated from all loci  suggesting that our estimation of IR values were not inflated by the presence of low allelic diversity at some loci. Observed maximum IR value was 0.504, 13% (7/52) of all offspring had IR values higher than 0.25 , and 36% (19/52) had o-IR larger than 0.125 : mean IR ± 95% CI = 0.037 ± 0.056 and mean simulated IR ± 95% CI = 0.009 ± 0.001, an 0.125 .R2 = 0.0629; P = 0.011). This was confirmed by a significant difference in R values between maturity status ; offspring IR values being higher than IR of mature sharks  and IR  monitoring and genetic survey used to construct the genetic network and to infer the mating output of an island adult shark population. This population is also unique since, unlike many places in the world, it has been preserved from any human exploitation, implying that low densities observed are normal for this population. Our results demonstrate that this small population displays patterns of connectivity throughout the archipelago and dispersal which are used as a strategy to avoid mating with closely related partners.Negaprionbrevirostris, appear to remain at their natal island, others disperse before reaching maturity [Negaprionacutidens, as among four resident female sharks of Bora Bora, three  appeared genetically linked to sharks sighted in Moorea  of juveniles, but a recent study showed that among immature sharks  the first sharks to leave the study area was about 150 cm TL [Negaprionbrevirostris [Carcharhinusmelanopterus [Although the mean relatedness was low among adults of the population, all sharks (but 1) were interconnected through close to moderate genetic relationships. First order genetic relationships accounted for 17.6% of all pairwise relationships, reaching 21.6% when excluding individuals from Bora Bora. The genetic network  illustramaturity  to colon50 cm TL . Individrostris  and in topterus . Howeveropterus . Therefoopterus . OverallNegaprionacutidens shares nursery locations with Carcharhinusmelanopterus in Moorea and Tetiaroa [From parentage analysis, we were able to investigate the mating system and recruitment patterns of lemon sharks in the archipelago. Like other reef sharks depending on nurseries for recruitment ,20,21, mTetiaroa . As femaTetiaroa ,20. ThisTetiaroa .Negaprionbrevirostris in the Bahamas  was not significantly higher than expected under random mating. However, maximum offspring IR values (IRmax = 0.50) were higher than those found in offspring of its sister species x = 0.05 ). IR val litters , althoug Islands . Negap success . Additiorostris ,48 suggeNegaprionacutidens in French Polynesia as: (1) a few males appear to dominate paternity , somaternity . AnotherIn addition, significantly higher IR values in juveniles than in adults  demonstrIndividuals from this local population of sickefin lemon sharks in the Society archipelago share many first-order genetic relationships providing evidence that population size in this species is fairly limited in the context of islands at least in French Polynesia, but likely in isolated islands system of the Pacific. This low genetic variability has encouraged sicklefin lemon sharks to develop different strategies to avoid mating between close relatives with evidence of migrations across islands and atolls. The highly isolated and fragmented environment of French Polynesia  may limiFigure S1Sampled nursery locations. (A) Map of French Polynesia with sampled islands. (B) Sampled nursery locations in Tetiaroa. (C) Nursery locations in Moorea. Circles are coloured according to the nursery location used in the study and black circles refers to adult sampling sites.(TIF)Click here for additional data file.Figure S2Distribution of internal relatedness values (IR) of adult (black bars) and juvenile (grey bars) lemon sharks.(TIF)Click here for additional data file.Table S1Description of the 16 microsatellite loci used to genotype sicklefin lemon sharks. Dyes: fluorescent Beckman Coulter dyes labels; Ta: annealing temperature (°C); N: number of individual scored; H0: observed heterozygosity; HE: expected heterozygosity; k: number of alleles; Fis: inbreeding coefficient; H–W: exact test for departure from Hardy-Weinberg.(DOCX)Click here for additional data file.Table S2Distribution of average relatedness values across categories (sex and socio-residency groups). Mean relatedness is displayed together with SD in parenthesis.(DOCX)Click here for additional data file.Table S3Underwater observations of male courtship behaviour in sicklefin lemon sharks in Moorea. For each year, the females that each male was observed to follow is indicated.(DOCX)Click here for additional data file.Table S4Underwater visual estimations of reproduction timing in sicklefin lemon sharks in Moorea. DBW: Dermal Bite Wound (dates of observations are indicated); P: Parturition indicated in grey .(DOCX)Click here for additional data file.Video S1Courtship behavior with female F01 is closely followed by male M10 in 23 October 2008. Female F01 was in near to term gestation in August 2008 (ust 2008  and presust 2008 . This feust 2008 . Note th(MP4)Click here for additional data file.


Arachis Hypogaea L.) cultivars which had been extensively planted in different regions in China. These SSR markers amplified 440 polymorphic bands with an average of 2.99, and the average gene diversity index was 0.11. Eighty-six rare alleles with a frequency of less than 1% were identified in these cultivars. The largest Fst or genetic distance was found between the cultivars that adapted to the south regions and those to the north regions in China. A neighbor-joining tree of cultivars adapted to different ecological regions was constructed based on pairwise Nei’s genetic distances, which showed a significant difference between cultivars from the south and the north regions. A model-based population structure analysis divided these peanut cultivars into five subpopulations . P1a and P1b included most the cultivars from the southern provinces including Guangdong, Guangxi and Fujian. P2 population consisted of the cultivars from Hubei province and parts from Shandong and Henan. P3a and P3b had cultivars from the northern provinces including Shandong, Anhui, Henan, Hebei, Jiangsu and the Yangtze River region including Sichuan province. The cluster analysis, PCoA and PCA based on the marker genotypes, revealed five distinct clusters for the entire population that were related to their germplasm regions. The results indicated that there were obvious genetic variations between cultivars from the south and the north, and there were distinct genetic differentiation among individual cultivars from the south and the north. Taken together, these results provided a molecular basis for understanding genetic diversity of Chinese peanut cultivars.One hundred and forty-six highly polymorphic simple sequence repeat (SSR) markers were used to assess the genetic diversity and population structure of 196 peanut ( Arachis hypogaea L., is an important oilseed and cash crop worldwide. It is one of the primary sources of vegetable oil and protein in developing countries. The peanut annual planting area is around 24 million hectares, with an annual production of nearly 35 million tons (http://apps.fao.org/cgi-bin/nph-db.pl?subset=agriculture). China is the largest peanut producer in the world with over 20% of planting area and more than 40% of production. During the last five decades, cultivated peanut had been subjected to intensive artificial selection, resulting in favorable changes in yield, disease resistance, biochemical composition and other agronomic traits The cultivated peanut, The previous studies paid more attention on morphological and agronomic trait variations Compared with a large quantity of morphological and agronomic variation in peanut cultivars Traditional cluster analysis could provide an easy and effective way in determining the genetic diversity of germplasm collections In the present study, the genetic diversity and population structure of 196 genotypes of peanut cultivar released in the last 5 decades from three ecological regions  in China were analyzed using 146 highly polymorphic SSR markers. Our objectives were to estimate the levels of genetic diversity, and to characterize the population structure of the Chinese peanut cultivars.The genotypes of 196 Chinese peanut cultivars  were ampThe average number of alleles per locus was the greatest in cultivars from the south, and was the least in cultivars from the Yangtze River. The average gene diversity index was the lowest in cultivars from the south, followed by that from the north . The avestF) could be interpreted as standardized population distances between two populations. The stF values would almost be in accordance with the genetic distances. A neighbor-joining tree of the thirteen province populations was constructed based on Nei’s genetic distances, showing significant difference between cultivars from the south and from the north (There was the largest genetic distance (0.40) in cultivars between the south and the north. Cultivars from the Yangtze River were very similar to those from the north (0.12). For different provinces, genetic distances between the populations from the south  and the north  were larger than those of others. Population of Shandong showed the smallest genetic distance with population of Henan (0.12), whereas population of Jilin showed the greatest genetic distance with populations of Anhui, Henan and Jiangsu (0. 55) . Pairwishe north .According to Nei’s genetic distance based on 146 SSR loci in different years, cultivars released before 1970 had greatest Nei’s genetic distance. Among other each decade, Nei’s genetic distances did not change much , which sK) values (The population structure of the Chinese peanut cultivars was inferred using Structure 2.0 software based on 146 SSR markers. The number of subpopulations (K) was identified based on maximum likelihood and delta K (▵) values . At K = ) values , Table 5) values , Table 5) values , Table 5) values , Fig. S2) values .Released years, geographic origins and population structures provided the organizing data for hierarchical AMOVA. In the total genetic variance among populations based on the released years, 34% was attributed to the released year, and the remaining 66% was explained by individual differences within populations . In the hypogaea germplasm was 4.0, and breeding lines was 2.2 SSR markers have been extensively used to detect the variability in peanut genotypes and to evaluate their genetic diversity. Compared with previous reports, the genetic diversity of the 196 Chinese peanut cultivars in this study was at a lower level, as reflected by the average number of alleles per locus (2.99) and the gene diversity index (0.11). Other researchers reported averages of 2.2 to 8.1 alleles per locus in various peanut collections hypogaea and ssp. fastigaeaIn this study, gene diversity index was 0.11, which was lower than those of previous peanut collections (0.15–0.18) fastigiata var. fastigiata that is rarely planted in China. Many unique rare alleles would be discovered in ssp. fastigiata, showing that ssp. fastigiata would have more genotype-specific bands than ssp. hypogaea do Some of the SSR markers generated genotype-specific bands, which could be used as molecular identity data for specific genotypes. Eighty-six rare alleles with a frequency less than 1% were identified in 196 genotypes peanut cultivars using 146 SSR markers. There was 0.45 in average rare alleles per SSR marker, which was lower than that of US mini-core collection (1.68) On the basis of geography origin of different peanut cultivars, peanut cultivars were divided in the three ecological regions: the south region, the north region and the Yangtze River region, which are the three ecological regions of the national peanut regional test in China fastigiata. Similarly, most of populations from the north were clustered together, and most of the cultivars were ssp. hypogaeafastigiata and hypogaeafastigiata var. fastigiata, which were clustered with those from the south together. AMOVA analysis revealed that, in the total genetic variance of the geographic origins, 31% were attributed to the geographic origins  was used to overcome the difficulty for interpreting the real K values ΔK for the 196 peanut cultivars is K  =  5  in STRUCTURE, LnP(D) and  K  =  5 . Cluster K  =  5 , Table 4=  5  method 2+, 1× Taq buffer, 0.5U Taq DNA polymerase (Fermentas) and 15 ng DNA. Amplification was carried out using a PTC-100 Thermocycler (MJ Research). The amplified products were visualized by 6% denaturing polyacrylamide gel (PAGE) followed by silver staining as described by Ren The SSR primer sequences were selected based on the previous reports stF) between populations were calculated using PowerMarker V3.25. The dendrogram was constructed based on genetic distance calculated using the Neighbour subroutine of PoweMarker V3.25 as described The polymorphism bands were recorded as 1 (present) or 0 (absent) for the same amplified fragments. Data format could be changed according to different analysis software. Major allele frequency, number of alleles per locus, gene diversity and polymorphism information content (PIC) were analyzed using PowerMarker V3.25 k) was assumed to be from 1 to 10, without admixture and with correlated allele frequencies, and the burn-in time and iterations for each run were both set to 50,000. Ten replications were used for each k. Due to the difficulties associated with finding the highest posterior probability before a large k value was examined, LnP(D) and Evanno’s Δkk value. It is calculated as Δk  =  M[|L(k – 1) – 2L(k) + L(k + 1)|]/S[L(k)], in which L(k) represents the kth LnP(D), M is the mean of 10 runs, and S is their standard deviation.based program Structure Principal components analysis (PCA) was a visualization technique commonly used in multivariate statistics, which also identifies eigenvectors and amounts of variance and cumulatively explained variances per component. The PCA analysis was conducted using the NTSYS package Figure S1The modal value of this distribution is the true K. The Δk of 10 repeats based on STRUCTURE calculation using SSR data. Δk calculated as Δk  =  m (| L (K+1) −2 L (K) + L (K−1)|)/S [L (K)].(DOC)Click here for additional data file.Figure S2Principal Component Analysis of five subpopulations of the 196 peanut cultivated varieties in China.(DOC)Click here for additional data file.Table S1Accessions, variety names, origin, region, released year of 196 peanut cultivars in China.(DOC)Click here for additional data file.Table S2Summary statistics of the 146 SSR markers used in this study. MAF: major allele frequency, AN: number of alleles per locus, GD: gene diversity, PIC: polymorphism information content.(DOC)Click here for additional data file.Table S3Summary genetic statistics of peanut cultivars in different years.(DOC)Click here for additional data file.Table S4Pairwise estimates of Fst and Nei’s genetic distance based on 146 SSR loci among different years.(DOC)Click here for additional data file.


Aphis nerii, though native to the Mediterranean region, is an invasive pest species throughout much of the world. Feeding primarily on Oleander (Nerium oleander) and Milkweed (Asclepias spp.) under natural conditions, these plants are unlikely to support aphid populations year round in the southern US. The objective of this study was to describe the genetic variation within and among US populations of A. nerii, during extinction/recolonization events, to better understand the population ecology of this invasive species.The importance of genetic diversity in successful biological invasions is unclear. In animals, but not necessarily plants, increased genetic diversity is generally associated with successful colonization and establishment of novel habitats. The Oleander aphid, A. nerii “superclones”. Genotypic variation was absent in all populations  or “clone”) and the genetic composition of only one population completely changed across years. There was no evidence of sexual reproduction or host races on different plant species.We used five microsatellite markers to assess genetic diversity over a two year period within and among three aphid populations separated by small (100 km) and large  geographic distances on two host plant species. Here we provide evidence for Aphis nerii is a well established invasive species despite having extremely low genetic diversity. As this aphid appears to be obligatorily asexual, it may share more similarities with clonally reproducing invasive plants, than with other animals. Patterns of temporal and geographic genetic variation, viewed in the context of its population dynamics, have important implications for the management of invasive pests and the evolutionary biology of asexual species. While studies are few, genetic diversity is believed to facilitate successful biological invasions Aphis nerii Boyer de Fonscolombe, is a pest of several plant families including Apocynaceae (Nerium and Vinca), Asclepiadaceae , Asteraceae, Convolvulaceae, Euphorbiaceae, and Rutaceae Aphis nerii has since become a common invasive species in warm temperate and tropical regions of the world Aphids are valuable study systems for investigating the roles of genetic variation and phenotypic plasticity on population, ecological, and evolutionary dynamics Nerium oleander (Apocynaceae), is a common ornamental plant in southern and coastal states and frequently grows along US highways A. nerii sequesters for defense, and are unable to support aphid populations throughout the year In the United States, the Oleander aphid commonly infests two plant families, Apocynaceae and Asclepiadaceae A. nerii inhabiting species of milkweed that differed in the amount cardiac glycosides that they possess A. nerii among different host plant families such as Milkweed and Oleander, to our knowledge, have not been examined.Aphid fitness tradeoffs among host plant species results in selection for host fidelity which can inhibit gene flow and result in the development of host races; i.e., host-associated population genetic structure Aphis nerii is believed to be an obligate parthenogen; males have never been found in natural populations Aphid species vary in their mode of reproduction from obligate to cyclical parthenogens, but less than 3% of species are strictly clonal A. nerii, produce winged forms in response to overcrowding and/or decreasing host plant quality Aphis nerii, however, is found in patchily distributed habitats throughout the United States, and both Oleander and Milkweed are unable to support aphid populations year round except in the southernmost latitudes of the US The genetic structure of aphid populations is shaped spatially and temporally by habitat distribution, dispersal capabilities, and life-cycle. Many aphids, including A. nerii with the goal of gaining insight into the population dynamics, life history, and ecology of this well-established invasive species. In so doing, we asked the following questions: 1) Is there any evidence of sexual reproduction in US populations of A. nerii?; 2) Do aphids inhabiting different hosts comprise host races?; 3) Does the patchy distribution of suitable habitat result in population genetic subdivision over small and/or large geographic ranges?; and 4) Is there temporal variation in population genetic structure? To address these questions, we used microsatellite markers to assess genetic diversity over a two year period within and among three populations separated by roughly 100 km and 3,700 km on two host plant species.The aim of this study was to characterize genetic variation within and among southern US populations of A. nerii. Whilst genetic variation was extremely low within and among populations, the genetic composition of one population was found to change drastically over time. Our findings suggest that A. nerii is an efficient colonizer that demonstrates true metapopulation dynamics.Here, we report no evidence of sexual reproduction or the existence of host races in N. oleander) at two locations in Georgia and one location in central California, June-August 2008, 2009. This “overwintering” period between samples allowed us to assess temporal genetic variation, and determine if sexual reproduction occurred. Sampling locations were: Statesboro, Georgia ; Tybee Island, Georgia ; and Concord, California . Sampling areas consisted of multiple patches of Oleander plants in close proximity but intermittently up to 2.5 km apart. To survey genetic diversity within each population, aphids were collected from different parts of the same plant and from as many different plants as possible. To assess genetic variation among aphids inhabiting different host plant species, aphids were collected from Milkweed (Asclepias amplexicaulis) at a second site in Statesboro, Georgia  in June 2008.Aphids were collected from Oleander  using primers originally designed for the Cotton/Melon aphid, 2. Reactions consisted of a 4 min denaturation at 94°C, followed by 35 cycles of 30 s at 94°C, 35 s at 58°C, and 45 s at 72°C, and a final 10 min extension step at 72°C.DNA was extracted by macerating individual aphids in 70 µl cell-lysis/proteinase-K buffer  followed by incubation for 1 h at 65°C and 15 min at 99°C (Lee and Frost 2002). PCR reactions were carried out in 20 µl final volumes with 2.5 mM MgClPCR products were run on small (15 cm×17 cm×0.8 mm), non-denaturing TAE  buffered polyacrylamide gels of either 9% or 10% concentration, with the lower portion of the gel supplemented with EnhanceIT polymer . Gels were run for 2 h at 30 mA, stained with ethidium bromide, and visualized on a UV light box. Allele sizes at each locus were estimated using the M3 size standard .FISFST and RST using the program FSTAT v. 2.9.3 F-statistics was tested using the randomization procedure available in FSTAT using 5000 permutations. Estimates of genetic distance between multilocus genotypes  (δμ2 and DSW) were calculated according to Shriver et al. O) to that expected under conditions of sexual reproduction (GE), as described by Stoddart and Taylor Observed and expected heterozygosities were calculated for each locus in each population. Deviations from Hardy-Weinberg expectations were tested according to Guo and Thompson A. nerii. The number of alleles was low; 3 to 4 alleles per locus. Three of the five loci each had an observed heterozygosity (HO) of 0.143, the two loci an HO of 1. Null alleles were not detected for any loci in any populations sampled.All five microsatellite loci used in this study were polymorphic in Aphis nerii. Only 2 multi-locus genotypes (MLGs) or “clones” were detected among the 319 individuals assayed across all populations and both years  suggesting that the MLGs do not share a close genealogical relationship Despite the low level of genotypic diversity observed, the genotypes of the two MLGs were divergent. That is, the two MLGs did not share any alleles, clearly indicating that they were not the product of recombination through sexual reproduction. Estimates of genetic distance, based on the stepwise mutation model, were large (δμO/GE) from 0.141 to 0.048 . Fourth, estimates of FIS were −1.0 for all populations.No evidence of sexual or mitotic recombination was found. First, no homozygous allelic arrangements were found at any of the heterozygous loci . Second,A. nerii from two common host plants (Oleander and Milkweed) from Statesboro, GA . All samples from both hosts consisted of MLG 1, indicating that there is no host associated subdivision at this location   .FST = 0, RST = 0)   . The Tyb= 0.787) . During A. nerii to better understand the ecology, life history, and population dynamics of this well established invasive species. We found that Oleander aphids are remarkably invasive throughout the southern United States, with extremely low genetic diversity.Small, genetically-uniform populations are subject to ecological and evolutionary forces  which threaten population persistence, in both native and introduced habitats Aphis nerii is believed to be obligately parthenogenetic, based on the complete absence of males under natural conditions A. nerii in the southern US.A. nerii. For example, long term asexual populations of the pea aphid, Acyrthosiphon pisum (Harris) in Japan were composed of five to seven MLGs, and cyclically parthenogenetic populations harbored much greater diversity than did asexual populations A. pisum, both within and among populations The most striking pattern observed in this study was the low level of genotypic diversity within and among populations over a large geographic area. We observed only two MLGs and a maximum estimate of genotypic diversity (G/N) within any population of 0.05. Several similar studies of other aphid species using comparable numbers of loci (4 to 7) have revealed higher levels of genetic variation than that observed in et al.A. pisum in Chile. Among the 432 individuals sampled over a 570 km range, 16 MLG's were identified with three MLG's comprising≈90%  of the diversity. Compared to previous studies, the pattern we observe in A. nerii is extreme even for “superclones”. Within any sampling year, each population consisted of a single MLG. In 2008, all populations sampled from both California and Georgia showed only a single MLG (MLG 1). In 2009, populations from Statesboro, Georgia and California comprised the same MLG (MLG 1), whilst a population from Tybee Island, Georgia comprised a single but entirely different MLG (MLG 2).Aphid populations are sometimes composed of a small number of dominant genotypes (“clones”) and many low frequency (rare) genotypes A. nerii has been assessed within and among populations from California, Iowa, and Puerto Rico Variation of morphometric and life history traits in A. nerii likely results from successive genetic bottlenecks or founder events. Subsequent clonal propagation and rapid spread of genotypes throughout the host range would be followed by rapid clonal competition/selection resulting in a few geographically-widespread and dominant clones  is the most common host of A. nerii in the northern US where N. oleander grows infrequently Asclepias species are perennial plants that sprout in Spring, bloom in early Summer, and then set seed and die-back in the Fall. The life history of this host plant would require A.nerii to have a secondary host (though this has not previously been reported) or to produce sexuals to produce overwintering eggs . On both Oleander and Milkweed, a regular pattern of colonization, rapid increase, and population extinction would be expected.Milkweed  Aphis gossypii Glover A. nerii populations, we would have expected to see genetic variation between populations inhabiting Oleander and those inhabiting Milkweed, regardless of geographic proximity. Our findings suggest that there is no selection for host specificity and that A. nerii is a polyphagous, i.e. generalist species, although more data are required to confirm this contention. This pattern is consistent with Lynch Groeters A. nerii surveyed in this study show the genetic signature of obligate parthenogenetic reproduction, supporting previous reports that the species is indeed obligately asexual. Within any sampling period, each population was composed of a single MLG. This level of variation is remarkably low compared to the variation observed in other aphid species presumed to be largely or completely asexual A. nerii is a generalist species with strong dispersal capabilities. As patches of host plants are colonized by few individuals that reproduce rapidly through parthenogenesis, founder individuals likely come from a source population(s) characterized by low genetic diversity.In summary, populations of A. nerii provides great insight for the management of invasive pests and the population dynamics of clonal organisms.Despite having extremely low genetic diversity Aphis nerii is a well established invasive species. Understanding the temporal and geographic genetic variation of


Isodon rubescens and some related species. While the bioactivity of oridonin has been well studied, the extent of natural variation in the production of this compound is poorly known. This study characterizes natural variation in oridonin production in order to guide selection of populations of Isodon with highest oridonin yield. Different populations of I. rubescens and related species were collected in China, and their offspring were grown in a greenhouse. Samples were examined for oridonin content, genotyped using 11 microsatellites, and representatives were sequenced for three phylogenetic markers . Oridonin production was mapped on a molecular phylogeny of the genus Isodon using samples from each population as well as previously published Genbank sequences. Oridonin has been reported in 12 out of 74 species of Isodon examined for diterpenoids, and the phylogeny indicates that oridonin production has arisen at least three times in the genus. Oridonin production was surprisingly consistent between wild-collected parents and greenhouse-grown offspring, despite evidence of gene flow between oridonin-producing and non-producing populations of Isodon. Additionally, microsatellite genetic distance between individuals was significantly correlated with chemical distance in both parents and offspring. Neither heritability nor correlation with genetic distance were significant when the comparison was restricted to only populations of I. rubescens, but this result should be corroborated using additional samples. Based on these results, future screening of Isodon populations for oridonin yield should initially prioritize a broad survey of all species known to produce oridonin, rather than focusing on multiple populations of one species, such as I. rubescens. Of the samples examined here, I. rubescens or I. japonicus from Henan province would provide the best source of oridonin.Oridonin is a diterpenoid with anti-cancer activity that occurs in the Chinese medicinal plant  Isodon (Schrad. ex Benth.) Spach (in vitro and in vivo activity against human cancer cells Isodon japonicus (Burm. f.) H. Hara whose common name in Japanese is oridoIsodon, most notably the Chinese medicinal plant Isodon rubescens (Hemsl.) H. Hara I. rubescens as the source of the compound Oridonin is a bioactive diterpenoid produced by plant species in the genus .) Spach . SeveralI. rubescens have traditionally been used as a folk medicine in Henan province, central China for the treatment of sore throat, inflammation and gastrointestinal problems donglingcao (???). The medicinal uses of I. rubescens were first documented in the 1977 edition of the Pharmacopoeia of the People’s Republic of China I. rubescens is a relatively recent introduction to the Traditional Chinese Medical literature. Although I. rubescens was not included in editions of the Chinese Pharmacopoeia after 1977, it was reintroduced in the most recent edition The aerial portions of I. rubescens and other species of Isodonbut see 23], or their variation in nature. The main goal of this paper is to summarize the results of a preliminary study of the natural variation of oridonin production using a unique combination of phylogenetic and population genetic approaches. This work was done to guide researchers in selecting populations of I. rubescens and related species that produce the most oridonin, since to-date there is no reported de novo synthesis of oridonin. This project was carried out in the context of a larger endeavor to evaluate more than 200 species of commonly used Chinese medicinal plants for their bioactivity Isodon was to be a used as an example that could be applied to any potential screening discoveries of promising bioactive chemicals that would require additional plant material for follow-up pharmacological studies and clinical trials.While much work has been done to characterize the chemical structure and diversity of diterpenoids in IsodonI. rubescens as compared to oridonin alone Isodon or for the total bioactivity of Isodon extracts.While oridonin has been extensively studied for therapeutic potential, it should be noted that it is not the only bioactive diterpenoid in Isodon is comprised of about 100 species and occurs primarily in Asia, with a few species in Africa Isodon has been well studied, recent molecular phylogenetic work has placed the traditional subgeneric classification in question Isodon in Japan using chloroplast sequence markers found low phylogenetic resolution and many species were polyphyletic. The phylogenetic patterns were attributed to some combination of a recent radiation, hybridization, or incomplete lineage sorting of ancestral polymorphisms in the genus IsodonIsodon species, published studies of the high diversity of diterpenoids in both IsodonI. rubescensIsodon and within I. rubescens. This variability would be indicated by multiple gains and losses of oridonin production in the evolutionary history of Isodon, and also by variation of oridonin production between individual plants, both within the same population as well as different populations of I. rubescens and also between parents and offspring. To evaluate these expectations, oridonin production was mapped onto a phylogeny of Isodon to determine the extent of evolutionary lability and to identify species or clades that might be the best sources of the compound. In addition, oridonin production was compared in microsatellite-genotyped individuals of wild-collected parents and greenhouse-grown offspring to determine heritability and populations with the highest chemical production.The genus I. rubescens and related species were collected from the People’s Republic of China in October and November of 2009 in Henan, Hubei, and Guizhou provinces. Samples were collected from a total of eight sites, with about 10 to 15 individuals collected at each site. Five populations consisted of the species I. rubescens, four of which were collected in the area of traditional use of donglingcao in Northern Henan province. One population of I. rubescens from Henan province represented a donglingcao cultivation site (JY). Two populations from Guizhou were determined to be the species Isodon enanderianus (Hand.-Mazz.) H.W. Li and one population from southern Henan province was identified as Isodon henryi (Hemsl.) Kudô. In addition, individuals of I. japonicus and I. lophanthoides var. micranthus (C.Y. Wu) H.W. Li were collected growing at the same collection sites of I. rubescens and I. enanderianus, respectively. Location details for each population are shown in Samples of donglingcao were purchased in Zhengzhou city, Henan province. Collection activities for this study were carried out under the auspices of a larger collaborative project between Harvard Medical School and the Beijing University of Chinese Medicine. Details about the collaboration agreements and the relevant Chinese government authorities that reviewed them are provided in a publication about the larger project Isodon are endangered or protected.The collection of each individual consisted of a pressed voucher specimen, leaves in silica desiccant for DNA and chemical analyses, and seeds, if available. Representative voucher specimens are deposited at the Gray Herbarium of Harvard University (GH) and are noted in I. rubescensSeeds from parents representing each population were planted on April 7, 2010 in seedling trays. Seeds were grown in 3B Mix potting soil, which includes peat moss, pine bark, perlite, starter nutrients, wetting agent, and limestone . A total of 1824 seeds were planted, averaging 27 seeds from each individual that was producing seeds at the time of collection. Seeds were collected from between four and 11 individuals per population. Of the total, 439 seeds germinated, and 308 plants survived to harvest. Seedlings were transplanted after two weeks to individual pots. Plants were arranged randomly with respect to parent population source, and re-arranged periodically during the growing period. Offspring individuals were harvested on August 10, 2010, which represents the typical harvest time of Between 200–500 mg of dried leaf samples were ground using a mortar and pestle to prepare for chemical analyses. The ground samples were put in a 40 mL vial  and 10 mL of 1∶1 dichloromethane : ethanol were added. The solution was sonicated for 30 min, centrifuged for 5 min, then filtered into a new 40 mL vial using Whatman Grade 1 Filter Paper  without disturbing the pellet. The filtrate was placed in a cold room at 4°C and the filtrant was resuspended in 10 mL of 1∶1 dichloromethane:ethanol and left at room temperature overnight. The mixture was then filtered, and resulting filtrate was combined with the filtrate of the first step. The combined filtrate was dried in a Savant SPD2010 Speedvac  for 5–6 hrs. After the extract was completely dry, 20 mL of LC/MS grade methanol was added and the resulting mixture sonicated for 30–60 min. Once fully dissolved, 150 µL of the resulting solution was added to a 1 mL syringe  with filter , and filtered into a 2 mL screw top vial  using a vial insert.3CN in water with 0.1% formic acid over 35 min at a flow rate of 0.7 mL/min. To quantify oridonin content, standard oridonin was run at seven concentrations from 1.5 ng/µL to 100 ng/µL. Oridonin content was quantified by comparison with a standard curve constructed from integrated peak areas at both UV 254 nm and MS+347. Oridonin content was expressed as percent dry weight (% dry wt.) of the plant.Ten µL of extracted and filtered samples were run on an Agilent 1200 Series HPLC/6130 Series MS , with a Phenomenex Luna C18 column  using a gradient elution of 15% to 100% CHopb) of offspring oridonin yield against parental yield was estimated and tested for significance using a t-test. The one parent-offspring heritability formula was used to calculate the narrow-sense heritability (2h) The heritability of oridonin yield was assessed by plotting the average offspring oridonin amount against parent oridonin amount. The regression coefficient  using a 1/4″ ceramic sphere  in a ThermoSavant Bio 101 FastPrep FP120 . Total genomic DNA was extracted using a Qiagen DNeasy Plant Mini Kit , following the instructions of the manufacturer. Two individuals for each population were sequenced for three phylogenetic markers: rps16, trnL-trnF, and ITS. One individual each of I. japonicus and I. lophanthoides was also sequenced. PCR mixes included 2 µL of genomic DNA, 1 µL of forward and reverse primer, and 15 µL of water in a Bioneer Premix tube . The rps16 region was amplified with the primers rpsF and rpsR2 trnL-trnF region was amplified with the primers TabC and TabF To prepare samples for DNA extraction, a small amount of leaf tissue, typically 1 cmColeus xanthanthus C. Y. Wu & Y. C. Huang was used as the outgroup, using Genbank sequences from another study Sequences were assembled in Geneious Pro 5.4.3 . In addition to 18 samples newly sequenced for this paper, 40 samples were included from a previous study Incongruence between the different gene regions was tested using the incongruence length difference (ILD) test Maximum parsimony (MP) analyses were done in PAUP using parsimony ratchet commands created in PAUPRat The best model of evolution for maximum likelihood (ML) and Bayesian analyses was determined using jModeltest 0.1.1 Isodon examined for diterpenoid diversity Isodon that were included in the phylogeny. Isodon, whether they produce oridonin, and associated references. The amount of evolutionary lability in oridonin production was estimated using the retention index (RI), and by comparing the parsimony tree length of the oridonin character  with a null distribution created by randomly reshuffling the oridonin presence/absence character among the samples included in the study. The random distribution was created in Mesquite Version 2.74 by reshuffling the character 1000 times. The tree length comparison was conducted when resolving polytomies as “soft,” since this is the more conservative option for detecting evolutionary gains and losses of a character.Oridonin is known to occur in at least 12 out of 74 species (16%) of Eleven microsatellites were amplified in both parent and offspring samples in multiplex mixtures using fluorescently labeled primers. The resulting amplicons were mixed with a GeneScan 500 LIZ Size Standard (Applied Biosystems) and separated on an Applied Biosystems 3730xl sequencer (Applied Biosystems). Alleles were scored using GeneMapper v3.0 (Applied Biosystems). Additional details about the microsatellite regions and the methods used to amplify them are provided in ISF) for each population. Due to a significant increase in ISF in several populations, analyses of population genetic variation involve only parents. Each microsatellite locus was tested for deviation from Hardy-Weinberg equilibrium (HWE) using chi-square tests. Due to the large number of tests (n = 99), the nominal level of statistical significance (α = 0.05) was adjusted by Dunn-Šidák correction (1−(1−α)n1/) to 0.00052. The number of alleles (AN), observed (OH) and expected heterozygosity (EH), and fixation index (F = 1−(OH/EH)) were averaged across loci for each population. Using the combined microsatellite data from the maternal parent and offspring, the Multi-locus Mating System Program  and the biparental inbreeding rate (m−TsT). The biparental inbreeding rate is estimated here as the difference between the multi-locus population out-crossing rate (mT) and the single locus out-crossing rate (sT).Statistical tests of population genetic variation were conducted within GENALEX v6.41 am MLTR;  was emplThe spatial pattern of microsatellite variation was examined in order to identify distinct population clusters and the relationship between genetic distance and geographic distance. A principal coordinate analysis (PCoA) was first employed to summarize microsatellite genetic variation among all individuals. The eigenvectors of the PCoA were calculated from a covariance matrix with data standardization using the program GENALEX. The clustering of individuals from each population was examined based on the first two principal coordinates. A Bayesian clustering algorithm implemented in the program TESS v2.3.1 Microsatellite genetic data were also examined for genetic isolation by distance (IBD) and compared to chemical distance. A matrix of genetic distance A total of 144 unique samples were tested for oridonin concentration, including 75 from parent populations, 64 from greenhouse grown offspring, and five of purchased herbal samples. Representative LC/MS chromatograms are provided in pob) of 0.381 (standard error±0.083). This coefficient was highly statistically significant based on a t-test (p = 0.00007). Narrow-sense heritability (2h) was estimated as 0.761. When a similar regression was performed using only populations of I. rubescens , the correlation between parent and offspring oridonin content was no longer statistically significant, yielding a regression coefficient of 0.157 .The dry weight of oridonin for each offspring was averaged among siblings and regressed on the value of the maternal parent  yieldingrps16, trnL-trnF) (P = 0.001), whereas the two chloroplast markers were not significantly incongruent from one another (P = 0.698). Therefore, chloroplast and ITS data were examined separately in all subsequent phylogenetic analyses.The aligned chloroplast data included a total of 1579 nucleotide characters, of which 1478 were constant, 51 were parsimony informative, and 50 were parsimony uninformative. The ITS dataset included 572 nucleotide characters, of which 404 were constant, 80 were parsimony informative, and 88 were parsimony uninformative. The ILD test indicated that the ITS partition was significantly different from the chloroplast partition . The ITS parsimony analysis yielded 960 unique equally parsimonious trees .In the context of the chloroplast MP strict consensus phylogeny, oridonin production has five evolutionary steps , which is within the distribution of randomly reshuffled characters . Oridonin production has three evolutionary steps  in the context of the ITS MP strict consensus phylogeny, however is lower than the values for randomly reshuffled characters . The pattern of oridonin presence/absence as mapped on the chloroplast and ITS strict consensus phylogenies is shown in rubescens (XX) population had significant tests. Based on the low mean number of alleles across loci and the high fixation index in this population (enanderianus (QD) and rubescens (JY) having the highest polymorphism, and rubescens (XX) having the least. Since rubescens (JY) is a cultivated population from wild collected parent material, the observation of higher polymorphism might be expected. All populations show a deficit of average observed heterozygosity relative to average expected heterozygosity. The populations rubescens (XX) and rubescens (YC) have the largest fixation indices, but F does not exceed 0.2 in any population. Only rubescens (QY) has a large maternal fixation index (matF), but the large standard error makes this estimate unreliable. Similarly, the estimated biparental inbreeding rates (m−TsT) have large standard errors, and only two populations, rubescens (QY) and rubescens (JY), have estimated rate greater than zero.Given the lack of phylogenetic resolution between populations collected for this study, the microsatellite data for all populations, regardless of species designation, were analyzed together. The microsatellite loci were first tested for deviations from Hardy-Weinberg equilibrium using chi-square tests . After cpulation , it is lrubescens (YC) and enanderianus (QD) can be distinguished along the first coordinate axis, while enanderianus (TR) is distinguished along the second coordinate axis. The DIC values in the Bayesian clustering analysis approach an asymptote at six clusters (enanderianus (QD), enanderianus (TR), henryi (XN), japonicus (XX), rubescens (YC), and a cluster where rubescens (JY), rubescens (QY), rubescens (HB), and rubescens (XX) are combined.The first two coordinates from the principal coordinate analysis account for 25% and 24% of the genetic variation, respectively. A plot of the first two components  suggestsclusters , with onxyr = 0.327, p = 0.001) and the correlation is stronger for log-transformed Euclidean distance . Similarly, when all populations in this study were included, genetic distance was significantly correlated with chemical distance in both parents  and offspring . However, when only populations of I. rubescens were compared  there was no statistical correlation between chemical and genetic distance in either parents  or offspring .A pattern of isolation by distance is evident in the Mantel test of microsatellite variation and geographic distance. There is a significant correlation with Euclidean distance  collected growing sympatrically with I. rubescens had the highest oridonin yield of all individuals. The southern population of I. rubescens from Hubei province (YC) had intermediate values of oridonin and was genetically distinct from I. rubescens in Henan province. Of individuals that produced oridonin, fewer than half were above the 0.25% oridonin dry weight limit specified by the Chinese Pharmacopoeia for samples of donglingcaodonglingcao were above the Chinese Pharmacopoeia limit, despite evidence of adulteration .Average yields per population show that oridonin is at higher concentrations in the area of traditional production of province . Four of2h = 0.761). This pattern is corroborated by the test of correlation between chemical and genetic distance. However, the contribution of additive genetic variance to the overall genetic variation is not clear, because our estimate of heritability assumes that there is no genotype-environment interaction and we are not able to test this assumption directly. The heritability of oridonin production is less clear when examining only populations of I. rubescens, either in the context of comparing average offspring values with parents to calculate heritability, or by examining correlations between genetic and chemical distance. The pattern of results in these two cases  is suggestive that when seeking populations that produce the most oridonin, the selection of the species with highest yield should be prioritized before selecting populations within species. In the case of Isodon, it would be important to first select a wide taxonomic breadth to test for oridonin content, as opposed to focusing on variation of oridonin production within only one species . However, the lack of heritability or correlation with genetic distance in oridonin content of only I. rubescens samples may be due to the smaller sample size. Additional samples of I. rubescens would be helpful to clearly determine whether heritability within I. rubescens is in fact low. This, in turn, would help to determine whether there is a genetic component to variation in oridonin production between populations of a single species, such as I. rubescens, and thus, whether additional sampling for high oridonin yield within species is necessary.Estimates of oridonin yield show that there is a strong heritable component to the underlying variation in production of the chemical . However, given the low resolution of the phylogeny, this number could be an underestimate. Further work with additional nuclear phylogenetic markers would be helpful for clarifying the patterns of oridonin evolution.The distribution of oridonin production on the chloroplast phylogeny cannot be distinguished from a random mapping of the character. By contrast, the distribution of oridonin production has less homoplasy than expected by chance in the context of the ITS phylogeny. The difference in these results is most likely because hylogeny . A phyloIsodon that have been found to produce the chemical  that have a reduced level of variation. Based on the average number of alleles in this population and heterozygosity patterns across loci, it is unlikely that null alleles are causing this deficit in variation. Instead, it is more likely that the reduction in variation in the rubescens (XX) population is due to biological factors, such as a recent population bottleneck (or founder event), or as a consequence of selfing. We used estimates of the maternal fixation rate and the biparental inbreeding rate Isodon, we are unable to clearly estimate inbreeding rates in these populations.Patterns of microsatellite genetic variability are fairly uniform across the Isodon, with clear separation of the three southern populations from Hubei and Guizhou provinces, rubescens (YC), enanderianus (QD), and enanderianus (TR). Notably, these three populations can be distinguished from one another based on the first two principal coordinates. Bayesian analysis of genetic structure suggests that the Isodon samples collected for this study are composed of six population clusters. In addition to the three southern populations, henryi (XN) and japonicus (XX) are also identified as distinct clusters. The clusters identified in the Bayesian analysis are geographically separated, with the exception that the sympatric sample of I. japonicus is distinct from I. rubescens at the Xinxiang (XX) site. One complicating factor of this analysis is that the populations also exhibit isolation by distance. Previous studies have shown that clustering methods can over-predict the number of unique clusters if there are large sampling gaps with respect to geographical distance Isodon appear to form distinct clusters, though this may simply due to the fact that with the exception of the sympatric species at XX, the different species are separated by large distances.A principal coordinate analysis indicates that there is extensive overlap in genetic variation in most populations of Isodon species examined for diterpenoids. Oridonin has arisen at least three times in Isodon, but further phylogenetic work is needed to clarify patterns of oridonin evolution.Oridonin has been reported in 12 out of 74 Isodon populations are characterized by high levels of gene flow, even between different species. Gene flow, however, is restricted geographically and genetic differentiation is observed among distant populations as evident in both the isolation-by-distance and structure analysis.Microsatellite genetic distance is correlated with quantitative difference in oridonin production when all individual samples of this study are considered.Isodon. The strong heritability is indicative that Isodon would respond well to selection and cultivation of species with high oridonin yield.Oridonin production is strongly heritable when comparing different species of I. rubescens are considered. However, additional sampling of multiple populations of I. rubescens is needed to confirm this.Oridonin yield does not appear to be heritable or correlated with genetic distance when only samples of Isodon populations for oridonin production should prioritize a broad survey of all species known to produce oridonin, rather than focusing on multiple populations of one species.Collectively, these results suggest that initial screening of I. rubescens or I. japonicus from Henan province would be the best source of oridonin.Of the samples in this study, populations of Table S1Sample collection information and Genbank accession numbers. Samples in bold collected by study authors.(XLS)Click here for additional data file.Table S2Summary of literature reports of oridonin as detected in species of Isodon.(DOC)Click here for additional data file.Table S3Chi-square test for deviation from Hardy-Weinberg equilibrium. Significant tests indicated (Dunn-Šidák corrected α = 0.00052) in bold with asterisk.(XLS)Click here for additional data file.Table S4Representative chromatograms of Isodon rubescens and Isodon japonicus.(DOC)Click here for additional data file.


Alosa alosa and A. fallax are anadromous species that have persisted through historical large-scale environmental perturbations and now additionally face an array of anthropogenic challenges. A comprehensive phylogeographic investigation of these species is needed to provide insights on both the historical processes that have shaped their extant genetic structure and diversity, and the prospects for their future management and conservation.Comparative broad-scale phylogeographic studies of aquatic organisms provide insights on biotic responses to the paleohydrological dynamics associated with climatic oscillations. These insights can be used to formulate a framework for understanding the evolutionary history of a species or closely related taxa as well as aid in predictive modeling of further responses to climate change. Anadromous fishes constitute interesting models for understanding the relative importance of environmental versus biological factors in shaping intraspecific genetic substructure on the interface between marine and freshwater realms. European shads, A. alosa and A. fallax are genetically divergent, congruent with previous studies. Three similarly divergent mtDNA clades were recognized within both A. fallax and A. alosa, most likely originating during common periods of isolation during the Pleistocene among the studied oceanographic regions. Periods of basin isolation apparently extended to the Black Sea as additional Alosa clades occur there. The present day geographic distribution of genetic diversity within European Alosa sp. suggests the existence of a strong but permeable barrier between the Atlantic and Mediterranean seas, as shown for a number of other aquatic species. Overall mtDNA diversity is considerably lower for A. alosa compared to A. fallax, suggesting that the former species is more sensitive to climatic as well as anthropogenic changes. For A. fallax, migration from the Mediterranean to the Atlantic was detected but not in the opposite direction, with colonization of the North Atlantic probably occurring after last glacial maximum.Despite introgressive hybridization, A. alosa and A. fallax have clearly responded differently to the hydrological dynamics of the Pleistocene, as reflected in their distinct demographic histories. As the species additionally occupy different ecological niches it should not be surprising that they differ in resilience to natural or human-mediated climatic changes. For A. fallax, it is further clear that its demographic response to large-scale hydrological events is not synchronized between the Atlantic and Mediterranean basins. These regional and species-specific differences should be incorporated into future predictive modeling of biological response to climate change as well as current management concepts.The similar haplotype network topologies between the two species support a common intraspecific history of isolation. Despite these similarities,  There is now an extensive literature on the phylogeographic structure of many European freshwater fish species, and these studies often relate to shifting hydrological river networks over several Pleistocene glacial cycles or dating even back into the Pliocene or Miocene epochs -7. KnowlThe combined effects of tectonic and climatic processes promotes abrupt sea level changes that alter ocean or sea interconnections, large-scale current patterns, salinity and temperature gradients, as well as water column stratification -13. In EAlosa comprise 16 species that are primarily anadromous representatives of the family Clupeidae, one of the world’s most commercially important fish families [A. alosa and A. fallax, exhibit largely overlapping ranges in the Northeastern Atlantic, which use to extend from Iceland in the north to Morocco in the south ; one endemic species (A. macedonica) described for Lake Volvi, a Greek freshwater lake; and five species  described for the Caspian Sea. Thus, these species occur across oceanic biogeographic regions where abiotic factors such as currents, salinity and temperature continuously change allowing episodes of long-distance dispersal, gene flow and alternatively isolation ..Alosa coA. fallax populations, some studies have a very regional focus [Despite their biological interest as a model to study the main factors involved in diversification in the region, their phylogenetic relationships have not yet been completely clarified ,26 and lal focus ,32, whilal focus ,30), higHybridization among European shad species is well documented  re-evaluating the genetic relationships among Alosa species; ii) testing if shared mtDNA haplotypes between A. alosa and A. fallax result from introgression or incomplete lineage sorting; iii) understanding how genetic variability is distributed among the Eastern Atlantic and Mediterranean basins, as well as inferring the major phylogeographic barriers to gene flow that may have been responsible for promoting differentiation; and, iv) understanding the impact of major Pleistocene climatic changes on the genetic composition and historical demography of A. alosa and A. fallax populations from different geographic regions. This study focuses on historical patterns and processes, but the inferences can contribute to the improvement of conservation measures in the future, as the species confront both natural and man-made ecosystem alterations.In order to obtain a better understanding of the roles of historical events and demographic processes in shaping genetic diversity of European cyt b (N = 335) and 975 bp for ND1 (N = 273), with no indels or stop codons, after removing the terminal stop codon for ND1. In 11 A. fallax individuals, more than one haplotype was observed within a single individual, corresponding to common haplotypes in our database. As the occurrence of heteroplasmy was reported previously in Alosa[The final alignment yielded 448 bp for yt b N = 5 and 975A. alosa and 42 in A. fallax individuals, from which 4 are shared among the two species. Maximum parsimony (MP) analysis revealed 60 equally parsimonious trees of 177 steps . The best-fit model was TrN + I + G with an estimate of invariable sites (I = 0.6479) and a discrete approximation of the gamma distribution . As similar topologies were obtained with all methods only the MP and Bayesian trees are shown  taxa form a large diverse clade with low support  of BSC was generally lower, except for the Bayesian analysis, where posterior probability is generally equal to 1.0  ranges from 0.85 to 1.25 Myrs (assuming 2% divergence rate), being lower between A. fallax and the BSC (0.45-0.75)  is always lower than interspecific divergence .There were a total of 127 variable positions, 92 of which were parsimony informative defining a total of 65 haplotypes: 11 found in A. alosa and A. fallax obtained with IMa2 did not show a well-defined peak, suggesting that there is not enough information in the data to confidently infer this parameter  in populations from the United Kingdom (Usk and Tywi) and Portugal (Lima and Tejo)  centers on the most common and widespread haplotype (Aa5) found in nearly all populations   but not in A. alosa . A SAMOVA considering k = 3 showed that this clustering indeed maximized ΦCT (0.51524), supporting our grouping for the AMOVA structure analysis.Within population variation explained the majority of the genetic variance in both European shad species (ca. 55% in Divergence time between the Atlantic and Mediterranean groups of populations estimated based on an isolation-with-migration model dates to 0.33 Myrs ago (0.25 if we exclude the haplotypes from clade 3). Although a 95% HPD upper bound could not be computed for the coalescent-based estimates of divergence time Table . Shared A. fallax  compared to A. alosa   with non-overlapping confidence intervals  showed higher levels of both nucleotide and haplotype diversity compared to clade 1  . Within A. fallax, a demographic expansion was supported for clade 1 (primarily in the Atlantic basin) and clade 2 (primarily in the Mediterranean basin) starting about 13,000 and 80,000 yrs ago, respectively  than in clade 1 (Atlantic basin).Within u) Table . Howeverly Table . Within go Table . DespiteA. alosa and A. fallax[A. fallax and BSC haplotypes  ca. 25–40 thousand years ago, which is at the height of the last glacial maximum (LGM) . ThuAlosa lineages. First, all species or clade splits are confined to the Pleistocene implying that these cold-tolerant taxa proliferated or radiated during glacier-mediated climatic oscillations and not during pre-Pleistocene (i.e. Pliocene) climatic conditions, which were considerably warmer [Alosa in the region under study. Second, all species or clade splits occurred over time periods older than the LGM and thus this most recent glacial cycle had little to nothing to do with the origin of major lineages. The divergence between A. fallax and the Black Sea lineages started roughly 0.45 to 0.75 Myrs ago. We speculate that a radiation occurred during this period when connections between the Mediterranean and Black seas (as well as Caspian) took place [Alosa into the Black and Caspian seas. These connections were subsequently interrupted several times , as suggested for other species in the Ponto-Caspio region [Using this framework, some general inferences can be drawn concerning the diversification of European y warmer , or perhok place , allowinmes (see  and refsmes (see ), promotmes , whereby the Lake Vistonis lineage has apparently gone extinct due perhaps to human-caused salt water intrusion [Alosa from Lake Volvi is considered a distinct species carrying out its entire life cycle in freshwater, whereas the Aegean Sea region, now with higher salinity harbors only A. fallax of Mediterranean origin.Salinity gradients or breaks have clearly played a role in the diversification of the genus ulations ,27,41,42gean Sea . Alosa cntrusion . Alosa fWhile ancestral polymorphism could in theory explain shared haplotypes between the two species, the evidence for hybridization and introgression is overwhelming, in agreement with previous published studies ,27,28. TA. alosa and A. fallax. Strong population substructure or complex demographic scenarios may provide violations of model assumptions. However, several lines of argument strongly support that introgression is occurring between the two species. First, it has been shown that migration estimates based on isolation-with-migration models are robust to population structure and departures from simple demographic scenarios [Coalescent-based estimates under a model of isolation-with-migration support gene flow between cenarios . Second,cenarios . And fincenarios .A. fallax populations of Usk and Tywi , although A. alosa basically disappeared from this region [A. alosa and A. fallax varies among river basins from 0 to 63%, reaching a maximum in Tywi . The causes of different levels of introgression among river basins are unknown but might be related to either differing degrees of anthropogenic disturbance, mainly the construction of migration obstacles such dams and weirs [Interestingly, the levels of introgression are particularly high in s region ,32. Thess region . The intnd weirs ,21, and/nd weirs . Severalnd weirs ,47 underA. fallax revealed by AMOVA and SAMOVA clearly relates to the major basins (Atlantic and Mediterranean), but also to smaller-scale patterns evidenced by regionally specific haplotypes. Therefore, it appears that differentiation was mainly shaped by historical processes of fragmentation between the Mediterranean and the Atlantic as described for many other species  act in restricting gene flow between the Mediterranean and Atlantic, as described for many other species [A. fallax, considering the existence of shared haplotypes (Af3 and Af10) between the two basins and the coalescent-based migration estimates, with significant gene flow from the Mediterranean into the Atlantic  is puzzling. Huge population declines and possible local extinction has been suggested for A. fallax in Morocco [The geographic origin of clade 3 in  Morocco , which cA. fallax . Future studies should thus make use of existing material (ancient DNA) and of A. alosa haplotypes “available” in populations of A. fallax through introgression, prior the local extinction of A. alosa, to identify the geographic origin of the clades identified here.Genetic variation of ax Table , clades ax Table . This paA. alosa is much lower than for A. fallax. The rapidly declining range of A. alosa, including its disappearance from the Mediterranean basin and Northern Africa can explain the differences found between the two species [Interestingly, although the two species share parallel intraspecific clade structures supporting parallel refugia, mtDNA haplotype diversity of  species ,21, and A. alosa and A. fallax lineages in the Atlantic basin, with roughly similar age estimates, presumably reflecting expansion after the LGM  seems to have started earlier in the Mediterranean than in the Atlantic , suggests that a southern refuge existed along the Iberian Peninsula (or further south), similar to that reported for many other species  are considered. Thus, Alosa’s comparative cold-intolerance may limit the potential range of glacial refugia in the Atlantic, which, together with the fact that contemporary climatic conditions appear to be causing problems for the species at the southern limit of their distribution, underscores, especially for A. alosa, the sensitivity of these species to climatic change. In contrast to the Atlantic, the Mediterranean seems to provide more long-term stable habitat for lineages of Alosa during colder periods, but the opposite may also be true during interglacials, as suggested by the recent disappearance of A. alosa and the distribution of A. fallax throughout the region, with lower abundance in areas with higher seawater temperature.The distribution of the genetic diversity of European s microps; freshwahispanica; anadrommo trutta; but seemo trutta for a re[S. salar and browS. trutta .The high population differentiation of A. alosa and A. fallax, suggesting a common intraspecific evolutionary history, their different demographic histories, present day distribution ranges, and spawning niche differentiation reflect different ecological needs and responses to natural or human-mediated climatic changes. Shads are clearly relatively vulnerable taxa due to their dependence on major river systems, which are some of the most heavily impacted habitats in Europe. Their responses to the remarkable flow and sea level changes experienced naturally in the past may become even more dramatic due to the additive effects of current human activities. The differences observed between these two species in terms of the genetic diversity must be considered by management actions to insure the maintenance of their declining populations.Despite the similar haplotype network topologies between A. fallax and nine populations of A. alosa across their entire distribution range as well as individuals from the Black Sea/Lake Volvi region presumably representing A. immaculata, A. caspia and A. macedonica; and from the western Atlantic coast representing A. sapidissima, used as outgroup (Table N = 384) were captured by angling or nets between 1991 and 2003. In Figure A. fallax, five subspecies were analyzed  , as previously described [We sampled 29 populations of up Table . Individo et al. ; i.e. < escribed . Individcyt b and the complete NADH-1 (ND1: 975 bp) by polymerase chain reaction (PCR) for 4–20 fish per population  in Alexandrino et al. [ND1 (Alond1f1 and Alond1r1) in Faria et al. [cyt b are described in Alexandrino et al. (2006). ND1 was amplified as follows: each 25 μl reaction contained 14.7 μl H2O, 2.5 μl of Ecotaq specific reaction buffer (Ecogen), 0.8 μl of each primer (10 mM), 3 μl and of 50 mM MgCl2, 1 μl of 10 mM dNTP’s, 0.2 μl Ecotaq Taq DNA polymerase (Ecogen), and approximately 100 ng of DNA template. The cycle parameters were as follows: initial denaturation at 94°C for 2 min; denaturation at 94°C (30 s), annealing at 62°C for cyt b and 63°C for ND1 (30 s), and extension at 72°C (30 s) repeated for 35 cycles; and a final extension for 3 min at 72°C. PCR products were purified with ExoSap-IT (USB) and sequenced following the ABI PRISM BigDye Terminator Cycle Sequencing protocols, in one direction using either Alocytbf1 or Alocytbr1 for cyt b, and using two internal primers . The sequences of the different haplotypes produced during this study are available in GenBank (accession numbers: JX080076-JX080177). Alignments were done separately for each gene using BioEdit 5.0.9 [Both genes were amplified using o et al.  and for a et al. . Amplifiit 5.0.9 .Dxy) and net nucleotide divergence (Da) between the haplogroups that correspond to the main taxonomic units here analyzed  as well as between intraspecific lineages  based on uncorrected p-distances. As there is no reliable calibration for mtDNA divergence rate in Alosinae, estimates are presented for a range of conventional rates (1-4%/Myrs), but discussions of phylogeographic scenarios are centered around an assumption of a conventional divergence rate of 2%/Myrs for the entire mtDNA [Interspecific relationships were primarily evaluated through the construction of bi-furcating trees. Maximum Parsimony (MP), Maximum Likelihood (ML); Bayesian (B) and Neighbor-Joining (NJ) reconstruction methods were used for both genes concatenated. We used MODELTEST 3.06 PPC  under thre mtDNA .A. fallax and A. alosa resulted from introgression or incomplete lineage sorting, we employed the isolation‐with‐migration model implemented in IMa2 software [t). The HKY model was used [A. fallax geographic groups of populations (Atlantic and Mediterranean), assuming a generation time of four years. This was done both including and excluding haplotypes from clade 3, to understand if hypothetical gene flow with a third population would substantially change our estimates.To test if shared haplotypes between software . We conswas used . Prior bwas used . A nucleA. alosa and A. fallax, including the ones shared among species, using the Median-Joining (MJ) algorithm [A. fallax sequences, three different nucleotides were observed in two of the entire set of variable positions, presenting a problem for RM algorithm, which only takes binary data. Because only two amendments were needed to transform the entire dataset into a binary format, we manually replaced the entry of these haplotypes in the input file, following the authors’ suggestions [h), nucleotide diversity (π) and mutation parameter (θ), were calculated using the software DNASP . A Redugestions . The corion 4.0, ). These A. alosa and A. fallax we performed a two-level hierarchical AMOVA [A. alosa, a two-level AMOVA was performed considering all the sampling sites, but also considering solely the northern Atlantic sampling sites , as well as the southern Atlantic sampling sites , separately. For A. fallax, a two-level AMOVA was performed considering all the sampling sites, but also solely for the sampling sites located in the Atlantic (1 to 24), as well as the ones located in the Mediterranean , separately.To evaluate the genetic variability within and among populations of al AMOVA  using ARal AMOVA ). For A.A. fallax exists among European Atlantic, Mediterranean and Moroccan coasts, populations were grouped in three main regions , and a three-level hierarchical AMOVA was performed. The SAMOVA software  set to three and a default number of simulated annealing procedures (100) was further used to test if the grouping we chose for the AMOVA analysis was the one that maximized the proportion of total genetic variance due to differences among groups, when k = 3. Population structure analyses were performed after excluding putative introgressed individuals.Additionally, to test if a major geographic barrier to gene flow for ion 1.1, ) with thD[FS[R2 statistics [0 = 2N0μ (before population expansion), θ1 = 2N1μ (after expansion) and the age of expansion , assuming a one-time sudden exponential growth model.To elucidate events of population expansion and decline, we used several neutrality tests, namely, Tajima's D, Fu’s FSD[FS and R2 satistics , which hA. fallax and A. alosa, except for those composed by only two haplotypes . However, as conclusions that can be drawn based on these powerful model-based coalescent approaches applied to a single locus are very limited [In addition, Bayesian skyride analyses  implemen limited , the res limited , as it s limited . The app limited  followin limited , and usi limited . Three iThe authors declare no competing interests of any kind.All authors participated in the experimental design and development of this research study. PA was the scientific coordinator of the research projects that supported this study. RF carried out the molecular and statistical analysis within the framework of his PhD studies. RF wrote the first draft of manuscript, which was substantially improved by SW. All authors read, revised and approved the final version.RF is a post-doc fellow with a diverse interest in evolutionary biology and is currently involved in the study of chromosomal speciation. SW is an associate professor specialized in the ecology, evolution and conservation management of freshwater fishes, especially salmonids. PA is an associate professor and the head of the Evolutionary Ecology and Genetics of Aquatic Organisms research group at CIBIO, with special interest on the study of the evolution and conservation of aquatic organisms and with a broad experience on the study of shads.Figure S1. This file contains figures supposed to be displayed as supplementary material. In these figures are presented the posterior probability distribution curves of the splitting times, effective population sizes and migration estimates using IMa2.Click here for fileFigure S2. This file contains figures that are supposed to be displayed as supplementary material. In these figures are presented the Bayesian skyride plots for the A. alosa and A. fallax clades for which changes in effective population size through time were detected.Click here for file


Semicossyphus, which includes the California sheephead, S. pulcher, and Darwin's sheephead, S. darwini. Using a phylogenetic approach based on mitochondrial and nuclear markers, and a population genetic approach based on mitochondrial control region sequences and 10 microsatellite loci, we compared the phylogenetic relationships of these two species, as well as the population genetic characteristics within S. pulcher. While S. pulcher and S. darwini are found in the temperate eastern Pacific regions of the northern and southern hemispheres, respectively, their genetic divergence was very small (estimated to have occurred between 200 and 600 kya). Within S. pulcher, genetic structuring was generally weak, especially along mainland California, but showed weak differentiation between Sea of Cortez and California, and between mainland California and Channel Islands. We highlight the congruence of weak genetic differentiation both within and between species and discuss possible causes for maintenance of high gene flow. In particular, we argue that deep and cooler water refugia are used as stepping stones to connect distant populations, resulting in low levels of genetic differentiation.In the past decade, the study of dispersal of marine organisms has shifted from focusing predominantly on the larval stage to a recent interest in adult movement. Antitropical distributions provide a unique system to assess vagility and dispersal. In this study, we have focused on an antitropical wrasse genus,  In marine fishes, population structuring at large scales is generally weak due to high effective population sizes and/or high migration rates. Similarly, speciation in the sea is thought to be counteracted by high gene flow enabled by dispersive larval forms and a rarity of strong physical barriers to dispersal and intermixing . Wrasses include a large number of predominantly coral reef species (approximately 600 species), with a basal tribe, the Hypsigenyines, that includes the temperate genus Semicossyphus, and its close relatives, the genera Bodianus and Clepticus, which are mostly found on coral reefs , Darwin's sheephead (S. darwini), and the California sheephead (S. pulcher). The Asian sheephead is found in Japan, Korea, and China  and for all samples from the other species via PCR using primers VF2T1 and VR1dT1, 16SAR and 16SBR, and GLUDG-L and CB3H, respectively , cytochrome oxidase I (CO1), and 16SrRNA (16S) were amplified for a subset of All amplifications were performed in 13 μl reactions containing 0.5 μl of DNA, 0.625 μl of each primer (forward–reverse) and 11.25 ml of Thermo scientific 1.1 × PCR master mix (2.5 mmol/L MgCl2). After an initial denaturation of 1 to 3 min, 30–35 cycles at 94°C for 45 s, followed by 45 s at an annealing temperature of 52–56°C and 60 s at 72°C were conducted, followed by a final extension of 3 min at 72°C. After purification following the manufacturer's protocol , sequencing was performed with the primers used in the PCR amplification on an ABI 3100 automated sequencer  at University of California Berkeley. The putative nature of each sequence was confirmed by BLASTN search. In the case of the nuclear markers, heterozygous individuals were scored using IUPAC ambiguity codes.Sequences were trimmed and aligned using the MAFFT  was generated in R using HaploNet in the APE 3.0-9 package  were considered.To explore and decompose the genetic variability of microsatellite loci into gene pools without providing prior information on the geographic origin of the samples, a Bayesian clustering approach implemented in STRUCTURE 2.2 was used  and 4.0% (16S). The sequence divergence between S. pulcher and S. darwini was 0% for the nuclear markers  and less than 1% for the mitochondrial markers . Considering a universal substitution rate of 1.5 to 2.5% per million year in fish cytochrome b sequences  that separated S. darwini from S. pulcher (corresponding to a sequence divergence of 2.1%). Considering a substitution rate of 10% per million years for fish control regions , had only 2 private alleles . We then clustered the California Channel Islands as a separate group, because these islands have also been considered a place where genetic differences may arise . Finally, we considered the southern Mexican islands of Cedros and Guadalupe as another group, as recruitment dynamics at Guadalupe Island have been suggested as potentially being linked with major oceanographic shifts (Cowen ct = −0.87) and was not statistically significant (P = 0.11). Within California samples, no pairwise comparisons of Φst or Fst performed in ARLEQUIN were found to be statistically significant and neither were measures of Jost's D using GENODIVE.Because  Present , we firsts Cowen . In thisSemicossyphus darwini were sampled, these were not included in the population analyses based on microsatellites. AMOVA analyses of S. pulcher microsatellite data did not result in any significant population structure. When we separated California and Sea of Cortez populations into two groups, we found that only 0.02% of the total variance could be attributed to this partition, a result that was also not statistically significant. Clustering the California Channel Islands or the Mexican offshore islands into separate groups also yielded nonsignificant differences, with 0.12% and 0.06% of the variance assignable to these partitions, respectively. Results from STRUCTURE were consistent with the AMOVA  larval stage of most benthic organisms, a life stage that is difficult to fully assess  at much higher rates than expected . The physical connection of continuous populations via deeper waters is therefore a likely conduit to genetic connectivity in this species, resulting in shallow population structure.As noted above,  Present , yet car Present . In factS. pulcher and S. darwini, two species that display antitropical distributions, may be due to several potential factors. Stepping stones as a mode of distant connectivity have been evoked for a long time . During these times, the water temperature in the tropical region was lowered, further increasing the potential for temperate fishes to breach that boundary  suggest that structure may be present. In other systems, such as hake or cod, population structure that was not uncovered in early surveys was shown to be present when more powerful molecular markers, such as SNPs, were used, and discrete stocks and fundamental population patterns were revealed , high productivity, similar fish assemblages, and kelp forests (Stepien Semicossyphus system offers a unique opportunity to understand which factors are most influential in shaping the structure of marine populations.While our study only examines a single system, it provides a rare example where macroevolution mirrors microevolution. The ability of  Stepien . In that