It is hypothesized that one of the mechanisms promoting diversification in cichlid fishes in the African Great Lakes has been the well-documented pattern of philopatry along shoreline habitats leading to high levels of genetic isolation among populations. However lake habitats are not the only centers of cichlid biodiversity - certain African rivers also contain large numbers of narrowly endemic species. Patterns of isolation and divergence in these systems have tended to be overlooked and are not well understood.Teleogramma depressum and Lamprologus tigripictilis, from a 100 km stretch of the lower Congo River using both nDNA microsatellites and mtDNA markers along with coordinate-based morphological techniques. In L. tigripictilis, the strongest genetic break was concordant with measurable phenotypic divergence but no morphological disjunction was detected for T. depressum despite significant differentiation at mtDNA and nDNA microsatellite markers.We examined genetic and morphological divergence among populations of two narrowly endemic cichlid species, The genetic markers revealed patterns of philopatry and estimates of genetic isolation that are among the highest reported for any African cichlid species over a comparable geographic scale. We hypothesize that the high levels of philopatry observed are generated and maintained by the extreme hydrology of the lower Congo River. Population boundaries are largely determined by local environment, the strength of barriers to migration, and the organism's inherent dispersal abilities. The interaction among these and genetic factors determines the potential for evolutionary divergence, and is at the heart of our understanding of ecological adaptation and ongoing speciation processes. When individuals can easily cross barriers between suitable habitat patches, gene flow acts to homogenize most neutral genetic diversity and may swamp the effects of any local phenotypic selection whereas low levels of migration may facilitate ecological divergence, even when selection is modest. Evolutionary divergence through fine-scale geographic isolation is believed to be one of the main mechanisms promoting speciation in the cichlid radiations of the African Great Lakes -3. Many Astatoreochromis alluaudi throughout the Lake Victoria Basin found little population differentiation[Oreochromis niloticus populations separated by more than 100 km. In the haplochromine genus Pseudocrenilabrus, Katongo et al. [Comparatively little is known about the population structure and extent of cichlid philopatry in river systems. In South American cichlids, Ready et al. ,11, haveentiation, whereasentiation detectedo et al.  detectedThe proximate causes and adaptive significance of cichlid philopatry have long been the subject of much speculation. In the African Great Lakes, proposed advantages that may indirectly promote philopatry include predator avoidance , microalSteatocranus mpozoensis, is known also to occur in the Mpozo River, a large leftbank tributary of the LCR. Thoracochromis demeusii occurs predominantly in the main channel but is also often found in lower reaches of numerous affluent streams, while the remaining LCR endemics are found exclusively in marginal habitats along the river's main channel. Interestingly there are also parallels in habitat type between the Great Lakes and the LCR system. The eddies along many sections of the LCR shoreline encompass patches of rocky, sandy, muddy, and weedy substrates that resemble the mosaics of habitat types described in the African Great Lakes [While considerably less rich than the African Great Lakes, the shoreline habitats of the Lower Congo River (LCR) nonetheless harbor a large number of cichlid species, and to date 21 narrowly endemic species have been reported from this short stretch of river. One of these, at Lakes . Unlike 2 section of equatorial Africa containing the largest continuous rainforest on the continent and a water volume that is five times larger than that of the Mississippi River. Near the twin cities of Kinshasa (Democratic Republic of Congo) and Brazzaville (Republic of Congo) the Congo River spills over a rocky sill that forms the eastern boundary of the LCR and plunges through a narrow gorge descending some 280 m in its 350 km journey to the sea [in situ depth measurements record the channel to be more than 200 m in some areas [vertical water velocities of up to 75 cm/second in some sections [The Congo River drains approximately 3.8 million km the sea . The rivme areas  forming The extreme hydrological conditions with persistent, high in-stream water velocities in the LCR are expected to hinder cross-channel passage and isolate small, epibenthic cichlid populations to shorelines. Furthermore, because cichlids have physoclistous gas bladders, adult fish have limited tolerance for rapid change in water pressure , suggestTeleogramma depressum, is a goby-like, benthic species that is confined to rocky, shoreline habitats and which guard fixed territories (Pers. Obs.). Teleogramma depressum appears to be restricted in its geographical distribution to a region approximately matching the present study reach. Lamprologus tigripictilis is a habitat generalist found over riffles and rocky habitats as well as over less complex substrates where sand or mud predominate. They have even been occasionally collected in flooded grasses, and are considerably less territorial than T. depressum (Pers. Obs.). The known distributional range of L. tigripictilis is considerably larger than that of T. depressum, and extends beyond the study reach from the region of Mbelo some 100 km downstream from Pool Malebo to Boma about 45 km downstream of Site E near Matadi.Here we report the first detailed estimates of interpopulational connectivity and phenotypic divergence among cichlids in a high energy African river. We selected two locally endemic species with distinct ecologies from a 100 km stretch of the LCR Figure . TeleogrL. tigripictilis, PCA . A grid display of deformations implied by PC 1 scores using Procrustes superposition highlights regions of shape variation concentrated on mouth size, paired fin placement, and body depth .The results of the coordinate-based morphometric analyses are graphically represented in Figure A Figure  indicateh Figure . In contm Figure  revealedm Figure  indicateT. depressum, the observed 10 locus mean heterozygosity ranged from 0.36 in the sample from Site C to 0.42 at Sites A and B. All loci were in Hardy-Weinberg equilibrium after using a Bonferroni correction for multiple comparisons, however one locus deviated significantly from Hardy-Weinberg expectations at the p=0.05 level in samples from Sites B and C. Population samples at Sites 2 and 3 were both fixed for single alleles at Locus TmoM25 and at Locus GM-125. L. tigripictilis populations showed a clear trend of decreasing heterozygosity from upstream to downstream, with Hobs ranging from 0.62 at site A to 0.47 at site E., and all loci were in Hardy-Weinberg equilibrium. Within site variation for mtDNA markers is shown in Table Sample sizes and genetic diversity summary statistics for the nuclear loci can be found in Table L. tigripictilis, p = 0.24 for T. depressum).The Bottleneck program detected no evidence of a recent bottleneck in any of the populations in either species  vs. (Sites B & C),  . Individuals from Sites A and B belonged to two distinct clusters, however these clusters were distributed between two collection localities and the probabilities of assigning individuals to one or the other cluster were often low. In an attempt to 'force' these individuals to assign to one cluster or another, a follow up Structure analysis was performed which initialized on the collection localities and which did not allow admixture. However the results from this subsequent analysis were similar to the initial analysis (results not shown). The estimated ϕst of 0.013 between L. tigripictilis samples from Sites A and B is modest but statistically significant (p = 0.035). The most dramatic difference in the data set was observed between Site B and Site D, with a ϕst estimate of 0.216 (p = 0.001). The structure results assigned individuals from Sites D and E to a single genetic cluster. The ϕst estimate between samples from these two sites is 0.132 and it is significantly different from zero (p = 0.004). A similar pattern was reflected in the mtDNA data set, with an average of 31 substitutions between individuals at Sites B and D, and two distinct haplotype clusters separated by 25 substitutions . A high level of gene flow between populations A and B caused a deviation from the relationship expected under an IBD model.Isolation by distance analysis could not be conducted on the T. depressum and L. tigripictilis we detected genetic isolation at small geographic scales. In L. tigripictilis, we also detected measurable phenotypic differentiation.Evolutionary divergence is a continuous and dynamic process in which landscape features may limit gene flow between populations. When local ecological factors select for distinct phenotypes in different and genetically isolated populations, or when genetic drift causes different phenotypic traits to become fixed, this process may lead to the development of reproductive isolation (speciation) between populations. Our data sets are the first documentation of high levels of both genetic isolation and phenotypic divergence owing to fine-scale philopatry in cichlids in a high energy fluviatile setting. In both T. depressum and L. tigripictilis are confined to relatively sheltered shoreline habitats that are in many ways similar to those along the shores of the Great Lakes with a linear array of rocky, sandy, and intermediate substrates. However the hydrodynamics of this powerful river system are strikingly different from those of the Great Lakes. Between the western edge of Pool Malebo and the Atlantic Ocean, the Congo River descends some 280m. This steep drop generates an array of sheltered, isolated habitat patches separated by variously sized, and often extremely powerful, rapids. In the study area, the width of the channel varies more than five-fold, from a maximum of 1.6 km in the Inga Reach to as little as 0.3 km in several intervening narrow chutes. Many of these constricted regions are associated with high-energy rapids that span the entire width of the channel and interrupt stretches of suitable cichlid habitat. Because of these geographic features and the high levels of endemism documented for this river reach, the Lower Congo River represents an important new biohydrologic model system for exploring the isolating mechanisms underlying patterns of species diversification. Consistent with the extreme hydrological forces operating in the LCR, our estimates of genetic isolation are among the highest reported for any African cichlid species on a comparable geographic scale ..T. depreT. depressum populations at Sites A and B. The rapids at Isangala and Fwamalo are both associated with a narrowing of the river channel . In both cases, an area of whitewater extends from bank to bank. Analysis of the modest number of available samples at sites B and C did not permit rejection of the null hypothesis of panmixia along the much larger, wider, and shallower rapids at Inga. This pattern may be a result of either reduced statistical power owing to modest sample sizes, or a genuine reflection of population connectivity along the complex shoreline in this stretch with its numerous potential stepping stone habitats.Two relatively small but intense rapids lie between the most strongly differentiated Lamprologus tigripictilis samples is more complex. Assignment tests support the presence of two distinct genetic clusters at Sites A and B, however the probability of belonging to either cluster is not related to the collection locality, and many individuals have intermediate assignment probabilities. A single individual collected from Site C also belongs to one of these two clusters. Almost all individuals at sites D and E assign to a single genetic cluster. Estimates of ϕst between each pair of adjacent collection localities were all significantly different from zero, although we treat this allele frequency based result cautiously because sample sizes were modest, particularly at sites D and E. The highest ϕst estimate was between samples from Sites B and D . This genetic break was also detected in the mtDNA data set. Our morphometric analysis detected phenotypic divergence across this genetic barrier as well, suggesting that the barrier is strong enough for selection or drift to maintain phenotypic differences between populations.Perhaps not surprisingly (given its broader distribution and habitat range) the pattern among T. depressum, the strongest genetic break is between Sites A and B, and the two sets of intervening narrow rapids likely play a role in isolating these populations. Lamprologus tigripictilis populations from these two sites are also significantly different from each other as measured using ϕst, however the level of genetic differentiation is lower than in T. depressum from the same sites or from L. tigripictilis populations from further downstream.Because the rugged terrain of the LCR did not permit us to collect samples along a formal transect, we cannot unambiguously determine which specific biogeographic barriers maintain population isolation. However the overall patterns of genetic divergence suggest that two species may not respond identically to the same barriers. In L. tigripictilis there is an intriguing general trend of lower genetic diversity at downstream sites relative to upstream sites. However, we detected no evidence of recent genetic bottlenecks in any of the populations, suggesting that the populations have existed in their current state long enough to reach migration-mutation-drift equilibrium. Relative to population B, population D has a large number of private alleles. Indeed, at 8 of the 10 loci, the most common allele in population D was not found in population B suggesting almost no recent gene flow between sites, consistent with long term isolation of these populations. The large number of mtDNA substitutions that distinguish upstream and downstream L. tigripictilis haplotypes further supports the idea that the populations have been isolated long enough to have achieved equilibrium.In The hotspots of species endemism of some African rivers may be less speciose and far less famous than those of the Great Lakes, yet they represent a substantial fraction of cichlid biodiversity and available habitat. It is likely that the observed relationship between landscape forces and population genetic processes observed here is comparable to those operating during the early stages of ecological divergence of many recognized endemic species in the LCR. They may even help illuminate the origins of the spectacular radiations within the Great Lakes. It has been suggested patterns of cichlid evolution and adaptation to high energy river environments may have had an influence on the spectacular patterns of philopatry and evolutionary divergence currently observed in rock-dwelling species in the African Great Lakes ,23. CurrTeleogramma depressum and Lamprologus tigripictilis were collected at multiple sites along a 100 km stretch of the Congo River between the settlements of Kinganga and Matadi  and five localities for L. tigripictilis. Tissue samples were field preserved in 95% ethanol, and voucher specimens were preserved in formalin and transferred to 70% ethanol for morphological analysis. Ethanol preserved tissue samples were stored at -80˚ C until DNA extraction. Collections and specimen processing was conducted in accordance with the American Fisheries Society's Guidelines for The Use of Fishes in Research [Sampling in these habitats is extremely challenging due to a combination of limited river access and extreme hydrological conditions. Nonetheless samples were available from three distinct localities for Research .Teleogramma by selecting landmarks from a stable dorsal projection, while in Lamprologus variation was captured in lateral projection  and ND2 . PCR reactions were performed with Ready-To-Go PCR beads  using a thermal profile of 94°C:30; 49-52°C 1:00 and 72°C 1:30 for 30 cycles. PCR products were cleaned with AMPure (Agencourt) prior to a sequencing reaction with Big Dye Terminator Reaction Mix (Applied Biosystems). Sequencing reactions were purified with CleanSEQ (Agencourt), suspended in 0.5 mM EDTA, and run on an ABI 3730xl automated sequencer (Applied Biosystems). DNA sequences used in this study have GenBank accession numbers from HM101344 to HM101461.Mitochondrial DNA haplotypes were determined by sequencing, using published primers, a combined 2044 base pairs comprised of partial cytochrome A panel of microsatellite loci developed for other cichlid species was screened using DNA from both species, and 10 polymorphic loci were identified for each species. Primer sequences and the GenBank source loci are shown in additional file PCR products were diluted ~1:20 in HPLC grade water and mixed with Applied Biosystems HiDi formamide and GeneScan-600 size standard according to the manufacturer's instructions. Samples were sized on a 3130 xl Genetic Analyzer and fragments were scored using GeneMarker software .a priori assumptions of population structure or group membership, and inferences derived using this approach do not rely on precise estimates of allele frequencies within habitat patches [As noted previously collecting fish in many sections of the LCR is logistically challenging and limits the number of individuals available for genetic analysis. Fortunately, the individually based assignment methods implemented in Structure 2.2 do not require  patches . Structu patches  and GenA patches . We test patches . Isolati patches .Interpopulation mtDNA distances were calculated using MEGA 4.0.2 . HaplotyAll three authors contributed to the writing of this paper and overall study design. Specimen vouchers and tissue samples were collected by MLJS and RCS during several expeditions to the study region in Bas Congo, Democratic Republic of Congo. JAM initiated the study, and conducted the nDNA microsatellite analyses. MLJS coordinated the project, undertook the morphological analyses and prepared the figures. RCS conducted the mtDNA analyses and prepared the haplotype diagrams. All three authors have read and approved the final version of this manuscript.S-1 Locations of morphological landmarksClick here for fileS-2 Microsatellite loci and primers used in these analyses.Click here for file


While the impact of climate fluctuations on the demographic histories of species caused by changes in habitat availability is well studied, populations of species from systems without geographic isolation have received comparatively little attention. Using CO1 mitochondrial sequences, we analysed phylogeographic patterns and demographic histories of populations of five species (four gastropod and one amphipod species) co-occurring in the southwestern shore of Lake Baikal, an area where environmental oscillations have not resulted in geographical isolation of habitats.B. turriformis, B. carinata and B. carinatocostata) exhibit rather stable population sizes through their evolutionary history, and their phylogeographic pattern indicates moderate habitat fragmentation. Conversely, species without strong habitat preference (gastropod M. herderiana and amphipod G. fasciatus) exhibit haplotype networks with a very abundant and widespread central haplotype and a big number of singleton haplotypes, while their reconstructed demographic histories show a population expansion starting about 25-50 thousand years ago, a period marked by climate warming and increase in diatom abundance as inferred from bottom-lake sedimentary cores.Species with stronger habitat preferences (gastropods In agreement with previous studies, we found that species reacted differently to the same environmental changes. Our results highlight the important role of dispersal ability and degree of ecological specialization in defining a species' response to environmental changes. Many studies have demonstrated the strong influence of climate fluctuations on the patterns of genetic diversity of species. Continental glaciations resulted in geographic isolation of terrestrial species by affecting habitat availability ,2. AfterAncient lakes are famous for their high level of biodiversity. Whereas many studies on speciation were devoted to the fauna of ancient lakes, reconstruction of their demographic histories received little attention (but see e.g. ). Lake BBaicalia carinata  is an abundant sand dwelling species with a circum-lacustrine distribution; Baicalia carinatocostata  is often found in sandy habitats together with B. carinata but usually in smaller numbers; Maackia herderiana dominates the rocky surfaces but is also found in lower abundance in sandy and silty substrates in the southwestern shore of the lake [Baicalia turriformis  inhabits rocks along the same shoreline as M. herderiana . The four species also use different egg-laying substrate: B. carinata lays its eggs on the surface of the shell of other conspecifics; B. carinatocostata lays its eggs in sand; M. herderiana uses cavities of stones; and B. turriformis attaches its eggs to smooth surfaces of rocks [Gmelinoides fasciatus . It is found in high abundances in sandy and rocky bottoms in almost all littoral zones of the lake at water depths between 0 and 5 meters. This species is a successful invader that rapidly increases its population size when introduced in new ecosystems [G. fasciatus are suspension feeders and have similar dietary preferences. Observations on the stomach content of the gastropods showed that they mainly consume planktonic diatoms Aulacoseira baicalensis, A. islandica, Cyctotella baicalensis and C. minuta [Recent studies comparing the demographic histories of multiple co-occurring species ,35,36 fothe lake ; and Baiof rocks ,41. Juveosystems ,43 and t. minuta . These dIn this study we investigated how demographic histories of several co-occuring species with different ecological preferences were affected by environmental changes in an ecosystem where these changes did not cause geographical separation of fauna. To this end we collected mitochondrial DNA data (CO1) from populations of five species from the southwestern shore of Lake Baikal. We examined phylogeographic patterns and performed comparative analysis of the demographic histories of these populations in view of the known past environmental changes.M. herderiana individuals carry the same haplotype and there is a number of singleton haplotypes, this suggests population growth. The same pattern is exhibited by G. fasciatus, with a dominating haplotype and a small number of less abundant haplotypes. Conversely, haplotypes found in B. carinata, B. carinatocostata and B. turriformis are very diverse and distributed throughout the network. In B. carinata and B. carinatocostata very different haplotypes were found in several localities, some of which were resolved in separate networks.Our taxon sampling included 222 individuals from the five targeted species collected from 13 localities. The list of sampling localities is shown in Table ST values and their significance levels for each studied species are shown in Additional file ST values between pairs of localities (G. fasciatus) to significant FST values between almost all of these comparisons (B. turriformis).Tables of pairwise FM. herderiana and G. fasciatus exhibit similar shape with most pairwise comparisons having small genetic distances, showing a relatively good fit to the expected mismatch distributions under the model of population growth. Conversely, the mismatch distributions of B. carinata, B. carinatocostata and B. turriformis are rather multimodal and ragged, and contain a higher proportion of comparisons resulting in larger genetic distances.Results of comparative analysis of mismatch distributions ,46 for eB. turriformis were less than 8 haplotypes recovered in this study). Despite small sample sizes, B. turriformis and B. carinatocostata exhibited high nucleotide diversity, while the lowest nucleotide diversity was found in M. herderiana (0.0019).Table Maackia herderiana is the only species for which classic tests rejected the hypothesis of neutrality, with Ramos-Onsins' R2 test against population expansion being significant . Sand dwelling B. carinata and B. carinatocostata show rather stable population sizes as does the rock-dwelling B. turriformis. Maackia herderiana shows a dramatic population expansion and G. fasciatus shows signs of population growth. Results of Bayesian Skyline Plots (BSPs) were not influenced by the change of the substitution model for G. fasciatus .Figure M. herderiana and G. fasciatus exhibit haplotype networks in which a central haplotype is very abundant and widespread, and to which several less common haplotypes are closely related. Baicalia carinatocostata and B. turriformis display very different pattern, with the same haplotype never found in more than one locality and lacking a central and most abundant haplotype. Baicalia carinata shows an intermediate picture, with some relatively abundant haplotypes being found in different localities and rare haplotypes restricted to single sampling localities.In this study we compared phylogeographic patterns and demographic histories of species with similar feeding preferences inhabiting the same geographical area of Lake Baikal. Overall, we found three different phylogeographic patterns in the five species investigated. Both M. herderiana, it should be noted that although inhabiting mostly rocky areas, this species has been found in both sandy and silted areas [M. herderiana resembles the generalist amphipod G. fasciatus, which lives in both sandy and rocky substrates [B. carinata and B. carinatocostata both live in sandy bottoms, but show rather different phylogeographic patterns. It was reported that B. carinata increases its dispersal by laying eggs on the shells of its conspecifics [B. turriformis displays high degree of geographical substructuring, even though significant geographical barriers between rocky habitats in the sampled shore seem absent. This more sedentary species mostly inhabits the surface of steep rocks and cliffs with individuals hanging on each other [B. turriformis due to the lack of suitable habitats available.While phylogeographic patterns often reflect habitat availability and connectivity ,48 our red areas . In thisbstrates ,50 and ibstrates ,43,51. Tpecifics ,41, and ch other  and is kch other . This hiM. herderiana we sampled most of the range of the shore where this species occurs [ST values between localities were non-significant. Baicalia carinata and G. fasciatus occur along whole shoreline of the lake and previous studies [G. fasciatus was found exhibited significant FST values. For B. carinata, significant FST values were found between some localities . Similarly, FST values estimated between localities of B. carinatocostata exhibited only few significant results. Conversely, genetic differentiation was higher in B. turriformis, despite the smaller sample sizes used in this study, and FST analysis revealed significant genetic differentiation between most localities. These results confirm that samples of M. herderiana, B. carinata, B. carinatocostata and G. fasciatus represent populations without strong geographical substructuring, and thus are appropriate for reconstruction of demographic histories. Given the higher genetic differentiation in B. turriformis, the reconstruction of demographic histories for this species should be interpreted with caution. Recent results  suggest that the co-occurring populations of B. carinata, B. turriformis and B. carinatocostata were not influenced by interspecific geneflow during the time period covered by the current study.For the reconstruction of the demographic history of populations it is important to identify if a sampling set represents a single population. For s occurs  and foun studies ,54 involM. herderiana. For G. fasciatus, results of neutrality tests were not significant, but had small p-values (p = 0.08 for R2 and p = 0.09 for Tajima's D test). The structure of the haplotype networks of M. herderiana and G. fasciatus, with a central abundant haplotype and a number of singleton haplotypes, also suggests population growth for these species.Classic neutrality tests did not detect significant departures from neutrality for any of the datasets. However, the most powerful Ramos-Onsins R2 test  detectedB. turriformis, B. carinata and B. carinatocostata were rather stable during their evolutionary histories. There are slight trends towards a decline for B. turriformis and B. carinatocostata as well as slight trend towards population growth for B. carinata. However, these slight trends cannot not be taken as evidence for changes in population size because as they appear, the posterior distributions widen. Conversely, BSPs suggest moderate growth for G. fasciatus, and dramatic expansion for M. herderiana. Figure M. herderiana and G. fasciatus are short, contrary to demographic histories of B. turriformis, B. carinata and B. carinatocostata. Long demographic histories of B. carinata, B. turriformis and B. carinatocostata do not show response to the climatic fluctuations that are known from the paleo-record of the lake, while shorter demographic histories of M. herderiana and G. fasciatus exhibit strong to moderate growth. It is thus plausible that M. herderiana and G. fasciatus are relatively recent colonizers of the southwestern shore of Lake Baikal, while the remaining species analysed represent more ancient inhabitants of this area. Alternatively, G. fasciatus and M. herderiana populations may have recently undergone strong bottlenecks, with the growth detected reflecting the recent recovery from such bottlenecks, while the remaining species could have maintained relatively constant population sizes throughout their histories. To elucidate this, future work could focus on the analysis of nuclear gene diversity, as autosomal and mitochondrial DNA diversity are expected to show different rates of recovery from bottlenecks [Our demographic reconstructions suggest that population sizes in tlenecks .M. herderiana and G. fasciatus by applying available rates of molecular evolution, we found that the start of expansion of populations of these species coincide, and could be estimated to 25-50 Kyr BP  model [G. fasciatus, we could not obtain values of ESS (Effective Sample Size) exceeding the recommended value of 200 using HKY model proposed by jModeltest, so we applied the GTR substitution model [G. fasciatus affects the recovered demographic history we compared BSPs for both substitution models. BEAST analysis was performed assuming selected substitution models but parameters were estimated from data. We ran chains of 150 million steps for M. herderiana, 20 million steps for B. carinata and G. fasciatus, and 10 million steps for B. carinatocostata and B. turriformis to obtain in each run ESS values > 200. For each species we performed at least 2 individual runs and compared the results to check for convergence. Data from two independent runs for each species was combined using Log Combiner v1.5.1 [Bayesian skyline plots were constructed using BEAST v. 1.5.1 ,74. SubsY) model  with a pon model . In order v1.5.1 ,74 in orG. fasciatus we used average from the reported rates (1.3-1.9%/Myr) of arthropods [To convert the time scale of demographic histories from substitutions per site into years, we used earlier suggested divergence rate of 1.83%/Myr (million years) for gastropods . Since tthropods . Once abthropods ,32.M. herderiana, performed the analysis and drafted the manuscript. BN participated in the analysis and helped to draft the manuscript. TP and JP carried out molecular genetic studies on Baicalia spp. and G. fasciatus. DS coordinated the design of the study and has been involved in drafting of the manuscript. All authors read and approved the manuscript.VF carried out molecular genetic studies on Detailed description of each individual used in the study. Description includes isolate identification, taxonomic status, locality and year of capture, collectors and accession numbers.Click here for fileST values between localities for each studied species, with the p-values given in parenthesesTables of pairwise F. Significant values before correction for multiple testing are marked with asterisk. Significant values after correction for multiple testing are shown in bold. Loc is locality number , N is number of samples.Click here for fileG. fasciatus using different substitution modelsBSP reconstructions for . Comparison of demographic reconstructions using GTR and HKY substitution models. Thick solid lines are median estimates, and thick dashed lines are mean estimates, shades show 95% highest posterior density limits.Click here for fileMaps of underwater landscapes of the study area. Types of bottom substrates at different depths of the lake. The maps were redrawn from Karabanov EB, Sideleva VG, Izhboldina LA, Mel'nik NG, Zubin AA, Zubina LV, Smirnov NV, Parfenova VV, Fedorova LA, Gorbunova LA, Kulishenko YuL. (1990) Underwater Landscapes of Baikal. Novosibirsk: Nauka Publ.,184 pp. (In Russian).Click here for file


Social insects dominate ecological communities because of their sophisticated group behaviors. However, the intricate behaviors of social insects may be exploited by social parasites, which manipulate insect societies for their own benefit. Interactions between social parasites and their hosts lead to unusual coevolutionary dynamics that ultimately affect the breeding systems and population structures of both species. This study represents one of the first attempts to understand the population and colony genetic structure of a parasite and its host in a social wasp system.Vespula squamosa and its primary host, V. maculifrons. Our analyses of genetic variability uncovered that both species possessed similar amounts of genetic variation and failed to show genetic structure over the sampling area. Our analysis of mating system of V. maculifrons and V. squamosa revealed high levels of polyandry and no evidence for inbreeding in the two species. Moreover, we found no significant differences between estimates of worker relatedness in this study and a previous investigation conducted over two decades ago, suggesting that the selective pressures operating on queen mate number have remained constant. Finally, the distribution of queen mate number in both species deviated from simple expectations suggesting that mate number may be under stabilizing selection.We used DNA microsatellite markers to investigate gene flow, genetic variation, and mating behavior of the facultative social parasite V. squamosa has not changed substantially from that of a typical, nonparasitic Vespula wasp. For example, population sizes of the host and its parasite appear to be similar, in contrast to other social parasites, which often display lower population sizes than their hosts. In addition, parasitism has not caused the mating behavior of V. squamosa queens to deviate from the high levels of multiple mating that typify Vespula wasps. This stands in contrast to some socially parasitic ants, which revert to mating with few males. Overall, the general similarity of the genetic structure of V. maculifrons and V. squamosa presumably reflects the fact that V. squamosa is still capable of independent colony founding and thus reflects an intermediate stage in the evolution of social parasitism.The general biology of  The complex, interactive communities displayed by highly social insects represent an important and highly successful evolutionary innovation ,2. The sIn hymenopteran social insects , social parasitism may take several forms ,7-10. ThVespula squamosa, commonly known as the southern yellowjacket, is a facultative social parasite  suggesting that genetic variability was not substantially different between the parasite and its host. In addition, the effective number of alleles did not show strong differences between species . Moreover, the variability at particular loci was similar in the two species. Specifically, the estimates of A50 and expected heterozygosity (h) were significantly correlated across loci in the two taxa .The standardized number of alleles  and V. squamosa . Thus, both of these Vespula species are apparently able to mate at random over the range in which sampling occurred.The overall estimates of genetic differentiation between colonies in V. maculifrons queens mated with 5.64 ± 1.27 males . The estimates of effective mate number (ke3) in V. maculifrons and V. squamosa were 4.96 ± 1.40 and 5.58 ± 1.66, respectively .As expected, we found that the genotype distributions of workers within colonies always were consistent with the presence of a single queen mated to multiple males. B = 0.0273 ± 0.0409 in V. maculifrons and B = 0.0433 ± 0.0411 in V. squamosa. These values were not significantly different from each other . However, the estimates of skew were significantly greater than zero in only 13 of the 36 V. maculifrons colonies, whereas the estimates of skew were significant in 10 of the 12 V. squamosa colonies. Thus, the proportions of colonies in which the skew was significant in the two taxa differed . Nevertheless, although paternity skew was statistically significant in many colonies, the overall magnitude of B was low and the actual mate numbers were similar to the effective mate numbers in both species.We then turned our attention to the reproductive skew of males mated to the same queen. The mean magnitude of skew was V. maculifrons and V. squamosa differed significantly from that expected under a Gaussian distribution . The failure of these distributions to fit the data arose from an observed excess of queens that mated with an intermediate number of males  overlapped zero. This indicated that inbreeding does not usually occur in either of these species. Furthermore, we found that relatedness of V. maculifrons males mated to single queens was not significantly different from zero .Nestmate ro Table . Howeverro Table , as judgro Table . But we The interaction between a parasite and its host may lead to an evolutionary arms race between the taxa. Microparasites often have large population sizes and fast generation times, and may be able to win the evolutionary arms race with their hosts . In contV. squamosa displayed significant differences in its mating system, levels of genetic variation, and population genetic structure from its host V. maculifrons. Subsequently, our investigation led us to more closely compare and contrast the mating system of the two species in order to understand what factors might affect mating behavior. Overall, we found strong similarities in the genetic structure and mating system of the host, V. maculifrons, and its parasite, V. squamosa. In addition, our investigation of mating biology uncovered patterns consistent with the effects of selection operating on mate number in both taxa.This study represents one of the first joint analyses of levels of genetic variation and breeding system in a parasitic social wasp and its primary host. The main focus of this study was to understand if the parasite V. squamosa and V. maculifrons. Population genetics theory suggests that the amount of genetic variation maintained within a population is related to the effective population size [One of the more striking results arising from our investigation of levels of genetic variation was that measures of allelic diversity were not appreciably different between ion size . TherefoVespula species. The absence of isolation by distance within both taxa suggests that these Vespula wasps exhibit substantial levels of gene flow within the sampling range of this study. This finding is consistent with the high flight capacity of Vespula wasps, which are naturally capable of dispersing over considerable distances [Vespula queens may readily be moved via accidental human transportation thereby increasing their effective migratory abilities [Furthermore, we failed to find evidence for genetic isolation by distance in either istances ,53. In abilities ,55.V. maculifrons and V. squamosa differs somewhat from that found in a previous study of the congener V. germanica in Australia [V. germanica was found to display genetic isolation by distance on approximately the same scale investigated in this study. We note, however, that V. germanica is a recent invader to Australia [V. germanica in Australia likely differ from those of V. maculifrons and V. squamosa in their native range in the United States. In particular, the observed isolation by distance in V. germanica populations in Australia may reflect non-equilibrium conditions that will ultimately fade as gene flow swamps local genetic differentiation [However, our finding of a lack of geographic structure in ustralia . In thatustralia . Thus, tntiation .V. maculifrons and V. squamosa stand in contrast to expectations derived from other studies of social hymenopteran parasites. For example, ant social parasites often have low population sizes and dispersal capabilities [The overall similarity of levels of genetic variation and low levels of structure in bilities . In accobilities  found thbilities  also fouV. maculifrons and V. squamosa in order to investigate if there were differences between the two species that might be associated with parasitic behaviors. As expected, we found that all colonies of both species were headed by only a single queen, and that all queens of both species were polyandrous [V. maculifrons and V. squamosa  are usually rare, restricted locally, and highly genetically structured. However, the genetic fingerprint of d Wilson  point ouV. squamosa should aim to determine the extent that this species is facultatively versus obligately parasitic. In particular, a study of geographic variation in parasitic behavior and frequency would be informative. Additionally, it has been suggested that social parasites that exploit multiple hosts may form host races. A previous study failed to find evidence for such host race formation in Polistes [V. squamosa, which parasitizes multiple hosts [Future research in the study of the social parasite Polistes . Howeverle hosts , would pEAH, JLK, and MADG conceived the study and participated in its design and coordination. EAH and JLK carried out laboratory analyses. EAH and MADG analyzed the data and drafted the manuscript. All authors read and approved the final manuscript.Utility of 43 microsatellite loci in V. maculifrons and V. squamosaa (Table


With the advent of high throughput DNA typing, dense marker maps have become available to investigate genetic diversity on specific regions of the genome. The aim of this paper was to compare two marker based estimates of the genetic diversity in specific genomic regions lying in between markers: IBD-based genetic diversity and heterozygosity.A computer simulated population was set up with individuals containing a single 1-Morgan chromosome and 1665 SNP markers and from this one, an additional population was produced with a lower marker density i.e. 166 SNP markers. For each marker interval based on adjacent markers, the genetic diversity was estimated either by IBD probabilities or heterozygosity. Estimates were compared to each other and to the true genetic diversity. The latter was calculated for a marker in the middle of each marker interval that was not used to estimate genetic diversity.2) of 0.26, comparable to those of real livestock populations. Genetic diversities estimated by IBD probabilities and by heterozygosity were positively correlated, and correlations with the true genetic diversity were quite similar for the simulated population with a high marker density, both for specific regions (r = 0.19-0.20) and large regions (r = 0.61-0.64) over the genome. For the population with a lower marker density, the correlation with the true genetic diversity turned out to be higher for the IBD-based genetic diversity.The simulated population had an average minor allele frequency of 0.28 and an LD (rGenetic diversities of ungenotyped regions of the genome (i.e. between markers) estimated by IBD-based methods and heterozygosity give similar results for the simulated population with a high marker density. However, for a population with a lower marker density, the IBD-based method gives a better prediction, since variation and recombination between markers are missed with heterozygosity. Conservation of genetic diversity in livestock is of vital importance to cope with changing environments and human demands . IntensiTo evaluate genetic diversity in livestock populations, several methods have been developed . These mr) calculated from pedigrees for the estimation of additive variance. Although theoretically both approaches should give similar results, in practice they are weakly correlated [Basically, there are two approaches to evaluate genetic diversity. In molecular and population genetics, heterozygosity of markers is the most widely used genetic diversity parameter . In quanrrelated ,8. As derrelated  and genorrelated ,11.r on the other hand is that the latter depend on a base population. Markers can be alike in state (AIS) but not IBD if they originate from different ancestors in the base population. With heterozygosity this distinction is not made. For example, in the case of QTL detection, IBD probabilities are used because they better predict whether two chromosome intervals carry the same QTL. The reason is that if an individual carries markers at two loci around an interval that are both AIS, but not IBD (i.e. originate from different ancestors), it is less likely that the interval between the markers is completely AIS and carries the same QTL. However, if both markers are IBD the interval will also be IBD (and AIS), unless a double recombination has occurred in the interval.A crucial difference between heterozygosity on the one hand and IBD probabilities and Both heterozygosity and IBD probabilities can be used to estimate genetic diversity in specific regions of the genome, in which it may deviate from the average diversity calculated over the whole genome. Heterozygosity and IBD probabilities as genetic diversity measures may also deviate from each other. It is unclear how substantial the difference is between the two approaches and whether it varies over the genome. These local differences may be averaged out if the average diversity is calculated over the whole genome. However, both approaches can be used to estimate the genetic diversity for sequences lying in between genetic markers. Because IBD probabilities are used specifically to predict the presence of QTL between markers one may expect that IBD probabilities better predict genetic variation between markers. Whether this is a substantial difference is not clear.The aim of this paper was to compare two different estimates of the genetic diversity of a region lying in between markers over the genome i.e. IBD probabilities between marker haplotypes and heterozygosity. Towards this aim, we generated genetic diversity over a genome by computer simulation of two populations each with a different marker density. IBD-based genetic diversity and heterozygosity were compared for the average diversity of regions in the genome containing several marker intervals, and for the genetic diversity at each marker interval. To evaluate how well these estimates predict the genetic diversity over the genome, both were compared to the true genetic diversity.A population was computer simulated with neutral SNP markers across the genome. Next, for each locus in the genome, the genetic diversity was estimated in three ways: (1) based on IBD probabilities with flanking markers; (2) based on expected heterozygosity with flanking markers; (3) the true expected heterozygosity of the marker itself. For (1) and (2), the marker at the locus itself was assumed to be unknown. In this way the predicted diversities (1) and (2) could be compared with true genetic diversity (3).Simulations were aimed at generating a population with a neutral genetic diversity varying over the genome. We avoided selection as this may cause specific patterns in genetic diversity (e.g. selective sweeps). Variation in diversity in the simulated population was generated by random mating, recombination, mutation and sampling of maternal and paternal chromosomes. The simulated population started with 1000 animals with an equal sex ratio, and this structure was kept constant for 1000 generations. Animals were mated by drawing parents randomly from the previous generation, and mating resulted in 1000 offspring  in each generation. A genome containing a single 1-M chromosome was simulated, starting with 2,000 SNP marker loci with positions on the genome determined at random. This density is roughly equivalent to the current SNP chips available for livestock species (e.g. 50 K SNP chip for the 30-M genome in cattle). In the first generation (base population), marker loci were coded as 1 or 2 and allocated at random, so that allele frequencies (p) averaged 0.5. This was comparable to the simulation used in the study of Habier et al. . During To determine whether marker density would influence the genetic diversity estimation with the different estimates, a second population was obtained with a lower marker density. This population was based on the first population, by changing only the number of SNP markers from 1665 to 166, by systematically deleting 90% of the SNP markers.Genetic diversity was estimated for each marker interval on the genome. A marker interval was defined as the interval between two genotyped markers, with one marker lying in between these two markers which was not taken into account for the genetic diversity estimation (ungenotyped marker) Figure . In the IBD probabilities were calculated between the existing haplotypes in the simulated population for each marker interval, by combining linkage disequilibrium and linkage analysis information, where both pedigree and marker information were used. IBD probabilities were first calculated for the first generation of genotyped animals, using the algorithm of Meuwissen and Goddard . In thisAfter calculating IBD probabilities for the haplotypes in the base generation, the haplotypes of the animals in later generations were added, and the elements in the IBD matrix for those descendant haplotypes were calculated using the algorithm of Fernando and Grossman . In thisThe genetic diversity for all marker intervals on the genome in the simulated population was estimated using haplotype frequencies and IBD probabilities between haplotypes. Haplotype frequencies (frequency of the different haplotype configurations in the population) per marker interval were obtained by:ci is a contribution vector with haplotype frequencies for all haplotypes on marker interval i, Nij is the number of haplotypes of type j on marker interval i, and Ni is the total number of haplotypes in the population on marker interval i.where Genetic diversity per marker interval was determined by calculating the average haplotype relatedness at each locus :(2)ri is the average relatedness for marker interval i, and IBDi is the IBD-matrix for marker interval i. The genetic diversity for marker interval i was calculated as:where This is the predicted probability that the marker in the middle of the interval is not IBD.exp_AVG), and heterozygosity for the interval treating both markers as a single two-marker haplotype (Hexp_HAP2). For the calculation of Hexp_AVG, first expected heterozygosity was calculated for the markers on the left and right of the interval separately  was calculated by taking the average of the expected heterozygosity for both markers left and right of the marker interval.where exp_HAP2 was calculated for the combination of the two markers on the left and right of the interval as a two-marker haplotype . To compare true genetic diversity (Hexp_TRUE) with GD_IBD and heterozygosity (Hexp_AVG and Hexp_HAP2), Pearson's correlations were calculated for each marker interval and for groups of marker intervals . Two correlations were estimated for each comparison: between true genetic diversity of the even markers and their estimated genetic diversity based on the uneven (flanking) markers, and the other way around. This was done because the genotyped marker in one marker interval became the ungenotyped marker in the next marker interval.To evaluate whether one of the approaches better predicts genetic diversity, a true genetic diversity was calculated for the ungenotyped marker lying within each marker interval. This marker was not used to estimate genetic diversity with GD_IBD, H2) between adjacent markers, calculated as the square of the correlation of allele frequencies [2 between adjacent markers is between 0.15 and 0.20 for an average marker distance of ~0.06 cM [In the simulated data, 1665 SNP markers were still segregating in generations 1001, 1002 and 1003. Marker distances ranged from 0.00 cM to 0.50 cM, with an average of 0.06 cM. The number of marker haplotypes used for GD_IBD after clustering varied from 1 to 56, with an average of 20.70 haplotypes. The average minor allele frequency over the 1665 SNP markers was 28%, ranging from 2 to 50%. The average linkage disequilibrium , ranged from 0.04 to 0.53 with an average of 0.36  resulted in systematically lower values with a smaller range compared to GD_IBD  , the level and range of values increased and were more similar to GD_IBD  .Genetic diversity estimated by IBD probabilities (GD_IBD) varied considerably over the genome, with values ranging from 0.00 to 0.75, with an average of 0.52 Figures  and 3b.  Figures  and 3c.  Figures  and 3d. exp_TRUE and GD_IBD was weak (r = 0.21), and comparable to the correlations between Hexp_TRUE and Hexp_AVG (r = 0.19) and Hexp_HAP2 (r = 0.20)   Table . These rk r = 0.2, and comexp_AVG and Hexp_HAP2 were averaged over groups of marker intervals, the correlations between Hexp_TRUE and these estimates increased. They were moderate when estimates were averaged over 40 marker intervals . However, when a population contains more variation, diversity in between markers can be missed by heterozygosity, as heterozygosity is only based on the variation of the markers itself. In that situation, GD_IBD also takes into account the variation and possible recombination in between markers, and is then expected to be a better estimator of the genetic diversity over the genome. Consequently, as shown in this study the method of choice will also depend on the marker density [How well the two methods predict genetic diversity depends on the variation in diversity between adjacent markers. In contrast to GD_IBD, the heterozygosity estimates assume that diversity is similar for adjacent markers and for instance ignore recombination. When regions of the genome form 'haplotype blocks', adjacent markers have (near) identical diversity. In this case, heterozygosity will better predict the genetic diversity. This was seen when we simulated a population with an effective population size of 100 instead of 1000, and 'haplotype blocks' occurred due to the loss of variation. In this population the correlation between the heterozygosity estimate H density , with hiIn conclusion, the IBD-based method and heterozygosity used to estimate genetic diversity of ungenotyped regions of the genome (i.e. between markers) give similar results for a simulated population with a high marker density. However, for a population with a lower marker density, the IBD-based method gives a better prediction, since variation and recombination between markers are missed with heterozygosity. IBD-based methods can provide more insight in the genetic diversity of specific regions of the genome, and subsequently contribute to select more accurately the animals to be conserved, for example, to construct a gene bank.The authors declare that they have no competing interests.KAE developed part of the programs used for analysis, carried out the simulations and analyses, and wrote most of the paper. MPLC developed most of the programs used for the simulations and analysis, and supervised and advised KAE. PB contributed to part of the discussion and supervised and advised KAE. JJW conceived the study, participated in its design and coordination, mentored and advised KAE daily, and contributed parts of the paper. All authors took part in useful discussions, and provided useful advice on the analyses and the first draft of the paper. All authors read and approved the final manuscript.


Elodea canadensis, Egeria densa and Lagarosiphon major are dioecious clonal species which are invasive in New Zealand and other regions. Unlike many other invasive species, the genetic variation in New Zealand is very limited. Clonal reproduction is often considered an evolutionary dead end, even though a certain amount of genetic divergence may arise due to somatic mutations. The successful growth and establishment of invasive clonal species may be explained not by adaptability but by pre-existing ecological traits that prove advantageous in the new environment. We studied the genetic diversity and population structure in the North Island of New Zealand using AFLPs and related the findings to the number of introductions and the evolution that has occurred in the introduced area.Elodea canadensis was introduced in 1868, and its populations showed more genetic structure than those of the more recently introduced of E. densa (1946) and L. major (1950). Elodea canadensis and L. major, however, had similar phylogeographic patterns, in spite of the difference in time since introduction.Low levels of genetic diversity were found in all three species and appeared to be due to highly homogeneous founding gene pools. The presence of a certain level of geographically correlated genetic structure in the absence of sexual reproduction, and in spite of random human dispersal of vegetative propagules, can be reasonably attributed to post-dispersal somatic mutations. Direct evidence of such evolutionary events is, however, still insufficient. Phalaris arundinacea L. in North America, the genetic variation is even comparable to that found in the native European range [Alternanthera philoxeroides (C. Martius) Griseb. [Eichornia crassipes (C. Martius) Solms-Laub. [Invasive plants are of evolutionary interest because they are successful in areas where they have been introduced, sometimes in spite of limited genetic variation compared to their native range. One reason for this success may be multiple introductions, representing genetically distant genotypes. Further, an introduced species can overcome the negative effects of genetic bottlenecks and create genetic variation in the introduced range when genotypes from different geographical areas are brought together, giving rise to genetic combinations resulting in novel genotypes not present in the native range. In the case of an range . Apparen Griseb.  and Eichms-Laub.  in theirClonal growth is often considered an evolutionary dead end, as defined by Stebbins  for selfElodea canadensis Michaux, Lagarosiphon major (Ridley) Moss and Egeria densa Planchon are invasive in NZ and in many other parts of the world. They are all dioecious Hydrocharitaceae and, like many submerged clonal macrophytes, rarely achieve sexual reproduction even in the native range [Elodea canadensis is native to North America and was introduced in NZ in 1868 [Egeria densa is native to South America  and was introduced in NZ in 1946 [Lagarosiphon major is native to South Africa and was first recorded in NZ in 1950 [E. canadensis [L. major [E. densa [E. canadensis from the Waingaro River  but this appears to be an isolated record, as male plants have not been featured in subsequent surveys of the aquatic vegetation throughout NZ. Even though both E. canadensis sexes might potentially co-exist in some waters, seeds have never been observed in any of the three species [E. densa and L. major [E. canadensis [E. canadensis was pointed out by Thomson [E. canadensis was already established at the time. In this study we attempt to determine the number of introductions and evaluate the role of somatic mutations and dispersal as possible sources of the genetic variation patterns observed. We have used AFLPs  is a suitable area for the study of genetic variation patterns of invasive species because, being an island, it is naturally delimited, and the site of many plant invasions that have been thoroughly monitored and documented. In addition, NZ has only recently been colonized by Europeans and therefore provides a unique opportunity to study recent invasion pathways and spread, as well as habitat adaptation. ve range . Elodea  in 1868 . Egeria  in 1946 . Lagaros in 1950 . Only fenadensis  and L. mL. major  and maleE. densa  have bee species . This liL. major , but thinadensis ,13, but  Thomson  who provorphism; ), becausE. canadensis, 0-10 in E. densa and 0-8 in L. major), whereas their frequencies showed different patterns  and most of the different samples had one pairwise difference. In E. canadensis a few samples were genetically identical and the spectrum of pairwise difference frequencies showed a normal distribution with highest frequency of samples differing from each other in 5 DNA fragments. Pairwise genetic differences with a genotype of E. canadensis from Denmark ranged between 14 and 20 DNA fragments, indicating an higher extent of genetic differentiation between the European genotype and all the NZ samples, and a net separation of their pairwise difference frequencies in the spectrum.Similar pairwise genetic difference ranges were found in the three species  and in L. major , whereas E. canadensis populations showed higher genetic structure . The population specific Fst indices . In E. densa the McLaren and Swan populations had a significant pairwise Fst of 0.82 . Population differentiation was not confirmed by the exact test of population differentiation in both species. Genetic differentiation between the NZ E. canadensis populations and the Danish genotype was 0.75 , indicating that the NZ genotypes were more similar to each other than they were to the Danish clone. Nei's unbiased minimum genetic distances ranged between 0 and 0.01 between all pairs of populations in L. major. In E. densa they ranged between 0 and 0.06; however, only one population (McLaren) had genetic distances over 0.02 with all the other populations. By excluding McLaren, the range decreased to 0 - 0.02. Pairwise genetic distances among E. canadensis populations ranged between 0.01 and 0.08. Nei'unbiased minimum genetic distance was 0.17 between the NZ E. canadensis clones and the Danish genotype. The genetic distance values appeared to better reflect the genetic similarities among the populations than the Fst values and confirmed differences in genetic structure in the populations of the three species.The analysis of molecular variance showed higher percentages of genetic variation within populations than among populations in all species. Similar levels of population differentiation were found in E. canadensis and E. densa showed similar levels of polymorphism  and higher than those of L. major (9.8%), the genetic pattern was different in the three species. In L. major the samples from the geographically close populations of Otamangakau and Taupo South appeared as a monophyletic group differentiated from a dominant genotype spread in every location  trees of Figures n Figure . The intn Figure . The otha Figure  showed as Figure . As in LL. major , indicating a major extent of genetic structure in E. canadensis populations. The trees also supported the distinction between the upstream Otamangakau and Taupo South populations and the downstream populations both in E.canadensis and in L. major.The UPGMA trees Figure  show theE. canadensis compared to E. densa and L. major and the similar geographical separation of Otamangakau and Taupo South populations both in E. canadensis and in L. major, indicate that AFLPs are appropriate for the study of genetic variation in these clonal species. If the observed genetic patterns were due to artefacts of the AFLP technique, similar results would have been expected in the three species, considering the similarly low levels of genetic variation.The DNA quality, the reproducibility of AFLP chromatograms, the different patterns observed in L. major, genetic variation within and between populations in the native range in South Africa was studied by Triest [Low levels of genetic diversity were found in all three species, which suggest one introduction or multiple introductions of similar genotypes for each of the three species in the northern part of the North Island, NZ. High levels of genetic diversity in invasive species may be the result of multiple introductions ,17. Howey Triest  using isE. densa in Oregon (USA) and Chile using RAPDs by Carter and Sytsma [E. densa cannot be ruled out in the North Island, NZ. A stepping-stone model of colonization could, however, explain the similar genetic patterns in Oregon and Chile and hide higher levels of genetic diversity in the native range. In a study using allozymes, Kadono et al. [E. densa throughout Japan, but as in the case of isozymes, the technique yields a limited resolution compared to RAPDs and AFLPs.Genetic diversity was studied in introduced populations of d Sytsma . Very lid Sytsma  shows tho et al.  found geE. canadensis populations. In the congeneric invasive species Elodea nuttallii (Planchon) St John, both multiclonal [E. nuttallii [E. canadensis. If this is correct, genetic diversity had to be very limited in the source populations, suggesting a stepping-stone model of invasion in NZ. We do not know the genetic pattern in Tasmania and how many source populations were involved. We can only move the discussion about the number of introductions from NZ to Tasmania. The differences with the Danish genotype suggest that higher levels of DNA variation are present within the species, but E. canadensis is invasive also in Europe and different evolutionary patterns could explain the divergence of allopatric gene pools.To the best of our knowledge this is the first study of the genetic variation pattern of ticlonal  and monoticlonal  were fouuttallii . A singluttallii  for E. cin situ by somatic mutations. The first scenario would entail either low genetic diversity in the native range or a stepping-stone model of colonization from other areas of the introduced range, as appears to be the case for E. canadensis. The second scenario, involving evolution of genetic diversity in NZ by somatic mutations is consistent with our results. A third model, which parsimoniously combines the previous two, limiting both the number of dispersal events and the number of mutations to have occurred in NZ, is that of ancestral polymorphisms retained in the three species [Not knowing the genetic variation of these species in their native range, we cannot draw conclusions about the number of introductions to NZ (or Tasmania). Two possible scenarios emerge, however, from this study: multiple introductions of genetically similar genotypes, or one single founding event  and evolution  species .Elodea canadensis was the first of the three species to be introduced in 1868 and its populations have higher Fst values and Nei's genetic distances than the later-established populations of E. densa and L. major. Also, the pattern of E. canadensis differs from that of the other species in showing more monophyletic groups, a continuum of genetic differences between individuals and populations, and differentiation between the geographically close populations of Taupo South and Otamangakau from the downstream locations. Strikingly the populations of Taupo South and Otamangakau were found to be a monophyletic group also in L. major, which was introduced in NZ as recently as in 1950. This can be explained either by recent "co-dispersal" of the two species (after 1950), or by independent dispersal histories shaped by the same geographic/physical constrains. In both cases multiple independent introductions appear very unlikely, as those would have blurred any common pattern. Considering that only one sex is present, or dominant, in each species, the absence of seeds and evident outcrossing events (recorded in the spectrum of pairwise genetic distances), somatic mutations seem to be a plausible source of genetic diversity in these clonal species.Several results of this study indicate that evolutionary processes occurred after these species were introduced in the northern part of NZ's North Island. The measures of population differentiation show that the time elapsed since introduction is reflected in the genetic structure of the populations. Posidonia oceanica (L.) Del. [Populus tremuloides Michaux [L. major and E. densa populations show the same pattern that has been considered indicative of clonal reproduction and somatic mutations. Contrastingly, the spectrum of E. canadensis populations does not fit with this model. The normal distribution of pairwise differences points to outcrossing genotypes, whereas the limited range of polymorphism, which is comparable to that of E. densa, points to somatic mutations. The comparison with a genotype from Europe shows higher numbers of polymorphic fragments with all NZ genotypes. In addition if the spectrum was the result of outcrossing events between the NZ genotypes, outcrossing would be more frequent than clonal propagation, as the frequency of "zero" pairwise differences is very low, and seeds would be common in the populations. Seeds have, however, never been observed in E. canadensis in the northern part of NZ's Northern Island [E. canadensis's spectrum, such as a pool of genetically similar/closely related founders, possible mutation-drift disequilibria and a longer introduction time.The spectrum of pairwise genetic distances has previously been utilized to study genetic diversity in populations of clonal outcrossing plant species, as it may be helpful in distinguishing between genetic variation produced by somatic mutations and genetic variation due to outcrossing. The spectrum of clonal outcrossing populations of the marine submerged L.) Del.  shows a  Michaux . Comparen Island . HoweverEgeria densa, which in the North Island of NZ has the typical spectrum of a clonally reproducing species that has accumulated somatic mutations, has more genetic diversity than the other macrophytes. This makes E. densa more adaptable. In invasive populations of the clonal aquatic species Hydrilla verticillata (L. f.) Royle, Albrecht et al. [Genetic diversity levels are different in the three species, in relation to the initial gene pools, possible ancestral polymorphisms, different somatic mutation rates and establishment times. t et al.  demonstrE. canadensis and L. major. Surprisingly, in L. major, these populations are isolated also from Lake Rotoaira, which is geographically close and connected by waterways to Otamangakau. A wider set of samples and populations covering also the southern part of the North Island and the South Island, combined with historic records of first introduction date in each location would help better understand the relationships between populations and reconstruct the introduction history of these species in NZ with higher resolution. It would also greatly enhance interpretation of nodes of dispersal vs. recruitment, providing a valuable tool for the local management of these aquatic weeds.In the absence of evident sexual reproduction, the distribution of genetic diversity in the populations documented in this study is found to be due to the dispersal of vegetative propagules, moderated by the presence of possible geographic/ecological barriers. The analysis of the geographic distribution of DNA polymorphic fragments showed more frequent dispersal events between neighbouring populations, even though long-distance dispersal was also common. Multiple introductions in lakes and rivers appeared also to be a recurring phenomenon, as well as down- and up-stream dispersal. The co-occurrence of more invasive species in the same localities and similarly complex dispersal routes in all three species suggest that human dispersal had a major role in the distribution of the genetic diversity. Johnstone et al.  ruled ouE. canadensis and L. major. Considering the lack of seeds and the random dispersal of vegetative propagules, which appears to be human-mediated, somatic mutations are supposed to be a potential source of genetic diversity in these clonal species. However, this study does not provide direct evidence of such evolutionary events. Genetic variation should be analysed in the native populations to conclusively shed light on the evolution of these species in the North Island of NZ.Very low levels of genetic diversity were found in three invasive aquatic species in the northern part of North Island, NZ. Even though it is not possible to quantify how much of the genetic variation can be attributed to each founding event, this study provides some evidence that evolutionary changes have occurred in the introduced range, even though sexual reproduction is not frequent or does not occur at all. Post-introduction evolution is supported by population genetic structure and similar phylogeographic patterns in E. canadensis collected in Denmark as a reference to evaluate the genetic similarities of the NZ populations against a different genotype.We sampled populations of the three species in a number of locations in the northern part of the North Island, NZ Figure . This saE. canadensis , representing 7 populations, 42 specimens of E. densa (14 populations) and 30 specimens of L. major (10 populations) were AFLP fingerprinted.At each location three samples were collected from different macrophyte beds randomly chosen at an average distance of about three metres. As clonal propagation is the main (or exclusive) form of reproduction in these species in North Island, we considered a geographical extension of the sampling area more informative than an increased number of samples collected at each location. The term population is used in this study in a broader sense, which includes perennial clonal stands that are not necessarily in a gene flow relationship. From each specimen we sampled the top shoot and stored it in a sealed bag with silica gel. Samples from the same locations were considered representative of one population. In total 21 specimens of DNA was extracted with the E.Z.N.A. plant kit from the apical parts of shoots. DNA quality was checked on a 0.8% agarose gel, run for 1 h at 120V. DNA consisted of a sharp band of the size of about 20.000 bp compared to the Lambda DNA, Hind III marker 2 (MBI Fermentas) and no signs of DNA digestion were visible.DNA concentration was measured with the Nano Drop Spectrophotometer ND-1000 (Saveen Werner) at 280 nm wavelength and was between 50 and 200 ng/μl in all samples, eluted with 50 μl E.Z.N.A. elution buffer.20 ng DNA were used for DNA restriction with 1.25 units each of EcoRI and MseI enzymes and ligation to 2.5 pmol EcoRI and 25 pmol MseI nucleotide adapters with 335 NEB cohesive end ligation units. Restriction and ligation were carried out simultaneously in a PCR . The program was 37 ˚C for 4 h followed by 0.1 ˚C decrease in 2 h and 10 min at 70 ˚C. Restricted and ligated DNA was diluted 4x prior to pre-amplification.2 μl of template DNA were added to 18 μl mastermix consisting of 10 μl 2x Mastermix (VWR Ampliqon), 6 picog EcoRI primer, 6 picog MseI primer and sterile water to reach the final volume of 20 μl. EcoRI primer and MseI primer consisted of one-nucleotide selective base. The PTC-200 PCR was programmed for 20 cycles, each with a 30 s DNA denaturation at 94 ˚C, 1 min primer annealing at 56˚C, and 1 min extension at 72 ˚C. Preamplified DNA was diluted 4x prior to selective amplification.E. canadensis  and E. densa  and four primer combinations for L. major  based on the presence of polymorphic DNA fragments and the scoring possibilities.2 μl of template DNA were added to 18 μl mastermix consisting of 10 μl 2x Mastermix (VWR Ampliqon), 1 picog EcoRI primer, 6 picog MseI primer and sterile water to reach the final volume of 20 μl. EcoRI primer was Cy (fluorochrome) labelled. The PCR program was 94˚C for 30 s, 65 ˚C for 30 s decreased by 0.7 ˚C/cycle for the subsequent 12 cycles, 72 ˚C for 1 min, followed by 23 cycles at 94 ˚C for 30 s, 56 ˚C for 30 s and 72 ˚C for 1 min. Several primer combinations were tested and many did not show polymorphism. Three primer combinations were selected for was run on a 7% acrylamide gel (Reprogel - Long Read) with 50-500 bp external sizers , in an ALF Express II DNA Analysis System (Amersham Pharmacia Biotech) at 1500 V, 55˚C for 400 min. 5 μl amplified DNA were added to 3 μl loading dye  and DNA was denatured in the PTC-200 PCR for 5 min at 94˚C prior to loading on the gel.E. canadensis , 127 DNA fragments were scored in 38 samples of E. densa and 142 DNA fragments were scored in 27 samples of L. major.DNA fragments between 50 and 500 bp were scored for presence or absence with the program ALFwin Fragment Analyser Software package (Amersham Pharmacia Biotech). Only peaks clearly above the detection limit (set with a peak shape of 10 and a minimum height of 1%), well amplified and resolved in all samples were scored for polymorphism. The AFLP chromatograms were virtually identical within each species, with the exception of some samples in which a few peaks were absent  compared to the rest of the sample set. DNA was extracted, digested, ligated and amplified twice from these samples, to make sure that missing DNA fragments were due to polymorphism and not to DNA fragmentation. The complete reproducibility of AFLP chromatograms excluded possible ligation  and PCR E. canadensis, 1% of the peaks scored in E. densa and 0.7% of the peaks scored in L. major. These levels of ambiguity are comparable to or lower than those quantified by Bonin et al. [E. canadensis nine polymorphic fragments, out of 22, had a frequency over 0.20 and one fragment had a frequency of 0.44. Polymorphic fragments were in most cases missing or present in all samples from the same population. In E. densa and L. major datasets only 3 and 1 DNA fragments respectively had a frequency over 0.20 and it was not possible to recognize any "population pattern" by scoring the gels.The scored polymorphic loci clearly showed either high peaks or no peaks. Low intensity peaks occurred in a very limited number of polymorphic loci and were scored as question marks. They corresponded to 1.5% of the peaks scored in n et al.  as typicE. canadensis, considered as one population, and the Danish genotype. Population differentiation was also evaluated based on Nei's [The resulting binary matrix was analysed for each species with the program Arlequin ver. 3.11  to estimon Nei's  unbiasedon Nei's  and unbion Nei's . The genon Nei's ). QuestiE. canadensis was used as the outgroup for the NZ samples. For E.densa and L. major the NJ trees were calculated as unrooted phylograms.Genetic relationships among clones were calculated with PAUP ver. 4.0b10  and witThe network of all maximum parsimonious trees was constructed with the reduced median algorithm . This alWe used the program TFPGA also to calculate UPGMA trees of the populations. Nei's genetic distances (unbiased minimum genetic distances) are based on pairwise common DNA polymorphic fragments and similarities between populations can be assumed to reflect a possible common vegetative origin and/or dispersal opportunities between populations. Statistical support was assessed with bootstrap analysis based on 1000 permutations and by changing the order of the samples several times in the data matrix, in order to alleviate the taxon entry-order problems associated with UPGMA method, as pointed out by Backeljau et al. .AWA: Awakaponga; HAM: Hamilton; HMU: Hamurana; KAI: Kaituna; McL: McLaren; NGA: Ngakeketa; ORA: Oraka; OTA: Otamangakau; PAR: Parkinsons; PON: Pongakawa; ROA: Rotoaira; ROM: Rotoma; ROT: Rotorua; Rout: Rotorua outlet; RSM: Rotoiti Sth. Man; TAR: Tarawera; Tout: Taupo outlet; TIK: Tikitapu.; Tsth: Taupo South; SWA: Swan; WAI: Waikato; WEA: Weavers; WHA: Whatihua.Localities abbreviations: CL ran the AFLPs, analysed the data and wrote the manuscript. TR coordinated the study and prepared the figures. TR, BO and JSC collected samples in New Zealand. All authors contributed to the scientific discussion of the results, commented the draft manuscript, and read and approved the final manuscript.


Urbanization is a major cause of habitat fragmentation worldwide. Ecological and conservation theory predicts many potential impacts of habitat fragmentation on natural populations, including genetic impacts. Habitat fragmentation by urbanization causes populations of animals and plants to be isolated in patches of suitable habitat that are surrounded by non-native vegetation or severely altered vegetation, asphalt, concrete, and human structures. This can lead to genetic divergence between patches and in turn to decreased genetic diversity within patches through genetic drift and inbreeding.We examined population genetic patterns using microsatellites in four common vertebrate species, three lizards and one bird, in highly fragmented urban southern California. Despite significant phylogenetic, ecological, and mobility differences between these species, all four showed similar and significant reductions in gene flow over relatively short geographic and temporal scales. For all four species, the greatest genetic divergence was found where development was oldest and most intensive. All four animals also showed significant reduction in gene flow associated with intervening roads and freeways, the degree of patch isolation, and the time since isolation.Despite wide acceptance of the idea in principle, evidence of significant population genetic changes associated with fragmentation at small spatial and temporal scales has been rare, even in smaller terrestrial vertebrates, and especially for birds. Given the striking pattern of similar and rapid effects across four common and widespread species, including a volant bird, intense urbanization may represent the most severe form of fragmentation, with minimal effective movement through the urban matrix. Habitat loss and the resulting fragmentation can have many impacts on wildlife populations. However, the effects of fragmentation may vary based on many factors including the size, configuration, and age of habitat patches, the vagility of the species in question, and the characteristics of the matrix between patches. Urban development may represent a particularly intense form of fragmentation for many animals. Species that are particularly sensitive to urban development may be quickly lost from urban areas Reptilia) and birds (Class Aves), and with radically different means of locomotion, such as flying and crawling.An increasing number of studies of the genetic effects of fragmentation have occurred in the past decade or so, although 30–40% of these have not shown significant effects and many are in non-urban landscapes such as fragmented forests Uta stansburiana), western skink (Plestiodon skiltonianus) and western fence lizard , and one bird, the wrentit (Chamaea fasciata) in Santa Monica Mountains National Recreation Area (SMMNRA), a national park near Los Angeles. The three lizard species have widespread distributions in California Iguanidae, but side-blotched lizards are considerably smaller and prefer more open habitat. Western skinks are in a distantly-related different family (Scincidae) and locally prefer grassland habitat, although all three species are broadly sympatric in the region.We investigated the genetic effects of urban fragmentation on three lizards, the side-blotched lizard  with a distribution that is limited to the west coast of North America and follows the scrub and chaparral habitat that they preferwww.biodiversityhotspots.org). Because it is in the Los Angeles area, SMMNRA is under intense development pressure and urbanization might increase to as much as 47% of the area by 2050, whereas only 11% was urbanized in 2000 The landscape of southern California continues to be rapidly altered by urbanization and the resulting habitat loss and fragmentation, even though it is part of the California Floristic Province and is one of Conservation International's world biodiversity hotspots . Among lizards, the level of differentiation was highest for side-blotched lizards, with an average pairwise FST of 0.073 (range −0.006–0.200), and very similar for western skinks  and western fence lizards . As a baseline comparison from continuous habitat, when we computed genetic distances between the sampling arrays within large and core patches and between several other sites outside of our urban study area , and fewer significant pairwise FST . Significant genetic distances between patches could also be caused by isolation by geographic distance. We found no significant correlations between genetic distance (FST) and geographic distance in any of the four species, suggesting no pattern of isolation by distance  and the mean number of effective alleles (NA) were not significantly lower in smaller patches for any of the four species . Rarefaction analysis indicated that the number of loci used produced consistent average relatedness results for all species and that the addition of the last locus added a 0.5% (fence lizards), 0.8% (side-blotched lizards), 1.4% (western skinks), and 0.1% (wrentits) change in relatedness estimates.Mean heterozygosity , as was NA  was shown to have small yet significant genetic structuring  over less than 500 meters on a recently fragmented island Plethodon cinereus), an even smaller and less mobile animal then the lizards we investigated, had pairwise FST between patches similar in value to the lizards in our study th Century urbanization.Pairwise Fnificant . This suST between patches on such a small scale. For example, a study of the capercaillie  in the Black Forest in Germany showed significant pairwise FST between sites, ranging from 0.007 to 0.036 Corapipo altera) showed similar results to the capercaillie. There was some genetic structuring shown between remnant forest fragments, however all significant pairwise FST could be attributed to one forest fragment. In addition, pairwise FST between forest fragments ranged from 0.001 to 0.029 for manikins, whereas in our study the pairwise FST were approximately 10 times higher. Finally, a study of great tits (Parus major) in forest parks within the city of Barcelona found many significant pairwise FST between parks (average 0.067), but the parks actually had higher genetic diversity than the surrounding forest, and there was significant gene flow both between parks and from the parks to the forest For birds, few studies have shown large pairwise FST values between sampling sites within continuous habitat), and therefore may be extremely restricted in a fragmented landscape. In one of the few other studies using Bayesian genetic clustering analysis, red-backed salamanders (Plethodon cinereus) were found to have two genetic clusters on either side of a large highway running through the urbanized study area The Bayesian clustering analysis confirmed the loss of genetic connectivity for each species in our study area. Similar analyses in other bird studies have consistently shown that one genetic cluster is most likely Stenopelmatus ‘santa monica’ and Stenopelmatus ‘mahogani’) across the same region Along with significant divergence between patches, we also found significant correlations between specific causes (roads) and measures (patch age) of fragmentation and genetic divergence in all four species . FurtherS. occidentalis, but not P. skiltonianus or U. stansburiana, will move through or persist in the residential areas of the urban matrix . However, reliable data on the urban movement and habitat use of these species does not exist. In fact, knowledge about use of the urban matrix by native animal species is extremely limited in general, but would be very valuable for understanding the conservation and management implications of urbanization. Urbanized areas may be dangerous places for these small vertebrates. Residential neighborhoods often introduce predators such as domestic cats, which may regularly prey on native vertebrates The significant genetic divergence and loss of genetic diversity over short geographic and temporal scales in these four vertebrates suggest that the urban matrix is relatively impenetrable for these animals. Anecdotal observations suggest that e) and fewer alleles (NA) in smaller patches  was strongly correlated with genetic divergence between patches, the effects on genetic diversity in these animals were significantly related to patch size and degree of patch isolation, but not to patch age. This would suggest that the habitat is still relatively suitable in habitat fragments, resulting in relatively stable populations that are not going through bottlenecks, such that more time since isolation is not as important a factor. But patches that are smaller from the outset simply cannot support as large a population, and therefore are more subject to the deleterious effects of genetic drift, specifically the loss of genetic diversity. Patches that are more isolated may in turn be less likely to receive new dispersers, i.e. they would benefit less from the “rescue effect” that could offset reductions in genetic diversity The extreme urbanization within the Simi Hills area has had a significant effect on lizard and bird population genetics. Unlike some other studies of landscape level genetic changes where a species' habitat is naturally patchy, this study examined genetic responses to species living in habitat that was likely once relatively continuous Phrynosoma coronatum) and whiptail lizards (Aspidoscelis tigris), whose distributions have already been reduced by urban development Rallus longirostris obsoletus; Belding's savannah sparrow, Passerculus sandwichensis beldingi; red-legged frog, Rana draytonii; least bell's vireo, Vireo bellii pusillus). It is also unknown how stressors, such as increasing local or global temperature and urbanization, might affect species in southern California. A recent study of Sceloporus lizards in Mexico found that 12% of local populations have gone extinct since 1975 For rarer species in the region, such as horned lizards , which is located in Los Angeles and Ventura counties, California, USA . Both freeways are mostly surrounded by commercial and residential development. Within the study area there are large core areas of relatively undisturbed habitat, although some low-impact human recreation does occur. Within the urban mosaic, habitat patches were surrounded by high- or low-density housing, highways and other roads, golf courses and other landscaped areas.Southern California is characterized by a Mediterranean climate with cool, wet winters and hot, dry summers. Vegetation consisted of coastal sage scrub, chaparral, riparian habitat, and oak woodlands. Our study site is within SMMNRA, the USA's largest urban national park  and degree of isolation (PROX) were calculated using FRAGSTATS We collected samples from habitat fragments which we characterized as “small” (75–450 ha) or “large” (3200–4400 ha) and from larger areas of continuous habitat which we called “core” areas . There wTo capture lizards we used arrays of pitfall traps and drift fencing. All samples for this study were collected between October 2000 and September 2005. Each array had seven 19-liter buckets buried in the ground with the lip of the bucket flush with the ground to act as a pitfall trap To capture birds, we used mist-nets. Trapping occurred from August 2004 to May 2006. Generally, we would open mist-nets  at sunrise and close them as the temperature increased to a potentially unsafe level in mid-morning. We targeted wrentits by playing male territorial songs with portable speakers placed at the base of the net. Once a bird was caught in the net, it was immediately removed and a U.S. Fish and Wildlife Service band was placed on its leg. We also took measurements of culmen length (mm), culmen width (mm), unflattened wing chord length (cm), tail length (cm), tarsus length (cm) and mass (g). Culmen length was taken from the anterior end of the nares to the tip of the beak using calipers. For genetic samples, we punctured the brachial vein on the wing of each bird with a small gauge needle and collected the blood that pooled there with a small capillary tube. Bleeding usually stopped after 10 seconds which yielded approximately 100 µl of blood. Blood was then placed in avian blood buffer All samples used in this study came from animals that were captured, handled, and released according to relevant national and international scientific guidelines. We used common field and handling methods that minimize stress and long-term effects of capture. We also researched methods alternative to toe-clipping of reptiles and determined that there were no less harmful yet permanent ways of marking individuals 5′-GTAAAACGACGGCCAG-3′) and a third primer with the complementary M13 sequence was dye-labeled We extracted genomic DNA with the Qiagen DNA mini kit (Qiagen Inc.). DNA samples were stored in TE buffer  at −20°C. We used six to eight microsatellite markers for each species . We usedWe used the computer program CONVERT to translate our microsatellite genotype files into the correct input format for various analysis programs ST values between patches using the infinite-allele model and 1000 permutations for significance ST between arrays within large and core patches with ARLEQUIN to show genetic divergence between sampling sites that were located within a patch of continuous habitat. For this calculation we also included some sampling sites from core areas of continuous habitat that were outside of the Simi Hills (our study area), but within SMMNRA, with an average of 4.28 km (range 1.8–6.6 km) separating these sites.We used the program ARLEQUIN to estimate pair-wise Fhttp://taylor0.biology.ucla.edu/struct_harvest/). To determine the most likely K, we calculated the posterior probabilities of the mean of seven runs at each K  distances using a Mantel test, was performed using IBDWS 3.14 ST; as calculated in ARLEQUIN, see above). The presence of major roads and the presence of Highway 23 were used separately in the analysis because the highway in our study area is larger and has more traffic than other roads. Also, several habitat fragments are only separated by major roads. Age of isolation was chosen because this measure incorporates not only when roads and freeways were built, but also when residential and commercial developments were erected.To test for the effect of major roads, highways, and patch age on genetic divergence, we performed partial Mantel tests ij) at each midpoint was calculated s; ij is 0 if individuals are completely genetically identical, and Dij is 1 if individuals are completely genetically dissimilar. We did not calculate the residual genetic distance, because we did not find a significant isolation by distance effect in the Simi Hills samples for any species (see We mapped genetic distance on the landscape using Alleles in Space (AIS) and the landscape shape interpolation cies see . By thiscies see .e), observed heterozygosity (Ho), number of effective alleles (NA), and relatedness (RLR) We used the program GENALEX We used the program STATA 9  to transform variables until they approached normal distributions and then to examine the relationship between the indices of genetic diversity and the size, degree of isolation, and age of the habitat patches. We used unpaired t-tests  and Bonferroni corrections to compare genetic diversity measures between small and large/core habitat patches. Degrees of freedom for t-tests were calculated using the Satterthwaite (1946) method Table S1ST between sample sites for 4 species. Significant pairwise FST values are in bold (see F(0.22 MB DOC)Click here for additional data file.Table S2ST and the number of significant comparisons between patches in and continuous habitat.Pairwise F(0.05 MB DOC)Click here for additional data file.Table S3Estimated posterior probabilities for K. Most likely number of genetic clusters (K) identified with the program Structure is shown in bold.(0.07 MB DOC)Click here for additional data file.Table S4A; relatedness, RLR; heterozygosity, He).Mean genetic diversity measurements within patches Click here for additional data file.Table S5e) and observed (Ho) heterozygosity.Spearman's Rho correlation coefficients. The number of individuals genotyped (N), the number of alleles (A), expected (H(0.04 MB DOC)Click here for additional data file.Table S6e) and observed (Ho) heterozygosity.Microsatellite primers used for each species. The number of individuals genotyped (N), the number of alleles (A), expected (H(0.14 MB DOC)Click here for additional data file.