Aedes albopictus, indigenous to East Asia, has colonized every continent except Antarctica. Its spread is a major public health concern, given that this species is a competent vector for numerous arboviruses, including those causing dengue, chikungunya, West Nile, and the recently emerged Zika fever. To acquire more information on the ancestral source(s) of adventive populations and the overall diffusion process from its native range, we analyzed the mitogenome variation of 27 individuals from representative populations of Asia, the Americas, and Europe. Phylogenetic analyses revealed five haplogroups in Asia, but population surveys appear to indicate that only three of these  were involved in the recent worldwide spread. We also found out that a derived lineage (A1a1a1) within A1a1, which is now common in Italy, most likely arose in North America from an ancestral Japanese source. These different genetic sources now coexist in many of the recently colonized areas, thus probably creating novel genomic combinations which might be one of the causes of the apparently growing ability of A. albopictus to expand its geographical range.In the last 40 years, the Asian tiger mosquito  Aedes includes five highly invasive species, A. albopictus, A. aegypti, A. j. japonicus, A. koreicus, and A. atropalpus. Of these, A. albopictus and A. j. japonicus are the most widespread across the globe, and A. aegypti and A. albopictus, being competent vectors for several human tropical diseases, have a major impact on human health. The North American species, A. atropalpus, arrived in Europe  through international trade , have both colonized central Europe and A. j. japonicus is also widely distributed in the US  in 1979, l Europe . It was l Europe  and intol Europe . Its abil Europe .A. albopictus mitochondrial DNA (mtDNA) surveys were restricted to short segments of the cytochrome c oxidase subunit 1 (COI) and/or NADH dehydrogenase subunit 5 (ND5) genes, suggesting a limited phylogeographic differentiation among populations, possibly also caused by the inclusion in these studies of laboratory stocks or sibling eggs  from Eastern and Southeastern Asian, American, and European populations. Our analyses reveal that only three of the five identified Asian haplogroups, which are differentially distributed in Asian populations living in temperate and tropical regions, were involved in the recent worldwide spread. These different ancestral sources from Asia now coexist in many adventive populations with possible implications for the adaptive capability of the species.To identify the ancestral source(s) of Table 1; Figure 1). Three were from the Americas (two from Virginia and one from Brazil). Nine were from Asia: three from Thailand , five from Los Baños, Laguna, Philippines and one from Wakayama prefecture, Japan. Ten were from Europe: two from Tirana (Albania), two from Athens (Greece), two from Cesena and two from Pavia , one from Cassino , and one from Reggio Calabria . This study also included three adult laboratory-maintained strain mosquitoes: two from the Italian Rimini strain (Table 1). The study did not involve protected species and specimens were not collected at sites protected by law.A total of 25 novel mitogenomes were included in this study. Twenty-two were from wild populations collected in Europe, Asia, and the Americas . The A. albopictus Reference Sequence (NC006817) was used to design primer sets. The coding region (nps 1-14893) was amplified by two long PCR fragments whereas the control region (nps 14894-16665) was amplified by two rather short PCR fragments . PCRs were carried out in 25 μl reaction with a standard reaction mix containing 1X White Buffer (1.5 mM MgCl2), 0.2 mM of each dNTP mix, 0.6 U of GoTaq G2 Polymerase (Promega), 0.2 μM of each primer and 20–30 ng of DNA template, using the following PCR conditions: 94°C (2 min); 35 cycles of 94°C (30 s), 55°C (30 s), 72°C (2 min); and a final extension of 72°C (5 min).Coding region long PCRs , were amplified with PCR #3 and PCR #4, respectively. Overall, the size of the amplified fragments ranged from ∼1,800 to ∼2,500 bp for PCR #3 and from ∼800 to ∼900 bp for PCR #4. As for one of the two mitogenomes from the Rimini strain , PCR #3 and #4 yielded products of ∼2,000 bp and ∼900 bp, respectively. The product of PCR #4 was directly cloned in the pCR2.1 TOPO vector (Invitrogen) following the manufacturer’s protocol. White colonies were PCR-screened for the insert length and desired clones were sequenced bi-directionally using the M13 universal primers. The procedure described above is inefficient for longer fragments such as that of ∼2,000 bp, therefore a different strategy was developed to sequence the region amplified by PCR #3. It was re-amplified in two overlapping PCR fragments (PCRs I and II), each with one primer within the tandemly repeated elements of type I (Supplementary Table Given the different copy numbers of repeated elements contained in the The obtained complete sequence of mitogenome #1 (accession number KX383916) was used to assemble and number, with Sequencher 5.0 (Gene Codes), the other 24 novel mitogenomes . We aligned the novel sequences and the two previously published mitogenomes – #17 from China  and #27 A. albopictus dataset. The ML tree was built using PAMLX .Most Parsimonious (MP) trees (1000 bootstrap replications) encompassing the 27 mitogenomes were built by using MEGA7 , employing PAMLX  and assung PAMLX  and runnA. albopictus partial COI and ND5 available in the literature for the presence/absence of these mutations. Of these, 284 mtDNAs are from Asia, 349 from the Americas, 32 from Europe, 153 from Africa, and 352 from Oceania (Table 2), thus encompassing populations from both native and non-native areas, and living at different climatic conditions.The identification of haplogroup diagnostic mutations allowed us to survey 1170 Table 1; Figure 1), whose nuclear genome was recently sequenced . Type I consists of a ∼190 bp repeat unit, whereas type II is made up of a short unit of ∼42 bp. The number of type I and type II tandem repeats that we observed in mitogenome #1 was different from those in sequence NC006817 from Taiwan  and variable among mtDNAs (Table 1 and data not shown). Moreover, between these two types of repeats, delimited by two conserved blocks, lies an A+T rich region of variable length. The overall length and tandem repeat composition make the PCR amplification and sequencing of the entire A. albopictus control region extremely difficult . For the reasons outlined above, and difficulties originating from the impossibility of distinguishing heteroplasmy from PCR artifacts (due to replication slippage), we restricted our sequencing of A. albopictus mitogenomes to the coding region  and nearby control region segments. This approach was employed to obtain the coding region sequences of the additional 24 mitogenomes.The first step in our study was to sequence the entire mtDNA (mitogenome #1) from one mosquito of the Italian (Rimini) laboratory-maintained strain . The overall tree structure is virtually identical with the two approaches and is supported by the ML tree  cluster into three major branches that we named haplogroups A1, A2, and A3. Haplogroup A1 includes 21 mitogenomes, A2 consists of the five Philippine samples, while A3 encompasses only one mitogenome (#27) from Taiwan. Haplogroups A1 and A2 are rather close to each other and to the A1′2 node. In contrast, the A3 mitogenome differs by 363 mutations from the same node.A. albopictus. Indeed, when calculated on the standard COI sequence (A. scutellaris (0.008) and A. aegypti (0.022), but much greater than the value previously reported for A. albopictus  and many of the mitogenomes from Italy, including one (#1) of the two detected in the Rimini laboratory strain. The mitogenome from Japan (#26) departs from the node A1a1a and its sister clade A1a1a1 contains a sub-branch, A1a1a1a1, of particular interest. It consists of four mitogenomes, two from Italy and the two from the US mentioned above. One of the US mitogenomes (#4) is identical to one from Northern Italy (#5) while the second (#2) is closely related to the mitogenome #3 from Southern Italy.The sister branch A1a2 is formed by mitogenomes from different regions of Southern Europe , including the second mitogenome (#14) from the Rimini strain, the previously published Chinese sequence  and theA. albopictus haplogroups and sub-haplogroups but also the definition of their distinguishing mutations . These include some diagnostic markers that are located in COI and ND5 partial sequences whose variation has been extensively assessed by published studies and can be retrieved from GenBank (Table 2).The phylogenetic analysis not only allowed the identification of Table 2 reports the haplogroup frequencies for A1a1a1, A1a2, A1b, A2, and A3 obtained from this survey, as well as a category termed “others” that includes mtDNAs that we could not classify and might encompass haplogroups not represented in our phylogeny. Figure 3 provides an overview of the worldwide spatial distribution of these haplogroups.By surveying these sequences for the presence or absence of these mutations, we were able to determine the most likely haplogroup affiliation for most of the 1170 tiger mosquito mtDNAs from populations worldwide. A. albopictus mitogenomes cluster into the three main haplogroups A1, A2, and A3. Intriguingly, the population screening of the COI mutations  found in mitogenome #27 from Taiwan, the only one belonging to haplogroup A3 in our phylogeny, did not reveal any match in the 1170 tiger mosquito mtDNAs from worldwide populations, not even in the population sample (N = 30) from Taiwan (Table 2). This suggests that A. albopictus mosquitoes with A3 mtDNAs were probably not involved in the recent worldwide spread of the species, raising the possibility that this haplogroup might be rare and/or with a restricted geographical distribution.Phylogenetic analyses revealed that our Table 2), but all in Insular Southeast Asia, suggesting that this haplogroup might be typical and possibly limited to the Philippines, Indonesia, Papua New Guinea, and Northern Australia (Table 2). Therefore, haplogroup A2 appears to have played a role in the spread of A. albopictus from South-East Asia  restricted to the context of Oceania. Indeed A. albopictus is thought to have spread from Indonesia by human-mediated transportation (Table 2) suggests multiple arrivals from distinct geographical sources  and shows high frequencies in Central America and Eastern USA. Haplogroup A1a2 is present with frequencies higher than 50% in Japan, Southern China, Singapore, Hawaii, California and Italy, whereas A1b is fixed or almost fixed in Thailand, Malaysia, the Papua New Guinea mainland, Cameroon, and Brazil, but present at much lower frequencies also in California and Texas (Table 2). Even though these geographical distributions are based on the limited population sampling reported in Table 2, some preliminary conclusions can be drawn.As for haplogroup A1a1, seen in the Japanese mitogenome #26, it is widely distributed in Italy . This may imply that genetic and physiological traits make populations with A1b most suited to the colonization of tropical areas  suggests that A1b mtDNAs arrived in Cameroon from a different tropical source. Interestingly, the absence of photoperiodic diapause in Brazilian mosquitoes supports their origin in tropical Asia, while the diapause in US populations is in agreement with the scenario of an ancestry in the Asian temperate regions , from all other mitogenomes in the phylogeny. The survey of these transitions in published data sets revealed that haplogroup A1a1a1 is the most common in Costa Rica, Panama, Texas, and New Jersey  and widespread in Italy (Table 2). This distribution suggests that A1a1a1 and/or its derivatives A1a1a1a and A1a1a1a1 arose recently in an adventive non-Asian population, probably from an ancestral Japanese source, and reached a high frequency because of genetic drift or founder events. From the earliest adventive non-Asian population(s) they then further spread to other distant regions. Such a possibility is in agreement with some previous observations, in particular with the suggestion that Italian tiger mosquitoes, whose first presence was documented in Northern Italy in 1990 . Finally, the scenario that the ancestral source of A1a1a1 might be a northern temperate area such as Japan is further supported by the presence of the COI transitions 1536 and 2435, which characterize mitogenome #26, in five published mtDNAs from Kyoto  the detection of four partial sequences from Texas with the transition at np 1823 , which dA. albopictus mitogenomes was charted, and the most likely Asian sources of some adventive populations were identified. The worldwide spread of A. albopictus appears to be associated with three mtDNA haplogroups , differently distributed in Asian populations living in temperate and tropical regions, whereas a fourth Asian haplogroup (A2) appears to be restricted to Insular South-East Asia. These ancestral genetic sources now coexist (and interbreed) in many of the recently colonized areas. This occurs not only in the field but also in the laboratory as attested by our detection of both A1a1a1 and A1a2a1 mitogenomes in the Rimini maintained strain, thus creating novel genomic combinations that might be one of the causes of the continuous and apparently growing capability of A. albopictus to expand its geographical range.Through our analyses, based on complete coding regions, the phylogeny of the A. albopictus populations that, by contrast, have not spread.Note that fine scale mitogenome surveys, encompassing multiple specimens from a wide range of East Asian populations might prove to be an essential pre-requisite to controlling the spread of this mosquito and limiting its social, medical, and economic implications. With a precise identification of the source populations in Asia it will become possible to evaluate the extent and nature of their nuclear genome diversity and the possible selective advantages  relative to other Asian VB, PG, AT, and AO conceived and designed the experiments. VB, PG, and SB performed the experiments. VB, PG, MRC, AA, LMG, AT, and AO analyzed the data. PG, PAJ, X-GC, AA, OS, LMG, ARM, GG, AT, and AO contributed reagents/materials/analysis tools. VB, PG, GG, AT, and AO wrote the manuscript. All authors contributed to interpretation of data, reviewed and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Plasmodium falciparum. This antigenic protein, encoded by the merozoite surface protein (msp-3) gene, is polymorphic and classified according to size into the two allelic types of K1 and 3D7. A recent study revealed that both the K1 and 3D7 alleles co-circulated within P. falciparum populations in Thailand, but the extent of the sequence diversity and variation within each allelic type remains largely unknown.An effective malaria vaccine is an urgently needed tool to fight against human malaria, the most deadly parasitic disease of humans. One promising candidate is the merozoite surface protein-3 (MSP-3) of msp-3 gene was sequenced from 59 P. falciparum samples collected from five endemic areas  in Thailand and analysed for nucleotide sequence diversity, haplotype diversity and deduced amino acid sequence diversity. The gene was also subject to population genetic analysis (Fst) and neutrality tests  to determine any signature of selection.The P. falciparum populations in Asia.The sequence analyses revealed eight unique DNA haplotypes and seven amino acid sequence variants, with a haplotype and nucleotide diversity of 0.828 and 0.049, respectively. Neutrality tests indicated that the polymorphism detected in the alanine heptad repeat region of MSP-3 was maintained by positive diversifying selection, suggesting its role as a potential target of protective immune responses and supporting its role as a vaccine candidate. Comparison of MSP-3 variants among parasite populations in Thailand, India and Nigeria also inferred a close genetic relationship between msp-3 gene diversity in P. falciparum in Thailand, providing the fundamental basis for the better design of future blood stage malaria vaccines against P. falciparum.This study revealed the extent of the The online version of this article (doi:10.1186/s12936-016-1566-1) contains supplementary material, which is available to authorized users. Plasmodium species are considered an indispensible tool for malaria control and eradication. To date, RTS/S is the most advanced malaria vaccine against the human malaria parasite Plasmodium falciparum, the most deadly of the human malaria parasites. The RTS/S vaccine is made from the circumsporozoite protein (CSP), the essential antigen of the sporozoite, and induces immunity against the pre-erythrocytic stage of the malaria parasite. Although the vaccine has completed the pivotal Phase III clinical testing, and subsequently received a positive scientific recommendation from European Medical Agency for immunization of children against malaria in 2015, the efficacy of vaccine against clinical malaria was less than 50 % in children and infants  resulted in synonymous mutations at positions 76 (A), 82 (S), 122 (A) and 148 (K), while eight codons  caused non-synonymous amino acid substitutions at positions 83 (E/Q), 90 (N/K/E), 145 (K/T/S), 146 (S/A), 147 (S/L), 149 (A/T), 150 (D/S/N), and 151 (S/E/A/E), respectively, , was detected between the two haplotypes. This point mutation resulted in a non-synonymous amino acid change at amino acid position 103 (A/S), demonstrating the relatively low genetic diversity of msp-3 among 3D7 alleles. In contrast, a separate analysis of the 42 sequences with the K1 allele  revealed 51 SNP loci (excluding gaps), resulting in 40 non-synonymous mutations and seven synonymous mutations. This result, therefore, clearly depicts the different levels of genetic diversity between the K1 and 3D7 msp-3 alleles.Furthermore, when the 17 nucleotide sequences with the 3D7 allele (H1 and H2) of dN/dS ratio was calculated using the sequences of the 59 samples as described in Methods. When the nucleotide sequences encoding the AHR region (nucleotide positions 232–570) of MSP-3 were analysed, the dN/dS ratio was 1.08 (greater than 1) (P = 0.818), although the value did not show a significant positive departure from zero. Thus, there was a tendency for positive diversifying selection. Furthermore, the three within-population tests for neutrality  were all significant, with values of 2.611 (P < 0.01), 1.998 (P < 0.02) and 2.644 (P < 0.02), respectively. To determine whether specific region(s) of the AHR region was under selection, a sliding window plot analysis was performed. The values of D, D* and F* were calculated on a window of 60 bp moving in steps of three sites. As shown in Fig. D test, Fu and Li’s D* test and Fu and Li’s F* test a window size of 100 bases and a step size of 25 bases (default setting) were also analysed [msp-3 with significant values from the sliding window plots with a window size of 100 bases and a step size of 25 bases and a window size of 60 bases and a step size of 3 bases were largely overlapped , rather than alanine (A) in the ion Fig. a, similaP. falciparum populations in Thailand are shown in Fig. P. falciparum populations in Mae Hong Son and Trat, variant 3 appeared to be more prevalent than variants 7 and 1, with a frequency of 42 and 40 %, respectively. The other variants (2–5 and 6) were, however, minor variants, each of which was represented at less than 10 % in each population.The overall prevalence and distribution patterns of the seven MSP-3 variants in P. falciparum populations based on the MSP-3 variants. Those in Mae Hong Son, Kanchanaburi, Ranong, and Trat had five different variants. In contrast, only three variants were present in the Ubon Ratchatani population suggesting that the genetic diversity of P. falciparum in the Thai–Laos border was lower than in the Thai–Myanmar and Thai–Cambodia borders.In addition, Fig. P. falciparum populations in Thailand, pair-wise inter-population comparison were conducted for each population using Wright’s Fixation index (Fst). The Fst values were significant (P < 0.05) between Ubon Ratchatani and the Trat, Ranong and Mae Hong Son populations, but not between those at Ubon Ratchatani and Kanchanaburi   website  carried variants 3, 4, 6 and 7 of MSP-3, all of which were derived from the K1 allele. The results demonstrate the diversity of MSP-3 variants in laboratory clones.The nucleotide sequences of the msp-3 sequences of natural isolates of P. falciparum in Thailand, India and Nigeria were obtained through the literature search at the NCBI and Scopus websites  had the same seven MSP-3 sequence variants as in this study of Thai populations, and so all these variants were re-discovered and confirmed in the present study. The Fst analysis also showed that the P. falciparum population from Tak province was similar to P. falciparum in other regions in Thailand, except for Ubon Ratchanani, in which the 3D7 allele was the dominant type  had five of the seven MSP-3 variants found in Thailand. This broadly similar distribution pattern of MSP-3 variants between P. falciparum populations in South Asia and Southeast Asia supports the existence of gene flow between these parasite populations or that the parasite populations are closely genetically related. In contrast, nine variants of MSP-3 were identified in P. falciparum populations in Nigeria (n = 51), of which five  possessed the 3D7 allele [msp-3. These results may support the view that the 3D7 allele of msp-3 originates from introgression from another Plasmodium species, or it recently emerged from the K1 allele, or a recent introduction of 3D7 alleles to the endemic regions [In addition to the allelic dimorphism, polymorphisms were also detected within each of the K1 and 3D7 allelic types. A large number of polymorphic sites and indel mutations were detected among six haplotypes of the K1 alleles, while only a dimorphic site was found between two haplotypes of the 3D7 alleles. This indicates that the levels of genetic diversity within the K1-allelic type are much higher than those of the 3D7-allelic sequences, and is probably due to the greater prevalence of the K1 allele in parasite populations in Thailand. Interestingly, in 7 allele . The stu regions , 37, 62.The sequence analysis presented here may be useful in predicting target sites in MSP-3 for protective immunity. The data presented here suggest that the AHR in the N-terminal region of MSP-3 contains allelic-specific epitopes that are immunogenic than the conserved sequences. This notion is supported by the clinical immunological analysis of anti-MSP-3 antibodies in Gambian children, which showed a bias in reactivity towards either the K1 or 3D7 type recombinant protein , suggestmsp-3 gene could infer the population structure of P. falciparum in Thailand, where the distribution pattern of MSP-3 variants depicts a differential genetic diversity among the populations in Thailand. In contrast to a previous study, in which msp-3 alleles were classified into two types by PCR [msp-3 showed that there were as many as five variants of MSP-3 in all of the parasite populations examined except for that in Ubon Ratchatani. This result indicates that the levels of genetic diversity of P. falciparum near the Thailand–Myanmar and Thailand–Cambodia borders were greater than that at the Thailand–Laos border. The Fst index revealed that the P. falciparum population structure in Ubon Ratchatani was similar to that at Kanchanaburi, since the two populations shared the same three MSP-3 variants. This finding may suggest the origin and gene flow between the parasite populations in Thailand. The Fst analysis of MSP-3 variants in Thailand, India and Nigeria also indicated that P. falciparum populations in Thailand and India were more closely related than those parasites in Nigeria. This finding was also supported by the previous genome-wide studies that demonstrated population subdivision by continent in P. falciparum natural populations [P. falciparum populations in India and Nigeria, despite different geographical origins. If the full-length MSP-3 was developed as a vaccine, it would be beneficial to include both allelic forms, i.e., variants 1 and 7 as the representative allele of the 3D7 and K1 types in a vaccine formulation. Such a vaccine might improve the efficacy against clinical malaria without increasing the selection pressure on the locus.Last but not least, the sequence analysis of the s by PCR , the sequlations , 64. TheP. falciparum in Thailand. The nucleotide sequence analysis of msp-3 revealed that the polymorphisms that define allelic classes of msp-3 are attributed to the indel and SNPs in the gene. The sequence information also aids in identifying regions under positive selection, thereby providing knowledge for a better design of any MSP-3-based malaria vaccine, as well as revealing the population structure of P. falciparum across Thailand, which is of use for monitoring and control of this parasite.This study extends the understanding of genetic variation in the MSP-3 protein and prevalence of MSP-3 alleles in natural populations of


Bemisia tabaci sibling species (sibsp.) group comprises morphologically indiscernible lineages of well-known exemplars referred to as biotypes. It is distributed throughout tropical and subtropical latitudes and includes the contemporary invasive haplotypes, termed B and Q. Several well-studied B. tabaci biotypes exhibit ecological and biological diversity, however, most members are poorly studied or completely uncharacterized. Genetic studies have revealed substantial diversity within the group based on a fragment of the mitochondrial cytochrome oxidase I (mtCOI) sequence (haplotypes), with other tested markers being less useful for deep phylogenetic comparisons. The view of global relationships within the B. tabaci sibsp. group is largely derived from this single marker, making assessment of gene flow and genetic structure difficult at the population level. Here, the population structure was explored for B. tabaci in a global context using nuclear data from variable microsatellite markers. Worldwide collections were examined representing most of the available diversity, including known monophagous, polyphagous, invasive, and indigenous haplotypes. Well-characterized biotypes and other related geographic lineages discovered represented highly differentiated genetic clusters with little or no evidence of gene flow. The invasive B and Q biotypes exhibited moderate to high levels of genetic diversity, suggesting that they stemmed from large founding populations that have maintained ancestral variation, despite homogenizing effects, possibly due to human-mediated among-population gene flow. Results of the microsatellite analyses are in general agreement with published mtCOI phylogenies; however, notable conflicts exist between the nuclear and mitochondrial relationships, highlighting the need for a multifaceted approach to delineate the evolutionary history of the group. This study supports the hypothesis that the extant B. tabaci sibsp. group contains ancient genetic entities and highlights the vast cryptic diversity throughout the genome in the group.The whitefly  The evolutionary processes leading to speciation in ecologically and genetically divergent populations have attracted the interest of many –3, leadiThe view that cryptic lineages are the outcome of recent speciation events has been contested in light of studies suggesting ancestral divergence of morphologically cryptic lineages, in some cases dating to the Oligocene i.e. 24 million years ago  for which the taxon was first described as Aleyrodes tabaci in 1889 (see references in  against K, Pr (K) plateaued at K = 9 , it was possible to identify a number of clusters K by examining both the posterior probabilities of the data against K and the ΔK estimator. Results from these sub-structure runs revealed some interesting patterns  and Sudan Q-like populations were genetically distinct from all other clusters. Despite the well-defined structure in this plot, some admixture and migration was detected between the Western and Eastern African clusters.In the analysis of African populations, we excluded Mozambique  in order to use as many loci as possible since this population deviated from HWE in an additional two loci. Biotype S, a South African haplotype, and some Uganda cassava-colonizers formed a single cluster, populations from Burkina Faso and Sudan formed another , while Cameroon and Ivory Coast haplotypes grouped together as a separate cluster. The Ugandan-sweetpotato  and the Riverside A haplotype, with the former being most genetically similar to the Guatemalan population , and the second with the populations from western Mexico.Moderate structure was observed in the B biotype-closest relatives analysis. The Arizona B biotype was genetically different from the Egypt, Greece-1 and Cyprus-2006 populations, which collectively formed a single cluster, with only fractions of individuals’ genotypes sharing ancestry with Arizona B. The B biotype sister clade relatives from Israel and Panama (exotic introduction there) were admixed between the Arizona B (prototype) cluster and the B-like cluster consisting of Egypt, Greece, and Cyprus collections. Finally, the population from Yemen was genetically distinct from all B biotype populations.aps identified 28 genetic clusters worldwide, was generally in agreement with the Structure results when examined at the biotype/geographic region level (results not shown). Because in Baps we included all populations, this analysis also identified as genetically distinct the samples from Reunion Ms, Reunion Ms-2009, Cyprus-Ork, Moorea-FP, and Australia, which were excluded from the sub-structure analysis carried out using Structure because they were not associated with a larger geographic group.The results of the analysis using BΦPT estimate via AMOVA indicated that most of the genetic variance in both biotypes was significantly partitioned within populations with less variance among populations (P<0.0001). This was particularly the case for B biotype where 73% of variance was within populations  and for Western Mediterranean Q biotype with 80% of variance partitioned within populations  (Genetic differentiation within the two invasive biotypes B and Q based on the <0.0001) .B. tabaci invasive haplotypes that group within the B and Q sister clades.Estimates of genetic diversity, measured as allelic richness and heterozygosity showed variable patterns for different biotypes. The highest levels of allelic richness and heterozygosity were found in the populations from Sudan and Burkina Faso, and Yemen  and 2 thLevels of allelic richness and heterozygosity in the populations examined may have been influenced by two additional factors: the effects of inbreeding in samples obtained from lab colonies, and because microsatellite loci can be more variable in biotypes from which they were isolated , 96. In B. tabaci populations revealed large genetic distances at the worldwide level, and suggests that this taxon consists of very divergent cryptic lineages that represent geologically old entities , predating the divergence of most other clades/genetic lineages within the species group, in line with our results [The microsatellite analysis of ties see , 4 and 5ties see . Althoug results , 27.B. tabaci emerging from our results is that of the 13 loci we used, seven failed to amplify in samples from some populations. Non-amplification of microsatellite loci by PCR is most often caused by poor primer specificity due to mutations in regions flanking the microsatellite repeat sequence, resulting to what is known as “null alleles” [a priori observation is also in line with conclusions herein, because microsatellite loci that failed to amplify did so systematically within a biotype or geographic group, consistent with the presumed genetic relationships among populations, with non-amplifying loci occurring mostly in individuals of the most divergent lineages (e.g. New World) (FST) using corrected and uncorrected datasets were very similar. In addition, the NJ trees constructed based on Dc corrected for null alleles gave the same tree topologies as uncorrected trees, suggesting that null alleles had minimal impact in the analyses and did not affect the conclusions.Another indication of the extreme divergence in alleles” , 99. Altalleles” . This a w World) . DespiteB. tabaci sibsp. group currently are the invasive haplotypes that group in the clusters contained within the main B and Q biotype ‘sister’ clades. Some B-like haplotypes have expanded their geographic range to a worldwide scale in the past 30 years, while the Q-like invasive types have done so more recently [The most well-known biotypes in the recently –105. Manrecently , 106 areper se have yet to be determined; but the most likely explanation is that the direction of global trade of infested plant products has facilitated these invasions into locations having both a similar climate and susceptible plant host species. From this and previous studies [The factors that have favored the worldwide expansion of the B and Q biotypes  studies , 19, 30, studies , 89. Fur studies , 107–110 studies , 101 wheJatropha and closely related euphorbiaceous species in the Caribbean Islands. So, despite the genetic bottlenecks induced by founder effects in these invasive biotypes, genetic diversity remains high, possibly either due to persistent ancestral variation or due to continuous and repeated migrations from multiple diverse source populations from different regions, or both. This latter phenomenon has been observed in other organisms [B. tabaci it would be facilitated by human-mediated transport on whitefly-infested plants around the world, from multiple source populations originating in relatively close proximity to one another, the upper northwestern portion of Africa and the Middle East-Arabian Peninsula.Here, results indicated that the invasive biotypes B and Q did not have lower genetic diversity when compared to that of all other biotypes Figs  and 2, wrganisms , 112, anB. tabaci. Interestingly, an invasive Q1 population reported in China has displaced the previously invasive B biotype in at least certain locations, particularly in certain vegetable crops [Several important patterns of population differentiation were evident within invasive types of B and Q. Within the Q biotype there exists a large split in the Western and Eastern Mediterranean lineages that correspond to what are known as the Spanish-Q (Western) and the Israel-Q (Eastern) , 114 or le crops . To whatle crops ; they orle crops , 117, anB. tabaci in the US during 1987–88, based on a unique, homogeneous esterase pattern. This was followed by a number of apparently parallel introductions on ornamental plants and by its rapid spread throughout the Americas during the 1990’s-onward (colonizing poinsettia plants), and subsequently to Japan [Within the B biotype/haplotype collections examined there was high gene flow among populations except for Arizona B, which, perhaps surprisingly, was well differentiated from the others. The Arizona B (prototype B) originated from poinsettia plants that were subsequently reared as a lab colony in Arizona (JK Brown laboratory) when it was first recognized as an invasive to Japan , China (to Japan ) and elsto Japan . The difto Japan , a locatThe exact origins of the invasive B-like and Q-like biotypes have yet to be determined, but it has been suggested that the so-called Spanish Q biotype originates in southern Spain and has relatives in Northwest Africa and elsewhere in the Mediterranean and Middle East, while the B biotype diversified in the eastern African Sahel-Middle East (see references and data in ). ConsisB. tabaci sibsp. group represents an excellent system for studies of cryptic speciation where evolution has favored a wide array of genetically, ecologically, and biologically diverse lineages around the world. One application is in understanding differences between invasive and non-invasive biotypes, and how these have shaped their evolution and adaptation.The Euphorbia characias in a high altitude area [B. tabaci lineage, but is clearly genetically related to the Asian populations, which agrees with existing literature based on mtCOI [Rubus ulmifolius and grapevine, referred to as Ru, whose haplotypes formed a sister clade to the Asian and Australian groups, and the clade consisting of T biotype with which they shared 10.7% pairwise genetic distance. Clearly the T biotype and its relative genetic variants in the Mediterranean make a very interesting case that should be further explored. With appropriate genetic analysis of these and other historical samples it would be possible to date their divergence from other close relatives and to determine whether they represent introductions or remnants of ancestral widespread distribution in the Mediterranean region.The T biotype was first identified in Italy in 2003 colonizing only ude area , 88, 119on mtCOI , 24, 120on mtCOI  found anIpomoea indica in Spain in 1995 [Wolbachia or Cardinium endosymbionts, known to at times drive uni-directional gene flow between infected and uninfected populations [The S biotype, which was first described from the weed  in 1995 , 122 is  in 1995 , 123. Th in 1995 , 120, IT in 1995  as well  in 1995 . In that in 1995 . Althougulations –130. Theulations , biotypeB. tabaci populations.In comparing patterns of population structure and history inferred from microsatellite and mitochondrial DNA some general patterns have emerged. Overall, there was agreement in the major lineages/clades/biotypes identified from our microsatellite analysis with those obtained from mitochondrial DNA sequences in several studies , 20 and Wolbachia or Cardinium infections. Therefore, a mtDNA phylogeny would have erroneously assigned the French population to the Eastern Mediterranean Q subclade, ignoring the background nuclear gene flow and provide a misleading picture of the species and more recent population histories.Some discrepancies were observed however that suggest the cautious use of mitochondrial DNA as a single marker for delineating species boundaries, and further in describing new species in this group, despite the methodological efficiencies involved , 26, 49,Wolbachia, Cardinium, Rickettsia and other endosymbiont infections in B. tabaci [Similar patterns, with conflicting assignments of populations to the Eastern and Western Mediterranean Q subclades between the microsatellite and mitochondrial DNA were observed in other populations we examined (Q biotype-Greece and S biotype–Spain) indicating that this was not an isolated case . In fact. tabaci , 131–133. tabaci , 51, 115. tabaci .B. tabaci are discussed in Gueguen et al [The possible effects of endosymbiont composition in the evolution of the mtDNA lineages in en et al , who sugen et al . Furtheren et al , 35 may Another case of conflict between nuclear-mitochondrial data is in the genetic relationships among biotypes. For example, in this study the Ms biotype is more closely related to the B biotype-like subclade, including its relative from Yemen, based on the observation that the two biotypes shared more multilocus genotypes compared to either of the two with the Q biotype. This result is in agreement with that obtained for the nuclear ITS1 gene phylogeny  in whichB. tabaci entities (biotypes or haplotypes) represent a collection of very old lineages, perhaps isolated from each other for millions of years as has been suggested for other cryptic taxa. The analysis using nuclear microsatellite markers from populations worldwide confirms extreme levels of genetic differentiation in the nuclear genome. Although the pattern of genetic/geographic structure is generally consistent to that obtained from the mtCOI phylogeny, some examples were identified in which there is a strong conflict between the two markers. In this light caution should be taken when using mtDNA as a single marker for phylogenetic reconstruction and species characterization for B. tabaci. Analysis of multiple genetic markers from different parts of the genome are likely better able to aid in corroborating biological (phenotypic) differences in populations, provide information about the relative time of divergence among biotypes, and identify loci under selection. Thus, a multifaceted approach that incorporates biological, ecological, and behavioral studies, crossing experiments, and better knowledge of the role of endosymbiont contributions to the obstruction of gene flow, together with genetic information is needed to understand and determine species boundaries in this complex sibling species group.The results of this study demonstrate that despite their cryptic nature, S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S6 Fig(TIF)Click here for additional data file.S1 TableaSamples with the same number were collected from neighboring locations and were pooled together for analysis after exact tests of population differentiation showed non-significant differentiation. bPopulation names refer to the geographic sampling location and were given a biotype alphabetic code  if they originated from a reference collection previously characterized. cGenBank accession number for mtCOI sequence of one individual from each of the populations sampled in this study. dNumber of adult females genotyped. * Populations that were excluded from the PCA analysis.(XLSX)Click here for additional data file.S2 TableaNumber of alleles. b,cMean expected (HE) and observed (HO) heterozygosities (averaged across populations) (±SE). dInbreeding coefficient within individuals relative to the population (FIS). eNumber of populations that deviated from HWE in each locus out of a total of 41 populations(XLSX)Click here for additional data file.S3 TableaSamples collected from neighboring locations, which showed non-significant differentiation (exact tests of population differentiation) were pooled together, as shown in bNumber of different alleles over loci. c,dExpected (HE) and observed (HO) heterozygosities over loci. eMean fixation index (F) over loci (±SE). fEstimate of null allele frequency using the EM algorithm as described in Materials and Methods.(XLSX)Click here for additional data file.


Orestias (Cyprinodontidae) represents a good model to observe the genetic differentiation that characterizes an incipient speciation process in allopatry since the morphospecies described inhabit a restricted geographic area, with present habitat fragmentation. The genetic diversity and population structure of four endemic morphospecies of Orestias (Cyprinodontidae) found in the Lauca National Park (LNP) analyzed with mitochondrial markers (Control Region) and eight microsatellites, revealed the existence of genetic groups that matches the fragmentation of these systems. High values of genetic and phylogeographic differentiation indices were observed between Chungará Lake and Piacota lagoon. The group composed of the Lauca River, Copapujo and Chuviri wetlands sampling sites showed a clear signal of expansion, with a star-like haplotype network. Levels of genetic differentiation were lower than in Chungará and Piacota, suggesting that these localities would have differentiated after the bottlenecks linked to the collapse of Parinacota volcano. The Parinacota sample showed a population signal that differed from the other localities revealing greater genetic diversity and a disperse network, presenting haplotypes shared with other LNP localities. A mixing pattern of the different genetic groups was evident using the microsatellite markers. The chronology of the vicariance events in LNP may indicate that the partition process of the Orestias populations was gradual. Considering this, and in view of the genetic results, we may conclude that the morphospecies from LNP are populations in ongoing differentiation process.During the Pleistocene and Holocene, the southwest Andean Altiplano (17°-22°S) was affected by repeated fluctuations in water levels, high volcanic activity and major tectonic movements. In the early Holocene the humid Tauca phase shifted to the arid conditions that have lasted until the present, producing endorheic rivers, lakes, lagoons and wetlands. The endemic fish  Speciation is a continuous process during which differentiation and reproductive isolation are established . In alloFishes exhibit great morphological and ecological diversity, inhabiting very diverse aquatic habitats. These characteristics have made them a speciose and diversified group. They are used for the study of processes implicit in the generation of species ,17,18,19Orestias is a speciose group of biogeographic interest due to its endemism and adaptation to the aquatic systems of the Altiplano [Orestias extends from Lago Lascha (9°S) in the center of Perú to the Salar de Ascotán in northern Chile (22°S) [Similar to the cichlids in Africa and Central America, ltiplano ,25,26. Tltiplano ,28,29,30Orestias [The long-term climate variations in this region, which have included alternation of wet and dry periods, generated a succession of paleolakes that covered extensive areas and included several vast salt flats and localized and fragmented watersheds . SimultaOrestias ; in thisOrestias .O. chungarensis Vila & Pinto 1986 in Chungará lake, O. piacotensis Vila 2006 in the Piacota lagoon, O. parinacotensis Arratia 1982 in the Parinacota wetland and O. laucaensis Arratia 1982 in the Lauca River and the Cotacotani Lagoon , which is mostly above 4000 m elevation; the paleolakes did not cover this region. However, advances and retreats of glaciers and volcanic events produced changes in the landscape and fragmentation of the watersheds ,36,37,38i Lagoon .Orestias have been mainly focused on morphological analysis in the context of traditional systematics, initially associated with taxonomic descriptions [Orestias that inhabit the Chilean Altiplano using linear and meristic  measurements; their results showed that some nominal species in the LNP were differentiated morphologically, (O. laucaensis and O. chungarensis), although meristic data showed high overlap between nominal species and could not distinguish them. Recent trophic studies of Orestias in the LNP watersheds showed low prey richness and dietary range, which was related to the reduced size and shallow depth of these systems, with mostly microinvertebrates inhabiting the macrophyte belt. Thus, the local populations of Orestias showed similar dietary habits associated with prey availability [Studies of the species of riptions , 40, 41.riptions  comparedlability , 43.Orestias in the southern Altiplano may be linked to historical vicariant events and contemporary variation in water level [From a phylogenetic point of view, a previous study suggested that diversification of er level . In thiser level . A prelier level . In spitIn summary, the different analyses of the nominal species of the LNP have shown contrasting results in terms of their differentiation. Morphometric measurements and karyotypes have allowed distinguishing them as separate entities, while meristic measurements and molecular phylogeny have indicated that these nominal species do not constitute distinguishable groups. This discrepancy between different morphological and genetic approximations has been interpreted to be the result of incomplete sorting , 44, thaOrestias morphospecies described in a restricted geographic area, each one occupying a different body of water, would therefore reflect an incipient allopatric speciation process. Under this hypothesis, we would expect to find genetic differentiation between the different bodies of water, at least for the most isolated ones, and an incomplete lineage sorting due to the short time elapsed since the fragmentation process.In the present study, we hypothesize that all four morphospecies would have been formerly part of a unique Lauca River basin population that was fragmented in different bodies of water due to an intense volcanic activity during Holocene time. The presence of four Orestias were described. These locations represent the diverse aquatic systems shaped by the fragmentation of the Lauca River (The samples (n = 270) were collected in eight localities of the Lauca National Park (LNP) including the sites where the four morphospecies of ca River , S1 File5’ ACC CCT AAC TCC CAA AGC T 3’) (Reverse 5’ TGA TAG TAA AGT CAG GAC CAA 3’) [2 50 mM, each primer 10 pm/μl, dNTP 2.5 mM, 0.5 μl Taq (Invitrogen), 9 μl ultrapure water, 1 μl DMSO and 10 ng/μl DNA.The control region of mitochondrial DNA (D-loop) was amplified by PCR using specific primers for the genus: (Forward  CAA 3’) . The PCRThe PCR cycle consisted of an initial denaturation for 5 min at 94°C, followed by 38 cycles of 45 sec at 94°C, 90 sec at 66°C and 90 sec at 72°C, with a final extension of 10 min at 72°C. PCR products were visualized in 2% agarose gel stained with ethidium bromide. Amplification products were sent for sequencing to Macrogen Inc. (South Korea). D-loop sequences were aligned, edited and assembled using the Proseq v.2.9.1 program . Using tST, based on the differences in haplotype frequencies and ΦST, based on the number of differences between pairs of sequences, with the Arlequin v.3.5.1.3 program [ST and ΦST was based on 10000 permutations; we used the false discovery rate [ST and NST among groups. NST takes into account the genetic distance between haplotypes, while GST only considers the difference in frequency. NST significantly greater than GST suggests phylogeographic structure. We performed 1000 permutations to determine its significance; if more than 5% of the permuted values of NST are greater than GST the null hypothesis GST = NST is rejected.The genetic structure as a function of the geographic component was analyzed using SAMOVA , which identifies the groups of genetically homogeneous and geographically close localities that maximize the variance of genetic diversity among these groups . To esti program . The staery rate  correctiery rate  to calcuWe performed the Tajima  and Fu  tests inτ = 2tμ, which is the time when growth began measured in mutational time. t is the time in generations and μ is the mutation rate per sequence per generation. The initial value of Theta is given by θ0 = 2Niμ where Ne0 is the initial effective population size estimated before population growth, while the final value is assumed to be infinite, θ1 = ∞, according with the Sudden demographic expansion model of Schneider & Excoffier [We used three different models to evaluate past demographic changes. First, we constructed the mismatch distribution for each group, and when pertinent we estimated the demographic parameters of the expansion using the Sudden demographic expansion model of Schneider & Excoffier  with thexcoffier .θt = θ0egt−, where θt is the value of θ at time t and θ0 is the current value of θ. Using these parameter s we estimated the time since the beginning of demographic expansion in units of mutational time. The time of the initiation of expansion was estimated as the moment when θt was 1% of θ0, as recommended by the author of the program [Second, we reconstructed the demographic history of each genetic group using the LAMARC 2.1.5 program . This pr program .Third, the demographic history of the groups was inferred using a Bayesian Skyline Plot (BSP) analysis with the BEAST program version 1.7.1 , which aThe only study for the estimation of freshwater fish mutations rates including time dependency in molecular rates was introduced by Ho (2005). He dates changes in river drainages and isolation of fish populations to document rates of mitochondrial DNA change over a range of temporal scales. He utilizes precise spatio-temporal disruptions of linear freshwater systems and hence avoids many of the limitations associated with typical DNA calibration methods involving fossil data.We used a mutation rate of 6% per million years proposed by Burridge et al. ; their sO. agassii of Bolivia by Esquer-Garrigos et al. [2 25 mM, BSA 0,1mg/μl, primers 50 pm/μl, dNTP 2.5 mM, 0.1 μl Taq (Promega Inc.) and 50 ng/μl DNA. The PCR conditions were: initial denaturation for 5 min at 94°C, followed by 35 cycles of 30 sec at 92°C, 30 sec at the locus-specific aligning temperature (T° a)  with individual genotype data using the software STRUCTURE V.2.0 . The proWe obtained a total of 270 samples in the eight sampling sites representing 890 nucleotide positions (GenBank Accesion Numbers BankIt1931003:KX498091-KX498358). The descriptive indices of genetic diversity showed 42 polymorphic sites (S); the number of haplotypes was K = 51; the haplotype diversity was Hd = 0.747 and the mean number of differences between pairs of sequences was Π = 1.593 . The samST and FST among groups were low but significant for the majority of the comparisons (The SAMOVA showed that the variation between groups was maximized with four groups (59.92%); the corresponding variance between sample within groups was low (2.14%) but significant. The four genetic groups in the LNP were: Group 1: Chungará; Group 2: Parinacota; Group 3: Piacota and Group 4: Lauca, Misituni, Cotacotani, Copapujo and Chuviri (Table B in parisons .The structure index Nst (0.576) based on the number of differences between pairs of sequences was significantly greater than Gst (0.462) calculated from the haplotype frequencies (p = 0.0034), indicating the existence of a phylogeographic structure among the groups of the LNP.e0 = 0 that indicate the reduced initial effective population size in the locality , the Lauca group  began from 8000–4300 YBP with effective sizes of one or two orders of magnitude lower in Piacota   indicated that the growth of the populations of Chungará (Ne= 60000) .The Bayesian skyline plots for the genetic groups also showed patterns of population growth and differences in expansion times. The expansion began approximately 5000 YBP in Parinacota, with an increase of population size of 40000 and a mean of 100000 individuals ; the expOrestias the samples for 8 loci  for any locus in any population . Intermediate values of heterozygosity (0.47–0.60) were found for all populations; Chungará had the greatest mean heterozygosity (0.60) and Piacota had the lowest (0.47).The descriptive indices of genetic diversity of the 8 microsatellite loci are shown in Individual assignment (GeneClass) to the populations delimited by pairwise Fst analysis showed that the greatest percentage of correct assignments was found in the Chungará and Piacota populations, with 100% and 96%, respectively. In the CCC and Lauca populations the correct assignments were 75% and 76%, respectively; the incorrectly assigned individuals of these two populations were assigned to the other population. Parinacota had the lowest percentage of correct assignments (68%); individuals incorrectly assigned were mainly assigned to CCC (12%) and Lauca group (12%).The STRUCTURE analysis found the lowest value of LnP for K = 4 , thus inO. chungarensis in Lake Chungará and O. piacotensis in Piacota lagoon.Our results showed the existence of various genetic groups in the localities of the LNP that coincide with the fragmentation of the aquatic systems due to the volcanic and climate events that originated closed systems such as Lago Chungará and the Piacota lagoon , 38. GenOrestias (S3 Table), thus genetic drift may have acted more strongly, retaining a smaller number of new haplotypes. This difference in diversity between lakes of different sizes has also been documented in cichlids in the region of Lake Victoria in Africa [The populations of these two localities show signs of growth after 12000 YBP, which may be related to the main volcanic events that occurred in the Pleistocene-Holocene boundary, which originated these lentic systems. The formation of the Chungará subwatershed (12000 YBP) as a product of the collapse of the Parinacota volcano isolated Lago Chungará from the Lauca watershed, which has probably lasted until the present. The low genetic variation of Chungará population, in particular the low number of differences between haplotypes, may indicate a founder effect during the separation and later appearance of new haplotypes during population growth in this large lake. The closed system of the Piacota lagoon has been disconnected from the Parinacota wetland during the arid period of the mid-Holocene (8000–4000 YBP). The small size of the lagoon would have limited the population size of n Africa , 23 and n Africa . In thesn Africa , 19.The analysis of mitochondrial sequences identified a third group including the two sites of the Lauca river with those of Cotacotani lagoon and the wetlands of Copapujo and Chuviri. In the same way as the previous groups, this group shows a clear sign of expansion illustrated by a star-like haplotype network. During the volcanic processes (12000–4000 YBP) the Lauca river was probably affected by landslides from the collapse of the volcano dome, the pyroclastic rain, lahars and inundations, which may have generated population bottlenecks as has been reported for recent eruptions in other parts of Chile . The anaOrestias have remained in situ in the wetlands that were separated from the river [The population of Parinacota showed a different population signal than the rest of the localities. Based on mitochondrial sequences showed greater genetic diversity and a disperse network with the presence of low-frequency haplotypes , severalhe river , safeguahe river , the ParThe results based on the microsatellite markers identified Parinacota as a singular population in terms of its genetic diversity. However, in contrast to the mtDNA information that identified Parinacota as a source population for the historical colonization of the other sites, microsatellites markers indicated that Parinacota would correspond currently to a point of admixture of the different genetic groups of the LNP . In the Orestias was stepwise. First, the volcanic collapse of Parinacota (12500 YBP) [O. laucaensis (Lauca) and O. chungarensis (Chungará) apparently occurred. Volcanic eruptions processes in the Holocene (8000–4000 YBP) maintained the populations separated, facilitating genetic differentiation between them. During this time there was also a drastic climate change with the arid episode (7000–6000 YBP), in which the connections between the rivers, wetlands and lagoons would have been affected. This is the case of the Piacota lagoon, which was probably separated from the Parinacota wetland by the decrease in water between them and also the eruption of the Ajata volcano, leaving the Piacota lagoon with an isolated population described as O. piacotensis. Finally, the return of wet periods after 4000 YBP allowed the re-connection of the Cotacotani lagoons with the Lauca river, which is shown by the presence of O. laucaensis in the different systems as one genetic group.The chronology of the vicariance events in the LNP determined that the separation of the populations of 500 YBP) –38 wouldOrestias in the LNP are illustrating an uncertain phase of speciation in which sudden changes in the environmental conditions, in this case mainly hydrographic, could reinforce or revert this speciation process, indicating the reticulate nature of evolution [Orestias from the LNP appear as an ideal model to exemplify the processes of genetic, morphological, ecological differentiation that accompany an incipient geographic isolation, and thus understand incipient steps of the allopatric speciation process.The combination of the chronology of the vicariance processes in the LNP and the genetic analyses allows us to conclude that although the localities of the LNP analyzed are not evolutionarily separated units, they are groups in the process of differentiation. The short time since the events that originated this process has not allowed a complete lineage sorting among these populations in spite of the morphological differences that have been described and that motivated the description of different nominal species. In spite of the genetic and phylogeographic differentiation detected in this study, up to now there is no evidence of reproductive isolation among the groups and they do not appear to have developed ecological specialization, at least not at the trophic level . There avolution . In thisS1 FileGoogle Earth Placemark file that contains latitudinal and longitudinal coordinates of our sampling sites.(ZIP)Click here for additional data file.S2 FileModified from Esquer et al. 2011. T°a: annealing temperature (°C) (Table A). SAMOVA analysis Spatial molecular analysis of variance (SAMOVA) of D-loop sequences in different geographic groups (Table B). TMRCA. Time to most recent common ancestor using Bayesian skyline analysis for the three genetic groups with expansion signal (Table C). FIS-values for eight microsatellite loci in populations of Orestias of the Lauca National Park (Table D). Genotypes of each sample in each localities of LNP (Table E).(DOCX)Click here for additional data file.


PPARGC1A, in particular the Gly482Ser variant (rs8192678), had been proposed to be subject to natural selection, particularly in recent progenitors of extant Polynesian populations. Reasons include high levels of population differentiation and increased frequencies of the derived type 2 diabetes (T2D) risk 482Ser allele, and association with body mass index (BMI) in a small Tongan population. However, no direct statistical tests for selection have been applied.The gene FST, integrated haplotype score (iHS), cross population extended haplotype homozygosity (XP-EHH), Tajima’s D and Fay and Wu’s H) were conducted on the PPARGC1A locus.Using a range of Polynesian populations  we re-examined evidence for association between Gly482Ser with T2D and BMI as well as gout. Using also Asian, European, and African 1000 Genome Project samples a range of statistical tests for selection (FST) was smallest between Asian and Pacific populations . When compared to European  or African populations  this differentiation was larger. We did not find any strong evidence for departure from neutral evolution at this locus when applying any of the other statistical tests for selection. However, using the same analytical methods, we found evidence for selection in specific populations at previously identified loci, indicating that lack of selection was the most likely explanation for the lack of evidence of selection in PPARGC1A.No statistically significant evidence for association between Gly482Ser and any of BMI, T2D or gout was found. Population differentiation  contains supplementary material, which is available to authorized users. The thrifty gene theory is based on the hypothesis that alleles causing increased weight, insulin resistance and type 2 diabetes (T2D) in contemporary populations were an adaptation to fluctuating food availability , for exaPPARGC1A transcriptional regulator has a central role in insulin signalling and mitochondrial regulation [rs8192678) variant (reviewed in [PPARGC1A gene is associated with body mass index (BMI) in a Tongan (Polynesian) population [FST values (a measure of inter-population difference at specific genetic variants) at the PPARGC1A locus [The gulation . The funiewed in ) in the pulation . This lepulation . This 481A locus . To datePPARGC1A gene were associated with weight and subject to selection in Pacific populations, then this would be important knowledge on the aetiology of these conditions in contemporary populations. Furthermore, evidence for selection during Polynesian population history would aid in the destigmatisation of obesity. Here, we tested for association between Gly482Ser rs8192678, BMI, T2D and gout in several Polynesian populations. We also examined evidence for recent positive selection at the derived 482Ser allele and in the region of PPARGC1A, by applying a recently developed analytical pipeline [The Māori and non-indigenous Polynesian populations of Aotearoa New Zealand (NZ) have a high prevalence of obesity, T2D and related metabolic conditions such as gout –8. The rpipeline  to genomNew Zealand (NZ) Māori, Cook Island Māori, Samoan, Tongan and NZ Caucasian sample sets were examined Table . SamplesFive additional populations were obtained from the 1000 Genomes Project phase 3 data release (see ): two AsPPARGC1A gene (dbSNP ID rs8192678 residing on chromosome 4 (GRCh37 chr4:23815662) and genotyped on a subset of the above populations (see Table Two genotypic data sets were used for this study: (a) a SNP-specific assay corresponding to a Glycine  -Serine  substitution at amino acid position 482 in the To determine associations between BMI and rs8192678 genotype, single marker linear regressions were undertaken using the R statistical software environment . Hardy-WFST [FST between sample sets, Tajima’s D, Fay and Wu’s H, and integrated haplotype score  for indre  to estire  previously reported by Voight et al.  as showiTests for association between BMI and rs8192678 genotype did not reveal any statistically significant association for any of the gene action models Table . SimilarD and Fay and Wu’s H for windows surrounding the Gly482Ser position, were within +/− 2.5% limits for all populations and window lengths  statistics for rs8192678 for each population using a genetic map. The iHS is a statistic to detect evidence of recent positive selection at a locus and is based on the differential levels of linkage disequilibrium surrounding a positively selected allele compared to the other allele at the same position. Only the Samoan population had |iHS| > 3.29 when computed using a genetic map   in specific ancestries. Here, there were 14 combinations of genes and populations  and NZ Māori and Tongan (0.069) populations greater than NZ Māori and CI Māori (0.000). The Samoan and Tongan populations had relatively small differentiation between them (FST = 0.003). Population differentiation was also estimated for the Gly482Ser variants in the genome-wide data sets  extent of population differentiation  or indirectly  to identify a potential functional role of Gly482Ser. To identify genomic evidence we used a recently-developed analytical pipeline to test selection by a combination of site frequency spectra based statistics,  as well as haplotype-length based measures that examine selection within populations (iHS) or between populations , which has also been used as an indicator of selection [FST only – no other statistics indicated consistent functional or genomic evidence for selection. However, reasons other than selection such as local co-ancestry can lead to outlier values for FST [FST results alone are not sufficient to conclude the presence of selection.The primary aim of this study was to directly test the hypothesis that the i et al.  as ‘A co. We alselection , as wellfor FST  thereforde novo mutation with strong positive effects on fitness, and ‘soft sweeps’ based on extant variants underpinning heritable characteristics that are typically under polygenic control. Power , above) to detect evidence of selection for the various methods used here differs depending upon these scenarios [a de novo mutation arising prior to ancestors of modern Homo sapiens migrating out Africa. On the other hand however, there is a relative dearth of evidence of hard sweeps in humans [PPARGC1A were indeed under selection, it would have been subject to concomitant selection in multiple Polynesian and other populations  the nature of positive selection being investigated, and (2) power of each of the statistical tests to reject the null hypothesis of no departure from neutral model of selection. Regarding (1), two intra-specific selection scenarios were frequently examined: ‘hard sweeps’ within populations based on selective advantages arising from a cenarios . For thecenarios  imply sen humans . In addis  and acts  allele frequencies for hard sweeps. In hard sweeps where ending allele frequencies were similar to those observed in Māori populations  ≈ 0.75 probability of at least one of the Samoan and Māori populations used in this study to show evidence of selection using the iHS test. However the (case–control) Samoan and Māori cohorts had (only) one and zero SNPs, respectively, exceeding the 1% iHS threshold. We also contend that, if anything, the use of a case–control design would be more likely to increase the frequency of haplotypes carrying the selected allele(s), and thus more likely to improve power should this locus be subject to selection. Therefore the lack of a statistically significant difference in 482Ser allele frequencies in the case control Māori cohort used in this study and the previously reported estimate by Myles et al. [To provide further insights into whether or not the lack of evidence of selection according to these statistics was due to inadequate sample sizes, we also analysed 14 population/gene combinations that had previously been identified as showing evidence of selection . In our PPARGC1A were a thrifty gene candidate that has been subjected to natural selection, selection would likely have occurred in all non-African ancestral populations due to factors such as climatic extremes, competition with other human groups, and/or the need to adapt to newly colonised environments - all of which are likely to contribute to periodic deprivations in food availability in progenitors of all populations studied.A further consideration is the elevated derived 482Ser allele frequency in all non-African populations compared to the African populations . These sD nor Fay and Wu’s H. Similarly, for the NZ Māori population, evidence of possible differential selection indicated by Tajima’s D in the PPARGC1A-encoding region exceeding the lower 97.5% quantile, , (b) later migration out of Africa by Homo sapiens progenitors of Polynesians subsequent to the mutation giving rise to the 482Ser allele, which drifted to increasingly higher frequencies in repeated migrations across the Pacific, and (c) followed by subsequent admixture between these Melanesian progenitors with ancestors of modern Polynesians [Existing 482Ser allele frequency distributions in the Pacific can be explained by a combination of (a) migration out of Africa by ynesians . The hign = 184, P = 0.014–0.037), but are consistent with the lack of association in a NZ Māori tribe  that was also reported by Myles et al. [Single marker regression analyses did not reveal any evidence of association between rs8192678 and BMI, gout affection status, or T2D in any of the populations investigated. Although each Polynesian population was small and likely to be underpowered for validating associations between small effect genes and conditions, no associations were found even when Polynesian populations were combined. We also found no evidence for sex-specific effects for any of the three traits in the combined Polynesian populations (data not shown). Our results contrast with those of Myles et al.  who repoPPARGC1A region in general. More extreme frequencies of the derived allele in the Polynesian populations can alternatively be explained by genetic drift associated with ancestral population bottlenecks during colonisation. Further, the lack of robust direct candidate gene-based evidence for the thrifty gene hypothesis in light of increasing evidence for selective influence of infectious disease-causing agents on genome composition [Based on the lack of evidence of association between the Gly482Ser variant with BMI and correlated diseases, as well as lack of evidence of association with any of the tests for selection, we conclude that this study does not support natural selection at either the Gly482Ser variant, or the position  and otheposition .


Georychus capensis). By adopting a range-wide sampling regime and employing two variable mitochondrial markers (cytochrome b and control region), we report on the effects that life-history, population demography and geographic barriers had in shaping genetic variation and population genetic patterns in G. capensis. We also compare our results to information available for the sister taxon of the study species, Bathyergus suillus. Our results show that Georychus capensis exhibits low genetic diversity relative to the concomitantly distributed B. suillus, most likely due to differences in habitat specificity, habitat fragmentation and historical population declines. In addition, the isolated nature of G. capensis populations and low levels of population connectivity has led to small effective population sizes and genetic differentiation, possibly aided by genetic drift. Not surprisingly therefore, G. capensis exhibits pronounced spatial structure across its range in South Africa. Along with geographic distance and demography, other factors shaping the genetic structure of G. capensis include the historical and contemporary impacts of mountains, rivers, sea-level fluctuations and elevation. Given the isolation and differentiation among G. capensis populations, the monotypic genus Georychus may represent a species complex.The subterranean niche harbours animals with extreme adaptations. These adaptations decrease the vagility of taxa and, along with other behavioural adaptations, often result in isolated populations characterized by small effective population sizes, high inbreeding, population bottlenecks, genetic drift and consequently, high spatial genetic structure. Although information is available for some species, estimates of genetic diversity and whether this variation is spatially structured, is lacking for the Cape mole-rat ( The life history of a species along with its habitat specificity, habitat matrix and the spatial and temporal variation in geological and climatic factors through evolutionary time, shape genetic patterns and consequently drives diversification and speciation  document genetic diversity at the population level as well as across the range of G. capensis, 2) investigate demographic stability (both within populations and across the range), 3) test whether genetic isolation is present across the disjunct distribution, 4) compare our results to a previous study on Bathyergus suillus  baited with peanut butter inside the burrow systems. The trap works in a similar way to the Victor Easy Set or Macabee traps, however uses a more powerful torsion mechanism which draws the animal into a pivot point at the front of the trap and applies ample power and pressure to quickly, consistently and humanely sacrifice rodents in the body mass- and size class of G. capensis (200 mm long and 400 g in mass). Collection of specimens was performed under ethical clearance from the Ethics Committee of the University of Johannesburg (Ethics number 215086650-10/09/15). Traps were checked every hour and the sacrificed animals were removed and immediately frozen at -10°C.calities  across ib gene  using a commercial DNA extraction kit  following the manufacturer's protocol. Two mitochondrial DNA fragments were targeted during PCR amplifications and sequencing using universal primers: 976 bp of the protein coding cytochrome  H15915; , 49) and and E3; ).b and 48°C for the control region). Amplicons were sequenced using BigDye chemistry following the protocols outlined in Jansen van Vuuren and Chown [PCR amplifications followed standard protocols (see ). Amplifnd Chown . Electrob accession numbers: MG496663—MG496927; control region accession numbers: MG496398—MG496662) as well as in concert (1819 bp); the results were largely congruent between fragments and the concatenated segments. Summary statistics  for each population was calculated in Arlequin version 3.5 [The two mitochondrial fragments were analysed separately  together. To assess the impact of range fragmentation on the spatial genetic diversity, we divided the samples into three groups . Finally, we assessed whether genetic diversity was significantly structured across the Western Cape region . Additionally, pairwise ФST values were calculated between all sampled localities. Significance for these tests were determined through 9 999 permutations of the data  all sampled localities . Isolation 3.5; . GeograpGeorychus, clustering analyses were performed in BAPS version 6.0 [A haplotype network was built using TCS 1.21 ; this nesion 6.0 , 59, 60 ST values) were sourced from a previous study on B. suillus [B. suillus sequence datasets. For both G. capensis and B. suillus, comparative statistics were calculated separately for both the control region and cytochrome b datasets  all sampled localities across the distribution of G. capensis  and 2) only including localities across the Western Cape Province . In addition, genetic structure within the two genera was only compared across their distributions in the Western Cape region. Statistical comparisons between values were performed using a non-parametric Mann-Whitney U test as implemented in IBM SPSS Statistics version 20.0.0 .Summary diversity indices (haplotype diversity and nucleotide diversity), population demography (Fu’s Fs) and population differentiation (pairwise Ф suillus  for whicced from ) as wellGeorychus individuals, 55 haplotypes were identified from the combined dataset , with only 14% of the variation situated within populations. When three groups were specified in the AMOVA analysis , 47% of the variation was partitioned among these groups, with 44% of the variation among populations within each group, and only 8% of the variation partitioned within these populations. In the Western Cape region, a similar scenario was found with 83% of the variation among populations with only 16% of the variation accounted for by within-population variation . Although there was significant isolation-by-distance in the Western Cape region , several barriers to gene flow were detected across the range of G. capensis  and across both the spatial scales  only for the cytochrome b dataset, with non-significant values for the control region and combined data  based on all datasets  and regional (across the entire distribution) scales. Indeed, G. capensis populations show significant declines in population size compared to B. suillus irrespective of the spatial scale considered. The overall population decline in the Cape mole-rat is in line with the fossil record , which suggests that Georychus was once more widely distributed [G. capensis populations. Isolation and fragmentation of this habitat may likely be attributed to several climatic oscillations which brought cooler and more arid climates during the Quaternary [Compared to tributed , 63, 64.tributed , 64, 65 tributed  resultinaternary , 68, 69.Spalax ehrenbergi [T. romana [Georychus is associated with mesic conditions and drainage systems . Lower G. capensis populations. Georychus populations are characterized by strong genetic structure across the distribution. Habitat specificity and a low dispersal capability may be the main factors driving spatial genetic structure. Suitable habitat is disjunctly distributed across the species’ range, and populations are spatially highly fragmented. Indeed, it was noted that G. capensis is less widespread and populations much more isolated compared to other South African bathyergid counterparts such as B. suillus and Cryptomys hottentotus [Bathyergus suillus occupies deep sandy soils [B. suillus is able to utilize mesic areas (with deep soils), thereby resulting in a range overlap with G. capensis. Given its more generalist ecology, B. suillus exhibits much larger and aggregated populations with a continuous distribution along the coastal regions of the Western Cape . Bathyergus suillus therefore maintains much larger female effective population sizes . Small effective population sizes are evident in all G. capensis populations , therefore representing larger numbers of unrelated mating individuals  and higher levels of genetic diversity . This tius , elevatG. capensis seems to be that of a physical barrier having a channelling effect; in B. suillus, mountains form a barrier between populations [The action of mountains on gene-flow in ulations . Within B. suillus). In addition, the action of the Breede River and Gourits River (barrier B on B. suillus [The isolation of Cluster 3 (the Struisbaai population) may be a result of isolation through marine fluctuations in sea-level which covered the Agulhas Plain , sea-level fluctuations and elevation along with biogeographic differences act in concert with contractions of the distribution of G. capensis populations along with possible adaptive differences across the distribution , 9089, 9ion (see ), it is S1 TableGeorychus populations as well as the B. suillus populations in Visser et al. [b and control region datasets separately, as well as for the combined dataset of these markers. An “a” indicates where reliable results could not be obtained due to too few samples from that population.Θ values Click here for additional data file.S2 TableG. capensis populations showing the haplotype diversity, nucleotide diversity and Fu’s F values in each population for the cytochrome b/control region datasets. For the Fu’s F values n.s. = non-significant,* = p<0.05, ** = p<0.01, *** = p<0.001. An indication is given where the analysis could not be performed due to “a” too few samples from that population and “b” all individuals within that population having the same haplotype.Genetic diversity of the sampled (DOCX)Click here for additional data file.S3 TableST values between the sampled G. capensis populations with values based on cytochrome b below the diagonal and values based on the control region above the diagonal. n.s. = non-significant,* = p<0.05, ** = p<0.01, *** = p<0.001.Pairwise ɸ(DOCX)Click here for additional data file.S4 TableB. suillus populations in [b/control region datasets. For the Fu’s F values n.s. = non-significant,* = p<0.05, ** = p<0.01, *** = p<0.001.Genetic diversity of the tions in  populati(DOCX)Click here for additional data file.S5 TableST values between the sampled B. suillus populations in [b below the diagonal and values based on the control region above the diagonal. n.s. = non-significant,* = p<0.05, ** = p<0.01, *** = p<0.001.Pairwise ɸtions in  with val(DOCX)Click here for additional data file.S1 FigG. capensis populations based on the A) cytochrome b dataset, B) control region dataset and C) combined (cytochrome b control region) dataset.Plot showing the Θ values for the sampled (TIF)Click here for additional data file.