Aspidiotus rigidus Reyne (Hemiptera: Diaspididae) are inflicting significant economic losses to the Philippines’ coconut industry, little is known about the population and dispersal history of this invasive pest in the country. Here, we examined the genetic diversity, structure and demographic history of A. rigidus sampled from localities with reported outbreaks from 2014 to 2017. We analyzed the genetic structure of the outbreak populations using mitochondrial COI and nuclear EF-1α markers. Both markers and all methods of population genetic structure analyses indicate clear differentiation among the A. rigidus populations separating the north from the southern regions of the Philippines. Very low or no genetic differentiation was observed within and amongst the populations per geographic region indicating two unrelated outbreak events of the pest originating from two genetically uniform populations isolated in each respective region. Historical data supports the resurgence of an established A. rigidus population in the south which could have been driven by sudden climatic changes or human-induced habitat imbalance. Our study provides valuable information on the genetic differentiation of the two A. rigidus groups that would be useful for developing and implementing biological control strategies against this pest in the Philippines.Despite the fact that massive outbreaks of the coconut scale insect pest,  Insect pest outbreaks are characterized by an explosive increase in an insect population occurring over a relatively short period . Large aAspidiotus rigidus Reyne (Hemiptera: Diaspididae) resides on the underside of the leaf, blocks the stomata and sucks plant sap strongly reducing the plant’s photosynthetic activity leading to a characteristic yellowing and drying of the leaves. Severely infested coconut palms dry up and die within six months or less [A. rigidus due to the outbreak in the Philippines, historical and observational data on the spread of the invasive coconut pest has been scarce. Other than the most recent observation published by Watson et al. [A. rigidus population may have been associated with natural enemies that, over time, regulated the pest population. However, the outbreaks from the localities infested with A. rigidus in the Philippines took longer to recover [In 2009, the Philippines suffered a devastating coconut scale insect outbreak damaging the coconut palms on the provinces of Luzon (northern region of the Philippines) and currently on some areas in Mindanao (southern region) inflicting significant economic losses to the country’s coconut industry. The diaspidid scale insect n et al.  the lastn et al.  with ful recover , e.g., sA. rigidus to the Philippines, and its spread was believed to be either by wind or accidental transportation of infested plants, coconut planting materials and products [The introduction of products . Infestaproducts  and wereproducts . The morproducts , implyinproducts , it is hproducts ,13.A. rigidus has been in the country as a minor pest and regulated by natural enemies. Based on historical reports, Lever [A. rigidus in the Philippines as long ago as 1969, and Velasquez [A. rigidus came from the island of Sangi in Indonesia given its relative closeness to Mindanao. In time, the immigrant A. rigidus could have established a resident population complemented by natural enemies limiting its colonization outside the area of introduction. Changes in anthropogenic, biotic interactions or climatic factors can influence a population’s rise to an outbreak level [A. rigidus population due to factors such as human-induced habitat imbalance e.g., excessive use of pesticide affecting the natural enemies controlling the pest population, or climate change such as a prolonged dry spell, which may induce changes in the local biotic community.It is also likely that s, Lever  reportedelasquez  recounteak level ,16. The A. rigidus in the Philippines needs further assessment and confirmation. Tracing the history of an invasion or identifying the geographic origin of a pest population can be done by characterizing population-level genetic variation using molecular markers e.g., [Inference of the source population, route and the mechanism of spread of rs e.g., ,18,19,20rs e.g., ,22, or irs e.g., ,24 of vaA. rigidus in the Philippines. Given the historical documentation of the pest in the southern region and the relatively extensive and rapid spread, but faster recovery, of the infestations in the northern region compared to the southern outbreaks i.e., Basilan and Zamboanga Peninsula, we hypothesize the presence of two distinct genetic groups for the outbreak events isolated within each geographic region. A population genetics approach is a useful tool to examine whether the northern and southern coconut scale insect outbreaks originated from immigrant or resident populations. To test this hypothesis, we utilized sequences of the mtCOI gene and the nuclear protein-encoding gene - EF-1α to investigate the genetic structure and diversity of A. rigidus populations from localities with documented outbreak-level infestations in the Philippines from 2014 to 2017. Furthermore, we employed a coalescent genealogy approach to provide additional evidence on the demographic relationship of the outbreak A. rigidus populations between the northern and southern geographic regions in the Philippines.Here, we aimed to assess the population genetic structure and demography of the outbreak populations of the coconut scale insect, Aspidiotus rigidus populations were sampled at seven localities with reported coconut scale insect outbreak across the Philippines from 2014 to 2017  located at De La Salle University (DLSU), Science and Technology Complex  on mangosteen (Garcinia mangostana L.), a differential host of A. rigidus observed not to support multiple generations of A. destructor. A phylogenetic analysis was employed  to confirm the identifications of the field-collected samples by comparing them to A. destructor and mangosteen-reared A. rigidus sequences.Mature female scale insects were identified as ect body . Non-par2, 10 pmol of each primer, 1 U of Taq DNA polymerase , and 2 to 50 ng of template DNA. PCR thermocycling was performed in a T100™ Thermal Cycler . Following the conditions from Park et al. [Genomic DNA was extracted individually using the DNeasy Blood & Tissue Kit  following the manufacturer’s guidelines. Extraction was performed by crushing the insect body of each individual in separate microcentrifuge tubes using a micropestle. DNA concentration and quality were assessed by spectrophotometry . The mitochondrial COI gene was amplified using the forward primer PcoF1 designed for scale insects by Park et al.  and the k et al. , the mtCk et al.  in whichS) and haplotypes (h), average number of nucleotide difference (k), haplotype diversity (Hd), and nucleotide diversity (Pi) of the two marker genes were calculated in DnaSP v. 6.10.04 [STF was computed in Arlequin v. 3.5.2.2 using 1000 permutations.Sequences were assembled using CodonCode Aligner v. 5.1.5 . Concatenated sequences were deposited in GenBank with the following accession numbers: MK862684-MK862988 for mtCOI and MN603810-MN603884 for EF-1α before the subsequent molecular analysis, the sequences were aligned via MAFFT v. 7.409 . Sequenc 6.10.04 . The hie 6.10.04 . Two geoD and Fu’s Fs statistic tests were estimated to infer demographic history and dynamics in each population, for the two geographic groups, and for all populations grouped together in Arlequin v. 3.5.2.2 for both mtCOI and EF-1α datasets. Also, Fu and Li’s D* and F* test statistics were computed in DnaSP v. 5.0 to determine departures from the mutation-drift equilibrium [0θ), and effective population size after expansion  between the observed and expected mismatches. The adjustment to a model of population expansion was estimated from the sum of squared deviation (SSD) and the raggedness index (r) in Arlequin v. 3.5.2.2.Tajima’s ilibrium . ParametTo test migration history between the two geographic groups, we calculated Bayes factors from the marginal likelihoods estimated in MIGRATE v. 4.4.0 ,35 basedBF” implemented in carlopacioni/mtraceR, a package for analyzing migrate-n outputs in R v. 3.5.2. Log Bayes factors (LBF) of all models were calculated by comparing against the model that has the highest log-likelihood. The models are ranked based on LBF and calculated model probability.A Bayesian search strategy was performed with the following parameters: one long chain  with a burn-in of 5000 iterations. A static heating scheme with 4 chains was applied using temperature parameters set by default with a swapping interval of one. Bayes factors were calculated via “A. rigidus haplotypes via PopART v. 1.7 [Median-joining (MJ) networks  for the T v. 1.7 .A. destructor collected from coconut palms, identified based on the circular distribution of egg skins were selected as an outgroup (accession number: MK862989 for mtCOI and MN603885 for EF-1α). The Akaike information criterion corrected for sample size (AICc) was implemented to find the best fitting evolutionary model for phylogenetic reconstruction via jModelTest v. 2.1.10 [Since no site variation was observed between the sequences in each population (except for the EF-1α sequences from Basilan), six representative samples per population, a total of 48 mtCOI sequences along with 42 plus all 16 Basilan EF-1α sequences were chosen for the phylogenetic analysis. Sequences of . 2.1.10  with 100A. rigidus by comparing the sequences with the mangosteen-reared A. rigidus, and A. destructor sequences. DNA sequence analysis of all the concatenated 647-bp mtCOI sequences of 305 individuals from seven A. rigidus outbreak populations collected from 2014 to 2017 in the Philippines, with the mangosteen-reared samples revealed only two distinct haplotypes (h), separated by 31 polymorphic sites (s) (Hd) was calculated to be 0.50 +/− 0.005 SD. Average number of nucleotide difference (k) was 15.5 and nucleotide diversity (Pi) was 0.024 +/− 0.00025 SD. No variation was found in the sequences of samples collected per populations. Reared A. rigidus and samples collected from the five populations of northern group, i.e., Orani, Bataan (BT), Nagcarlan (NG) and San Pablo (SP), Laguna, Tanauan (TN) and Talisay (TL), Batangas are grouped into one haplotype. Samples from the two populations of southern group, i.e., Basilan (BS) and Zamboanga (ZB) were grouped together in the second mtCOI haplotype (s), with four haplotypes (h). Like the mtCOI sequences, all samples from the northern group clustered into one haplotype. For the southern group, samples from ZB grouped into one haplotype, while BS were separated into two haplotypes. Haplotype diversity (Hd) was calculated to be 0.48 +/− 0.064 SD. Average number of nucleotide difference (k) was 5.7 and nucleotide diversity (Pi) was 0.0057 +/− 0.00061 SD. Except for BS, all other localities have no sequence variation per locality.All samples identified based on the characteristic distribution of egg skins were confirmed as ites (s) . Haplotyaplotype . For theHd of 0.688 +/− 0.039 SD, with a k value of 0.087 and a low Pi value of 0.00086 +/− 0.00010  and a non-significant r of 0.2871. Other demographic parameters such as Tau, 0θ and 1θ index, are presented in For both datasets, neutrality test computation for all samples showed positive values for Tajima’s rameters . EstimatResults of the analysis in migrate-n were presented in A. rigidus from selected localities in the Philippines with reported heavy infestations collected from 2014 to 2017. Both the mtCOI and EF-1α markers and all methods of population structure analyses revealed strong differentiation among the A. rigidus populations separating the northern (Luzon) outbreak from the southern (Mindanao) region. The separation of the populations by geographic region and the observed lack of genetic variability within populations were represented graphically in the median-joining network and phylogenetic analysis employed in the study . Genetic population clusters result from multiple source populations contributing to an insect pest outbreak . Howeverrclones”  in the PAspidiotus nerii Bouche [A. rigidus was observed to reproduce parthenogenetically [Acyrthosiphon pisum Harris was the main reason influencing the demographic success of the pest in central Chile [Tuta absoluta Meyrick (Lepidoptera: Gelechiidae) spreading through South America and ultimately reaching the Mediterranean Basin, with only a single genetic type. Likewise, a well-established invasive population of the oleander aphid, Aphis nerii Fonscolombe were reported as having extremely low genetic diversity in the southern United States, with a “superclone” population supposedly obligatorily asexual [Nematus oligospilus Forster  dominating willows in three countries in the southern hemisphere i.e., South Africa, New Zealand, and Australia.Armoured diaspid scale insects were observed to have unusual genetic systems such as diverse types of paternal genome elimination and parthenogenesis . Specifii Bouche ,22. Accoetically ,10. Yelletically . Parthenetically ,42. As eetically . A highlal Chile . Cifuental Chile  found no asexual . Caron e asexual  reportedA. destructor rigidus (now A. rigidus) reported by Reyne [A. rigidus in Tanauan, Batangas, Luzon was in 2009 [Based on historical reports, Lever  claimed by Reyne  in Indonby Reyne  did not  in 2009  with no A Bayesian search strategy was performed to assess the migration history between the northern and southern populations. However, our results from the mitochondrial and nuclear datasets are difficult to reconcile. We reiterate that in this historic gene-flow analysis, the two groups were assumed to have reached mutation-migration-drift equilibrium. Despite the contrasting results, both models indicate that sequences from the two groups do not belong to one panmictic population. Even so, given the difference in the divergence or migration pattern of the models for each marker, we were unable to unequivocally infer the source of each outbreak population. Methodological assumptions  in the pOn the other hand, lower genetic variation is expected for younger populations due to founder effects and genetic bottlenecks during colonization and establishment . InvasivA. rigidus population was relatively older than the northern one. Alongside previous historical reports, the level of genetic variation between the geographic regions supports our hypothesis of an existing resident A. rigidus population in the southern part of the Philippines. Local insect populations have the potential to outbreak due to anthropogenic and environmental changes [Stenotus rubrovittatus Matsumura (Heteroptera: Miridae) in Japan were induced by changes in the agro-ecosystem without invasion of populations from other areas. Populations of the pest were also genetically isolated by distance and separated into genetic clusters occupying spatially segregated regions. Additionally, temporal fluctuations of pest insects in agroecosystems could be driven by various factors [Dendroctonus simplex LeConte (Coleoptera: Curculionidae) on Tamarack (Larix laricina (Du Roi) K. Koch) in Minnesota, USA are climate-driven, being specifically associated with warmer and dryer years, more likely in areas with prior defoliation. Schwartzberg et al. [Malacosoma disstria Hübner (Lepidoptera: Lasiocampidae) in relation to the phenology of its host trees. These findings illustrate the mechanisms by which anthropogenic and climatic changes induce outbreaks from native insect pests.Genetic variation was already low amongst the southern populations for the nuclear DNA, but in comparison to the uniformly genetic northern population , our fin changes ,3. Simil changes  reveals  factors . Pestici factors ,53. Weat factors  reportedg et al.  simulateComperiella calauanica Barrion, Almarinez, Amalin (Hymenoptera: Encyrtidae) parasitizing A. rigidus infesting the coconut palms in a forest-based resort in Calauan, Laguna  with no history of chemical insecticide applications. The parasitoid has been observed to be host-specific, with up to 80% parasitisation rate. The use of C. calauanica for biological control implementations through conservation and augmentation in the field have been conducted in the Philippines [The information regarding the amount of genetic variation in pest species could be useful because insects with reduced genetic variation have been reported to be more easily controlled than those with more genetic diversity . For inslippines ,59. UtilA. rigidus from other countries of native range and spread via wind dispersal or importation of infested planting material from the northern region to the south given the timeline of the outbreak reports. However, our results strongly suggest the separation of two distinct groups, the northern and southern A. rigidus from the outbreak populations collected from 2014 to 2017 in the Philippines. Very low or no genetic differentiation was observed within and amongst the populations per geographic region, indicating two unrelated outbreak events of the pest species originating from two genetically uniform or “superclone” populations currently isolated in each respective region. Historical data supports our assumption on the current resurgence of an established A. rigidus population in the south. Given the lack of historical information supporting the existence of an established A. rigidus populations in the northern region, we disregard the possible resurgence of a persisting native population and suggest that the outbreak possibly resulted from a recent introduction of a non-native population. Assessment of the possible source population of the northern outbreaks needs further exploration.The current opinion about the origin of the coconut scale insect outbreak in the Philippines was a recent introduction of A. rigidus populations. Other robust and more informative genetic markers such as microsatellites could provide further genetic information during study of the invasive coconut scale population. Further studies should also include more expansive sampling, taking into consideration other possible sources of A. rigidus such as Indonesia [A. rigidus in the Philippines. Nevertheless, our findings provided an initial important genetic basis and information for designing and implementing biological control strategies against the invasive coconut scale insect pest A. rigidus in the Philippines.The use of mtCOI and the nuclear EF-1α markers showed no or very low genetic differentiation for all ndonesia  and Vietndonesia . This wo


Platycephalus are economically important demersal fishes that widely inhabit the continental shelves of tropical and temperate sea waters. This genus has a long history of taxonomic revision, and recently four Platycephalus species  in the northwestern Pacific Ocean (NWP) have been recognized and redescribed. However, many aspects of their systematics and evolutionary history are unclear.Flathead fishes of the genus Platycephalus species in the NWP with well-supported clades in which Platycephalus sp. 1 and Platycephalus sp. 2 were the closest, clustered with P. cultellatus, while their genetic relationship with P. indicus was the furthest. There were significant genealogical branches corresponding to P. indicus but not to other Platycephalus lineages. We further examined the phylogeographic patterns of 16 Platycephalus sp. 1 populations along the coastlines of China and Japan. A total of 69 haplotypes were obtained, with 23 shared among populations. One dominant haplotypic group, with a modest lineage structure and low levels of haplotype diversity and nucleotide diversity, was observed among Platycephalus sp. 1 populations. The demographic history reconstruction suggested a Platycephalus sp. 1 population expansion event dating back to the late Pleistocene.A total of 411 individuals were sampled from 22 different sites across their distributions in the NWP. Three mitochondrial loci were sequenced to clarify the phylogeny and phylogeographic history of the fishes. The results showed significant differentiation of four Platycephalus. Reproductive schooling and potential egg/larval dispersal ability, coupled with the effects of ocean currents, are responsible for the present phylogeographic pattern of Platycephalus sp. 1.Distributional rang variations may be the crucial factors shaping the genetic relationships of the genus The online version of this article (10.1186/s12862-019-1477-1) contains supplementary material, which is available to authorized users. In marine environments, the phylogeography of organisms may be shaped by complex interactions of extrinsic ecological factors  and intrinsic life-history characteristics  , 2. MoreDuring glacial cycles, continental shelves became alternately exposed and submerged under sea water, driving demographic extinction or expansion in numerous marine species . In the Platycephalus, belonging to Family Platycephalidae, Order Scorpaeniformes, are widely distributed in tropical and temperate areas of the Indo-Western Pacific (IWP) and eastern Mediterranean [Platycephalus with COI barcoding by Puckridge et al. [P. indicus with eight separated lineages distributed across large expanses of the IWP. The possibility of further cryptic speciation in this and other apparently widely distributed flatheads in the IWP needs to be considered.Flathead fishes of genus erranean –17. Theyerranean . They weerranean . The taxerranean –17, 19. erranean . For exaPlatycephalus, P. indicus, recorded and studied in the NWP [Platycephalus sp. 1 in China and Platycephalus sp. 2 in Japan were documented based on morphological characters and DNA barcoding, proving the validity of these species at the genetic level [P. cultellatus and P. indicus were also redescribed from the northern South China Sea [Platycephalus species distributed in the NWP are very similar in morphological and meristic values, with almost all characters overlapping [Platycephalus species in the NWP by regional ichthyologists. Given the glacial cycles mentioned above and the recent anthropogenic pressures, the phylogeographic history of the four Platycephalus species recorded in the NWP remains unclear. Moreover, no systematic research has been performed to study the population genetics of the newly recognized Platycephalus sp. 1, which is the only species widely distributed along Chinese coastal seas.There has long been only a single species of  the NWP , 22. Recic level –24. Thishina Sea . All fourlapping , 22, 25.Platycephalus were sampled along the coasts of China and Japan, as well as from extra-IWP reference locations. The specimens were identified as four species to investigate the phylogeny and evolutionary history of the genus Platycephalus. The phylogenetic relationships were reconstructed with multiple mitochondrial loci. These data were also used to characterize the population structure of the Platycephalus sp. 1 throughout its range. The population history of Platycephalus sp. 1 was inferred, and the results were used to illuminate how this species responded to the severe climatic fluctuations in the Pleistocene ice ages. Such information may assist in fisheries and ecosystem conservation and investigations of cryptic speciation in the genus Platycephalus in the NWP.In the present study, populations of the genus COI sequences and 402-bp Cyt b sequences from 151 specimens of 22 populations of the four Platycephalus species  of 0.91 ± 0.02. These haplotypes were not shared among species. The numbers of haplotypes observed within each species were 30 for Platycephalus sp. 1, 16 for P. indicus, six for P. cultellatus, and five for Platycephalus sp. 2. Moreover, P. indicus exhibited the highest level of haplotype diversity (h = 0.90 ± 0.03), and P. cultellatus exhibited the lowest haplotype diversity (h = 0.60 ± 0.15), with Platycephalus sp. 1 (h = 0.76 ± 0.05) and Platycephalus sp. 2 (h = 0.71 ± 0.14) in between. The most common haplotype in Platycephalus sp. 1 (P1SH1) was observed in 46.6% of the 16 sampled Platycephalus sp. 1 populations, and most of the other haplotypes (72.4%) exhibited only one nucleotide difference.We obtained 651-bp COI and Cyt b sequences were used to infer the phylogenetic relationships of the sampled Platycephalus species. The concatenated tree topologies from the Bayesian and ML phylogenies were generally congruent .The haplotypes from concatenated ent Fig. a revealeCOI sequences from other Platycephalus species were utilized to assess the phylogeny and taxonomy of the genus Platycephalus; 510-bp COI sequences were collected from GenBank and this study , covering samples from Korea, Rongcheng (China), Taiwan (China), and India. Moreover, 12 of the 67 sequences actually belonged to P. cultellatus (haplotypes PCSH1 and PCBH3) from the South China Sea, and one sequence belonged to Platycephalus sp. 2 from Japan (haplotype P2SH1)  and east of Java (haplotype PV5) were more closely related to the population from Okinawa, and they further clustered with populations from the northern South China Sea, while the P. indicus samples from Australia, the Persian Gulf, and the Mediterranean were grouped as their own distinct clusters  was quantified as corrected average pairwise differences, ΦST values, and AMOVA test results. Except for that between the BA and KU populations from the Persian Gulf, other pairwise values all exhibited large and significant divergence among populations from the South China Sea, Okinawa, and the Persian Gulf, with ΦST values ranging from 0.7794 to 0.9495 , with 93.3% of the total genetic variation partitioned among groups. There was a high and significant ΦST  for P. indicus populations. Scatter plotting from a Mantel test indicated a significant relationship (P = 0.0398) between FST/(1-FST) and geographic distance among the four P. indicus sample sites . The results indicated distance-related genetic differentiation among P. indicus populations. Considering the large geographic distance between populations as well as the limited sampling size for P. indicus, more samples across their intervening range are required to assess geographic patterns of genetic variation among P. indicus populations over their geographical expanse, i.e., whether gradual or with breaks at critical geographic sites.The genetic relationships among the four 95 Table A. An AMOions n = 8 was quaPlatycephalus sp. 1 in the NWP, we amplified the mtDNA control region of additional specimens (n = 348) sampled from 15 Chinese coastal populations and one extra Tokyo Bay population from Japan , and the Xiamen population exhibited the highest level of nucleotide diversity (π = 0.0082 ± 0.0048). The Tokyo Bay population represented both the lowest haplotype diversity (h = 0.26 ± 0.12) and the lowest nucleotide diversity (π = 0.0012 ± 0.0011). Both the corrected average pairwise differences and the ΦST values between the Chinese coastal populations were low, nonsignificant and partially negative  to 0.626 (Weihai) (P < 0.05) , which indicated that there was only a modest population structure among populations, with 91.6% of the total genetic variation apportioned within Platycephalus sp. 1 populations. When we took the Tokyo Bay population as one group and the Chinese coastal sea populations as another group, there was a significant genetic structure difference between groups , with 37.9% of total genetic variation appointed between groups. A total of 61.2% of the total genetic variation was appointed within populations. Moreover, when we further split the Chinese coastal sea populations into the Bohai and Yellow Sea group, the East China Sea group, and the South China Sea group based on potential geographic isolation features, there was a lower, but significant ΦCT value , with only 11.1% of total genetic variation appointed among groups. The constructed group with the Tokyo Bay population splitting off was identified as the optimal group due to the highest significant ΦCT value (Table P > 0.05) between FST/(1-FST) and the geographic distance among the Platycephalus sp. 1 populations .One-group AMOVA revealed a low but significant ΦPlatycephalus sp. 1 populations also was demonstrated by the haplotype distribution network  and Fs  statistics, which indicated a recent population expansion event. Moreover, the sequence mismatch distribution analysis illustrated a unimodal, smooth distribution pattern, which did not differ significantly from the pure demographic and spatial expansion model with a sum-of-squares deviation of 0.0015. This result suggested a typical expansion event in Platycephalus sp. 1 populations , as well as the approximate nucleotide substitution rate (3.0–10.0% / Myr) [Platycephalus sp. 1 populations along the coastlines of the NWP could be dated to approximately 30,800 to 103,400 years ago.A test of the demographic history of rate 3.0–.0% / Myr% / Myr) , the demPlatycephalus sp. 1  have recently been recognized and redescribed in the NWP [Platycephalus within the NWP. Phylogenetic analysis revealed significant differentiation among four Platycephalus species in the NWP. Variation of mtDNA sequences showed significant genealogical structure differences among P. indicus populations across a large expanse, but this variation was not observed among populations of the other three species. There was genetic homogeneity among most Platycephalus sp. 1 populations along the coast of China, while significant differentiation was observed between populations of China and Japan. The inferred demographic history of Platycephalus sp. 1 suggested a population expansion event tracing back to the late Pleistocene.In addition to their center of diversity in Australia –17, 19,  the NWP –23, 25. P. indicus, was recognized in the NWP, while Platycephalus sp. 1 and Platycephalus sp. 2 were only recently distinguished from P. indicus in China and Japan, with strong evidence for species validity at the genetic level [Platycephalus species recognized in the NWP have overlapping or similar morphological metrics, which resulted in frequent species misidentification, as shown by the few Platycephalus sequences in GenBank having been misidentified as P. indicus  to the South China Sea (Beihai), whereas Platycephalus sp. 2 was geographically restricted to the coastal seas of Japan. This finding was consistent with those of reports from Japanese ichthyologists [P. indicus and P. cultellatus in China were confined to only the northern South China Sea. The abundant distribution of Platycephalus sp. 1 and the infrequent presence of P. indicus and P. cultellatus along the Chinese coast sharply contrasted, which suggested that the latter two species might prefer warmer water. The phylogeny was very shallow, with no clear phylogeographic patterns for Platycephalus sp. 1, Platycephalus sp. 2, and P. cultellatus populations in the NWP. However, a significant lineage structure related to geographical distribution was observed for P. indicus populations of Okinawa, Beihai, and the Persian Gulf , P. indicus (Japan), P. cf. indicus (Hongkong), and P. indicus (Persian Gulf), which may indicate cryptic speciation. Regarding results of the Puckridge et al. study [Platycephalus sp. 1 and Platycephalus sp. 2 may correspond to the P. indicus (China/Korea) and P. indicus (Japan) lineages, respectively. Analysis of more samples across the intervening range is warranted to assess whether there might be gradual variation in P. indicus over a geographical expanse.The phylogenetic relationships revealed distinct genetic structural differences among the sampled NWP Fig. . Platyceologists , 24, 27 ulf Fig. a. This sons Fig. a, and thus study  in whichl. study , the newPlatycephalus is widely distributed in tropical and temperate areas of the IWP. This distribution suggests that the lineage could have originated in the tropical region, then dispersed north and became isolated in the NWP during glacial cycles. During the last glacial maximum (LGM), various refugia emerged, and geographic marine obstacles contributed to genetic isolation, which drove the differentiation that resulted in lineage structure divergence related to geographic distribution. For example, P. cultellatus was sampled only in the northern South China Sea, whose glacial refuge was the central South China Sea during the LGM. After the LGM, seasonal changes in the coastal currents in the northern South China Sea and the long developmental period of pelagic larvae contributed to the dispersion of the species.Geographic boundaries formed during the Pleistocene ice age have also played an important role in species dispersion. The genus Platycephalus species in the NWP. Intrinsic life-history divergence may also have contributed to speciation processes, and hence further investigation into the life-history characteristics of these species is warranted.In summary, distributional range variations, probably resulting from the divergence of thermal adaptation and the natural history effects of glaciation, may be the crucial drivers that shaped the genetic relationships of Platycephalus sp. 1 populations  across most of its distribution, as well as a shallow star-shaped haplotype network   Table B. Platycuveniles . The larg season . AccordiPlatycephalus eggs or larvae could be transported northward during summer from the South China Sea to the East China Sea and the Yellow Sea by the Taiwan Warm Current and the Yellow Sea Warm Current [Platycephalus sp. 1 can travel long distances despite their limited adult dispersal capabilities. Several other fish species from Chinese seas, such as Liza affinis and Sillago japonica, with pelagic early-life stages, also could be mixed by ocean currents and present genetic homogeneity among Chinese coastal populations [Platycephalus sp. 1 populations of the Chinese coast. The Tokyo Bay population apparently receives limited recruitment from the Yellow Sea and the East China Sea, likely due to the isolation by the Japanese landmass as well as the strong Kuroshio Current and Tsushima Warm Current . The samples were collected by trawl net in offshore waters from local fishermen or from fish market of local fishing ground, thus ensuring that the samples collected were representative of the local populations. Detailed information about the locations and numbers of specimens collected is listed in Additional file Specimens of the genus ns Table : PlatyceCR), cytochrome b (Cyt b), and cytochrome c oxidase I (COI). PCR was carried out in 25-μl reaction volumes with the primers listed in Additional file Genomic DNA was extracted from muscle tissue by a standard proteinase K digestion and phenol-chloroform extraction method . PCR amph), and nucleotide diversity (π), were obtained in ARLEQUIN [Sequence concatenations and file format conversions were implemented in Sequence Matrix . SequencARLEQUIN .CR and in PartitionFinder 2 [COI and Cyt b sequences. Protein-encoding sequences of COI and Cyt b were partitioned based on three codon positions, and the best codon substitution model was determined. To select the optimal model parameters and the best phylogenetic tree, all partition schemes were compared with the likelihood ratio test [Various partition methods were employed to find the best-fit combination of substitution model parameters for the phylogenetic analysis. The best-fitting nucleotide substitution model was determined using the Bayesian approach implemented in jModelTest  for CR aFinder 2  for COI tio test  for the tio test , 57 in Mtio test ; the anaPlatycephalus species. If the sampled specimens were fewer than six at a certain location, then all individuals were included. For phylogenetic analysis, 1) concatenated COI and Cyt b sequences of 151 specimens from populations of the four Platycephalus species were analyzed, and 2) homologous COI regions for other Platycephalus species were searched for using BLAST and downloaded from GenBank (https://www.ncbi.nlm.nih.gov/), excised and included in the combined COI phylogeny. The species and their COI-sequence GenBank accession numbers are listed in Additional file Cociella crocodile were used as an outgroup.To construct phylogenies based on the mtDNA sequences, six individuals were selected from each population of the sampled CR were analyzed for P. indicus and Platycephalus sp. 1, but not for P. cultellatus and Platycephalus sp. 1 due to their limited sample sizes. Pairwise population differences within P. indicus and Platycephalus sp. 1 populations were estimated with ΦST, and corrected average pairwise differences were calculated in ARLEQUIN. The statistical significance of these parameters was tested as table-wide significance (P < 0.05) with 10,000 permutations. Population structure was evaluated by a hierarchical analysis of molecular variance (AMOVA) [Platycephalus sp. 1 populations. One group was designated with all Platycephalus sp. 1 populations. Two groups  and four groups  were designated based on potential geographic isolation factors, such as the Tsushima Strait, Yangtze River estuary, and Taiwan Strait  were designated based on their geographic distance. Genetic split between populations was identified by finding the largest significant values of ΦCT among population groups. Haplotype networks were generally better than phylogenetic trees to describe population relationships [Population structures based on the  (AMOVA)  using 10ionships , which wionships .FST/(1-FST) were plotted against geographical distance between sample sites of Platycephalus sp. 1 , while pairwise values of FST/(1-FST) were plotted against the logarithm of distance between sample sites of P. indicus  considering the wide distribution range of P. indicus. The strength and significance of the relationship between genetic distances and geographic distances was assessed using reduced major axis (RMA) regression and Mantel tests using the software ARLEQUIN.To test for isolation by distance, pairwise values of Platycephalus sp. 1 populations. The neutrality mutation test was calculated using Tajima’s D [FS [Ƭ = 2 μt, where μ is the mutation rate [The historical demographic pattern was examined for jima’s D  and Fu’s’s D [FS , with si’s D [FS  or spati’s D [FS  expansio’s D [FS , and theion rate .Ne) across time and to create Bayesian skyline plots [BEAST v.1.7.4 was employed to estimate the changes in effective population size Additional file 2:Figure S1. Phylogenetic relationships of the genus Platycephalus based on concatenated COI and Cyt b sequences. Partitioned maximum likelihood tree and codon maximum likelihood tree are on top and bottom, respectively. (TIF 1372 kb)Additional file 3:Table S2.COI sequences of the Platycephalus from GenBank. (DOCX 44 kb)Additional file 4:Figure S2. Phylogenetic relationships of the genus Platycephalus based on COI sequences from this study and from GenBank. Bayesian inference tree, partitioned maximum likelihood tree, and codon maximum likelihood tree are from left to right. (TIF 1953 kb)Additional file 5:Table S3. Distribution of haplotypes shared among populations of the Platycephalus sp. 1. (DOCX 17 kb)Additional file 6:Table S4. Comparison of selected morphological characters of four Platycephalus species from Chinese coastal seas and Pacific coast of Japan, as found in bibliography. (DOCX 20 kb)Additional file 7:Table S5. Primers information of mitochondrial DNA for the Platycephalus. (DOCX 19 kb)


Rhopalosiphum padi (Linnaeus) is a major pest of cereals and pasture grasses worldwide. Here, we report the genetic features of populations of R. padi that colonize different cereal crops in central Chile.Aphids (Hemiptera: Aphididae) are insects with one of the highest potentials for invasion. Several aphid species are present globally due to introduction events; they represent important pests of agroecosystems. The bird cherry-oat aphid Rhopalosiphum padi individuals were collected in central Chile and genotyped at six microsatellite loci. The most frequent multilocus genotype (MLG) was then studied further to assess its reproductive performance across cereal hosts under laboratory conditions.R. padi in Chile are characterized by a low clonal diversity (G/N = 62/377 = 0.16) and the overrepresentation of a few widely distributed MLGs. One of the MLGs constituted roughly half of the sample and was observed in all sampled populations at high frequencies. Furthermore, this putative aphid “superclone” exhibited variations in its reproductive performance on cereals most commonly cultivated in Chile. The sampled populations also exhibited weak signs of genetic differentiation among hosts and localities. Our findings suggest that (1) obligate parthenogenesis is the primary reproductive mode of R. padi in Chile in the sampled range and (2) its introduction involved the arrival of a few genotypes that multiplied asexually.Populations of  Worldwide, roughly 30% of crops are lost due to pests and pathogens. Worryingly, these yield losses are expected to increase due to global warming, particularly for major grain crops  in temperate regions . Insect Aphids (Hemiptera: Aphididae) are insects with one of the highest potentials for invasion. Several pest aphids are present globally due to introduction events, and they accordingly represent important pests in agroecosystems worldwide. The biological features that explain the aggressiveness and invasiveness of aphids include: (i) parthenogenetic reproduction that enables rapid growth of the population, (ii) the global availability of cultivated and wild hosts, and (iii) their ample phenotypic plasticity in response to changing environments .Rhopalosiphum padi (Linnaeus) is a major pest of cereal crops worldwide , particularly in important areas of wheat production in Asia where this aphid has rapidly become the most frequent cereal pest  obligate parthenogenesis, with only asexual lineages produced all year on Poacea, and (3) androcyclic, with several parthenogenetic generations on Poacea followed by the production of only sexual males in the autumn. As described for several pest aphids, the predominance of a certain reproductive mode in R. padi relies on geographic and climatic factors  cyc factors . For exaen found . While cen found .Understanding the genetic features of pest populations can help inform decisions about appropriate pest-management strategies. By using high-resolution molecular markers , it is possible to determine basic aspects of pest biology and anticipate their evolutionary responses . SpeciesR. padi in the primary cereal-growing region of Chile. By sampling R. padi individuals from main cereals and genotyping with six microsatellite loci, we showed that these populations are characterized by low genetic diversity, strong indications of the presence of obligate parthenogenetic as a unique reproductive mode, and the dominance of one single genotype with features of a superclone. We also investigated this single genotype in the laboratory to determine whether this genotype, collected from different locations and host plants, exhibited phenotypic plasticity in reproduction across different cereal hosts. Our results may facilitate effective implementation of pest-management strategies of invasive pests, particularly those with predominant clonal reproduction.Here, we studied genetic diversity in populations of Rhopalosiphum padi individuals were collected from 17 different cereal fields located in central Chile , barley (Hordeum vulgare), oat (Avena sativa), and maize (Zea mays). Aphids were collected from colonies growing on a single host plant (one single leaf per plant). Samples were separated by at least 10 m from the next sampled plant, thus limiting the chance of taking individuals from the same parthenogenetic colony. Samples were gently taken with a paintbrush and stored in tubes of 1.7 mL filled with absolute ethanol. They all were identified under a binocular microscope following taxonomic keys in the laboratory . All aphid colonies were reared in a system composed by potted plants enclosed in a plastic-transparent cylinder covered with mesh cloth fabric on top that facilitate air circulation and avoid aphid from escaping. Each lineage was maintained in triplicated in these plastic cylinders inside closed acrylic cages (50 × 40 × 30 cm). Aphids were then left to multiply by parthenogenesis, as they were reared under conditions that ensure their asexual reproduction. These units were maintained under controlled environmental conditions in a 4.0 × 5.0 m growth chambers . Standard fluorescent tubes combined with 600W high pressure sodium lamp were used to ensure constant radiation. For the study of the performance of the most common genotype (see below), clonal lineages of these genotype were maintained separately in the laboratory according to the samples site, generating different clonal lineages of the same genotype, which were compared in terms of the reproductive performed across different cereal hosts.al Chile . Samplinal Chile . The samboratory . This wa sampled . Only onR1.35, R5.10, R2.73, R5.29b, R6.3, R3.171, R5.138, and R5.50 were tested  protocol was set-up according to ++ free reaction buffer, 50 mM of MgCl2, 0.5 U of Platinum Taq DNA Polymerase  five U/μL, and three to six ng/μL of DNA. Dyes were assigned to each locus in a way that allowed us to amplify all six loci in only three multiplex PCRs (multiplex 1: R5.50 (FAM) and R5.138 (VIC); multiplex 2: R5.10 (FAM) and R5.29b (VIC); multiplex 3: R3.171 (FAM) and R2.73 (VIC). The thermal cycling conditions consisted of 2 min of initial denaturation at 94 °C followed by 32 cycles of 20 s at 94 °C, 20 s at 58 °C, and 20 s at 72 °C, with additional eight cycles of 30 s at 94 °C, 30 s at 53 °C, and 45 s at 72 °C, and a final elongation step of 2 min at 72 °C  for each individual aphid was obtained by combining the alleles amplified from all six microsatellite loci. We assume that individuals carrying the same combination of alleles  have inherited its genetic architecture from a genetically identical asexual ancestor ; eventually, however, random mutations may produce changes in the length of short sequence repeats at a certain locus  within asexual lineages . This mef MLGsim  facilitaR. padi was computed using different indices. To assess the magnitude of the distortions that the over representation of some aphid clones can produce on measuring the genetic diversity, data analyses were performed on both the whole sample  and on one single copy per MLG, and then compared among populations. Thus, we focused on the frequency of each MLG rather than the frequency of microsatellite alleles  and clonal evenness index  were computed using the software GENCLONE 2.0 (http://genepop.curtin.edu.au/). The linkage disequilibrium (LD) between pairs of loci and the inbreeding coefficient (isF) over all loci were computed in the same software , using only one copy per MLG (http://biology-assets.anu.edu.au/GenAlEx/Welcome.html). The allelic richness was calculated using FSTAT version 2.9.3.2 (http://www2.unil.ch/popgen/softwares/fstat.htm).The genetic diversity of  alleles . The groLONE 2.0 . In orde per MLG . In addiFST (K) was set from 1 to 10 and repeated 20 times. Each repetition consisted of a burn-in period of 100,000 iterations and 1 million Markov Chain Monte Carlo iterations. The online program STRUCTURE HARVESTER (K) using the Evanno method (http://tree.bio.ed.ac.uk/software/figtree/). This tree was associated with the Bayesian clustering analysis in order to establish the phylogenetic relationship between the resulting genetic clusters.Analyses of the partition of the genetic variance (AMOVA) were performed using ARLEQUIN v 3.5.1.3  from theFST  implemenFST  using thARVESTER  was usedo method . The grao method . The phyR. padi found at the highest frequency in all samples (Rp1) was further studied in terms of its reproductive performance across different cereal hosts under aforementioned described laboratory rearing conditions (see “Aphid Collection and Laboratory Rearing” section). This would allow estimating any effect of the location and host of provenance. Thus, five clonal lineages of Rp1 were obtained: Rp1 genotype collected in Licantén from T. turgidum, Rp1 collected in Docamávida from barley, Rp1 collected in Limávida from T. turgidum, Rp1 collected in Villa Alegre from T. turgidum, and Rp1 collected in Cato from T. turgidum. These clonal lineages were maintained on barley for several generations before subjecting each clonal lineage to performance assessment. The reproductive performance was assessed on seedlings of oat (Avena sativa cv. Supernova), barley (H. vulgare cv. Sebastián), winter wheat (T. aestivum cv. Pantera) and durum wheat (T. turgidum subsp. durum cv. Llareta). When all seedlings attained growth stage 13  as (ln(fN) – ln(iN))/((ft – it)). Here, iN and fN indicate the initial and final number of individuals, respectively, while ft–it correspond to the difference in days from the beginning to the end of the experiment . Regarding the genetic diversity, the observed heterozygosity (HO) ranged between 0.400 and 0.667, whereas the expected heterozygosity (HE) was between 0.354 and 0.684. Both indices of genetic diversity were homogeneous among locations.A total of 377 The combination of all six microsatellites allowed the identification of 62 MLGs. Among them, 23 MLGs were found more than once in the whole sample and were considered as multicopy, while 39 MLGs were unique ; Fig. 1.Psex values, suggesting very low chances of arising from sexual reproduction . Considering one single copy per MLG, however, the AMOVA resulted in low non-significant genetic differentiation among all localities . Because using multiple copies generates that distortions, the latter AMOVA was preferred. Consistent with this result, most of pairwise FST comparisons exhibited non-significant genetic differentiation among samples from different localities . The pairwise FST between samples from maize and wheat were the only statistically significant .Considering all copies for each MLG, the AMOVA showed significant genetic differentiation among all localities . Analysis of the population genetic structure considering one copy per MLG from all localities, revealed the best partition of the dataset involves two genetic clusters (K = 2) according to the Evanno method , with significantly higher values on T. turgidum and lower values on T. aestivum , with significantly higher values exhibited by Rp1-Cato and Rp1-Licantén and the lowest values exhibited by Rp-1-Limávida , which was produced due to a lower number of nymphs produced by Rp-1-Villa Alegre lineage on T. turgidum , with significantly higher values on T. turgidum and lower values on T. aestivum , although no interaction between factors was found .Reproductive performance was characterized in the most frequent genotype Rp1 collected from four different hosts. Notice that performance was also assessed separately on Rp1 individuals generated from colonies collected from different sites but similar host (wheat). Number of nymphs produced by the most frequent genotype varied among hosts  specific climatic conditions, which are characterized by mild winters, (2) the absence or low abundance of the primary host in the areas where cereals are grown, (3) spontaneous sex loss due to mutations or hereditary distortions  gene flow of asexuality genes from asexual populations to sexual populations. Reproduction between sexual and androcyclic individuals has been reported for R. padi  inttortions  possibly R. padi . All of R. padi lineages present in Chile were made in the 1960s , which suggests the occurrence of a single mutational step. Similarly, the differences between Rp1 and Rp3 are small and again restricted to only a single locus R510 , which, in this case, suggests two mutation steps (269 -> 271 -> 273 -> 275). These events probably took place in Chilean populations of the grain aphid Sitobion avenae and the pea aphid Acyrthosiphon pisum  of R. padi were distributed in clines along a climatic gradient  collected in Scotland and found a predominant genotype (genotype E) on three different cereal hosts. These results suggest that asexual reproduction persists as the primary mode of reproduction. In Spain, where the winters are not very cold and the primary host is absent, R. padi has been reported to overwinter parthenogenetically . Specifically, only samples collected on maize in the locality RA exhibited a distinct set of genotypes  dominated the sampling and was found in two consecutive seasons (2013–2014 and 2014–2015) in three different localities . Genotypes Rp2 and Rp3 were also found consecutively in two seasons, but only in one location (Licantén). This situation has also been noted in other introduced aphid species such as Myzus persicae and S. avenae . Although R. padi is present in Chile in a wider range than even at more southern latitudes  on xazinoid . Interesturgidum . This fiturgidum . Thus, aS. avenae and R. padi revealed the presence, albeit at very low frequency of the bacteria Regiella insecticola  aphid pests can take advantage of the oversimplified design of current agroecosystems, (2) subjecting aphid populations to strong anthropic selection  may result in the predominance of selected superclones that appear to be more aggressive, (3) factors such as life cycle strategies, environmental conditions , behavior , and selection  affect aphid genetic variability, and (4) the evolution of that genetic diversity and population structure in time and space may be faster or slower depending on the intensity of the factors described above.R. padi are characterized by very low levels of genotypic and genetic diversity, suggesting that obligate parthenogenesis is the primary reproductive mode in the sampled range. Weak signs of genetic differentiation among localities and host-based differentiation were also observed. Among the MLGs found, one was present in all of the sampled populations at high frequencies and exhibited variations in reproductive performance on most common cereals cultivated in Chile. Chilean R. padi populations appear to be similar to those from Australia; both are composed of a single widely distributed superclone that likely resulted from a recent introduction. These results highlight the value of asexual reproduction during early stages of introduction to new regions in aphids, a distinctive feature that needs to be considered when implementing pest-management strategies.Chilean populations of 10.7717/peerj.7366/supp-1Supplemental Information 1P-values that are below the significance value 0.000368, obtained after applying Bonferroni’s correction for multiple tests.The range of colours from blue to white indicates decreasing pairwise genetic differentiation. Asterisks in bold are indicated the Click here for additional data file.10.7717/peerj.7366/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.7366/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.7366/supp-4Supplemental Information 4Allelle size for each locus, host plant collected, location and date of sampling.Click here for additional data file.10.7717/peerj.7366/supp-5Supplemental Information 5Locus name, central repeat motif, locus-specific hybridization temperature (Ta), and the number of alleles amplified with in each microsatellite locus.Click here for additional data file.10.7717/peerj.7366/supp-6Supplemental Information 6P-value for each locus pair across all populations (Fisher’s method). Analyses were performed on dataset without repeated genotypes (one single copy per MLG). Asterisks indicates the P-values that are below the significance value 0.00033, obtained after applying Bonferroni’s correction for multiple tests.Click here for additional data file.10.7717/peerj.7366/supp-7Supplemental Information 7P < 0.05).Number of nymphs, apterous, alates and population growth of rate after 14 days of infestation under controlled conditions. Different letters within columns represent statistically significant differences according to Tukey’s HSD test (Click here for additional data file.


Podarcis guadarramae in northern Portugal, one of the European regions with the highest percentage of burnt land. This species is a small saxicolous lizard as it inhabits natural outcrops and artificial stone walls, likely in recurrent-fire landscapes. We genotyped nine microsatellites from ten populations selected according to a gradient in fire recurrence, and compared genetic diversity indexes and demographic patterns among them. At the population level, we hypothesize that a high level of mortality and population bottlenecks are expected to reduce genetic heterozygosity in sampled localities affected by recurrent fires. Alternatively, genetic signatures are expected to be absent whether fire did not cause high mortality. Regardless of levels of mortality, we expect a gain in genetic diversity whether recurrent fires facilitate lizard dispersal and migration due to the increased quality of the habitat for wall lizards. At the regional level, we examine whether a recurrent fire regime may disrupt the spatial structure of populations. Our results showed an increase in genetic diversity in recurrently burnt populations, and a decline in longer-unburnt populations. We did not detect bottleneck effects in repeatedly-burnt populations. High genetic diversity in recurrent fire populations suggests a high dispersion rate between adjacent metapopulations and perhaps immigration from outside the fire boundary. At the regional level, lizard populations show low differentiation and weak genetic structure, suggesting no effects of fire. This study confirms field-based censuses showing that recurrent-fire regimes give ecological opportunities to wall lizards that benefit from habitat openness.Socioeconomic and climatic factors are modifying fire regimes with an increase of fire frequency and extension. Unfortunately, the effects of recurrent fires on biological processes that ultimately affect the genetic diversity of animal populations are mostly unknown. We examined genetic patterns of diversity in the wall lizard  More than a local ecological disturbance, fire is a global ecosystem process2, with approximately one third of the land mass experiencing intensive burning3. In the last decades, fire-prone regions as the western Mediterranean are experiencing a major shift in the fire regime with an increase of fire frequency and extension4. The main driver of this shift is socioeconomic, i.e. increased amount of fuel due to rural abandonment5, in parallel to a global climate warming7. Shifts in particular fire-regimes can have devastating impacts on the sustainability of many ecosystem components8.Wildfires have played a determining role in shaping the evolution and function of many ecosystems around the world9. Understanding the emergent genetic patterns of diversity resulting from habitat disturbance is critical in predicting how species will adapt to and persist in changing environments. However, the role of fire-regime as a driver of the patterns and distribution of genetic diversity is poorly understood11. Due to their mobility, the response of animals to fire is complex12, and the lack of information about the genetic patterns resulting from responses of organisms to fire is especially relevant13. To understand this issue, one must consider that the demographic impact of fire may depend on particular life history traits, such as dispersal ability, environmental requirements14, and resistance to fire15. The complexity builds up when both mortality and recruitment interact to determine the consequences of fire on within-population genetic diversity11. On the one hand, high mortality translates into population bottleneck, thereby reducing effective population sizes and producing a random loss of genetic diversity16. On the other hand, post-fire population recovery can stem from either survivors or through colonizers from adjacent unburnt areas17, which may also have an impact on the distribution patterns of genetic diversity. The loss of allelic diversity will be low if there is either a high survival rate18 or if recruitment comes from multiple sources20. Post-fire population dynamics, such as genetic drift and/or migration, can affect genetic differentiation between populations22. Conversely, if post-fire migration is facilitated between populations, increased gene flow can reduce genetic differentiation24. This is the case in open-habitat or early colonizer species, which increase their local density and genetic variation in recently burnt sites20. In contrast, genetic diversity in long-unburnt specialists may decrease due to habitat fragmentation patterns imposed by fire25.Fire shapes community composition for many taxonomic groups especially due to its major effect on the habitat structurePodarcis guadarramae26 in northern Portugal. Wildfire is one of the most important agents of landscape change in Portugal27. The study area, in particular, is one of the European regions with the highest percentage of burnt land due to a combination of socioeconomic  and climate  factors. Podarcis guadarramae is a small-size saxicolous wall lizard28 that uses rocks, trunks, and bare ground for thermoregulation, foraging and shelter29. In northern Portugal, this lizard has a patchy distribution with populations located in natural open rocky outcrops and artificial stone walls surrounding agriculture fields26. The forested and bush matrix isolates lizard populations and hampers inter-population contact due to the low dispersion ability of this species through vegetated areas. Wildfire reduces vegetation cover, increases the extent of open outcrops, and allows lizard dispersion among patches with favourable (open) habitats. These life-history traits can explain the positive population responses of P. guadarramae to fire observed in northern Portugal31. However, the demographic processes  underlying the positive response of wall lizards to fire are unknown. For this reason, we have collected lizard samples in ten populations selected according to a gradient of disturbance level (i.e. number of fires and time since the last fire). The objective of this study was to examine how recurrent fire regimes affect: i) at the population level, the genetic diversity of each lizard population sampled, and ii) at the regional level, the geographic/genetic structure of the sampled P. guadarramae populations. Emergent genetic patterns of diversity can help to understand demographic processes linked to fire recurrence and habitat use. Based on P. guadarramae life-history traits, we have addressed the following hypotheses.In this study, we have investigated the effects of a recurrent fire regime on the genetic diversity and spatial structure of the wall lizard At the population level, if fire causes high mortality rate, wall lizard populations located on rocky outcrops that have experienced higher number of fires are expected to exhibit genetic signatures consistent with recent demographic bottlenecks and low genetic diversity. Alternatively, such genetic signatures are expected to be absent (i.e. no diversity loss) when fire does not cause severe mortality. Moreover, if recurrent fires improve habitat quality and facilitate lizard dispersal and migration, genetic diversity gains are expected regardless of lizard mortality caused by fire.At the regional level, we hypothesize that fire can shape population structure due to the expected fire impacts over the genetic diversity and ultimately over population differentiation. Accordingly, we expect fire to disrupt the spatial structure of populations by decreasing the naturally expected correlation between genetic and geographic distances.From the initial battery of nine microsatellite loci genotyped, one (Ph17) showed presence of null alleles in seven out of 10 populations  of the eight loci showed moderate values, ranging from 0.663 to 0.732 (average 0.694 ± 0.019), while the observed heterozygosity averaged 0.647 (±0.026). The allelic richness (AR) per population ranged from 5.922 to 7.763, whereas the mean number of alleles (NA) per population varied from 6.125 to 8.625  were compared, a clear difference was reported for NA and PA . Overall, the expected heterozygosity ) and FIS were positively correlated with the number of fires that occurred at each population . Moreover, these measures were negatively correlated with the time since the last fire occurred at each population .All genetic diversity measures . With exception of Moledo pair, all remaining locations showed no significant differentiation regarding the pairs of populations. AMOVA results revealed that all levels of genetic differentiation were significant  and 4Neµ = 5  to no evidence of bottleneck in any population (when assuming the SMM). When fitting the TPM, the evidence varied along the proportion of single step mutations included in the model. Assuming the default parameters incorporated in BOTTLENECK , only two unburnt localities showed significant heterozygote excess, as suggested by the Wilcoxon test. In summary, comparing the various tests, only the unburnt population located at Leonte showed consistent evidence for the occurrence of a bottleneck. Even though results are not robust to changes in the mutation model, it is clear that the evidence is not larger for bottlenecks in repeatedly burnt, when compared to unburnt, localities.Bottleneck test results were not robust to changes in the assumptions regarding mutation models Table . M-ratio13. We demonstrated emergent genetic patterns of diversity resulting from demographic responses to fire by the wall lizard P. guadarramae at population level (i. e. genetic diversity indexes), but not at regional level (i. e. relationship between genetic and geographic distances). Due to its environmental requirements of open outcrops32, P. guadarramae benefits from recurrent burned landscapes dominated by granitic rocks where lizard populations achieve high effective population sizes33. The positive response of P. guadarramae to fire in northern Portugal31 showed that recurrent-burnt landscapes give ecological opportunities to this wall lizard that seem to be related with the increase of genetic variability in the repeatedly burnt populations.Our study design (a gradient of recurrent-fire histories) provided the opportunity to perform tests to enlighten which genetic patterns and processes are driven by the occurrence of recurrent wildfires, and to extend the knowledge in this research field that is currently lacking information17. Some studies have documented a short-term post-fire decline of fire-sensitive species34, although studies on vertebrates have shown that fires rarely cause complete mortality and that residual populations may remain in situ in burnt areas18, specifically cliff- and rock-specialist species for which habitat is not destroyed by fire34. Biological processes, such as mortality, survival, and post-fire recolonization can affect genetic diversity in contrasting ways:11 reductions in heterozygosity and allelic richness resulting from population bottlenecks22; increases in genetic diversity and reduced regional genetic differentiation resulting from high immigration rates20; and intermediate values of unchanged genetic diversity when mortality is low or is compensated by immigration18.Population recovery following major disturbances such as wildfires is likely a reflection of two non-mutually exclusive biological processes: i) abundance of survivors; and ii) post-fire recruitment P. guadarramae. Our results clearly showed that fires altered the genetic diversity of wall lizards. Our first expectation was that fires could alter patterns of genetic diversity in burnt populations by decreasing it through successive bottlenecks and founder effects. These results have been found in populations of butterflies25 and lizards23 and have been further attributed to bottlenecks in several taxa such as mammals35, birds22, and lizards36. Consequently, burnt populations would become more differentiated from unburnt populations by means of genetic drift. However, our results did not meet this expectation. Interestingly, we obtained the opposite pattern: significantly increasing allelic diversity with number of fires, and conversely, a significant decreasing of allelic diversity with time since the last fire. This trend was valid for the overall correlation between fire and genetic diversity parameters, and also, in general, to local pairwise comparisons.Analyses of genetic diversity helped us to unravel how recurrent fires affect the population dynamics of P. guadarramae can be explained by a combination of both high survival rate, mobility, immigration, and a post-fire increase in the carrying capacity of disturbed habitats.We suggest that the pattern of higher genetic diversity  found in repeatedly burnt populations of 37. Refuges enhance immediate survival during a fire event but can also facilitate the post-fire persistence of individuals and populations within the burnt landscape by providing resources in the short-  or long-term (resident habitat) from a series of disjunct isolated populations to a metapopulation, and to a patchy population linked by frequent movements39.The likelihood of immediate survival of an individual during a fire will be influenced by the severity of the fire, its distance to potential refuges and behavioural mechanisms the organisms may use to avoid direct flames and heatP. guadarramae to fire has been demonstrated in several demographic studies in the region31. Physiological experiments41 and habitat preference studies have highlighted that this lizard copes very well with post-fire habitat conditions. These studies and our personal observations in recently burnt spots support the increased dispersal ability of individuals in post-fire habitats that brings new diversity . Increased dispersal activity in recently burnt habitats was the cause of increased genetic diversity in the agamid Amphibolurus norrisi in Australia42. Schrey, Ashton, et al. (2011) also found increased genetic diversity in burnt areas, in one of the examined lizard species. They tested a metapopulation source/sink model based on the habitat preferences of each species, by examining the direction of gene flow (i.e. immigration into or out of preferred habitat). The burnt area indeed received influx of individuals from open-habitat species, increasing genetic diversity. In other taxonomic groups with higher dispersal capability, a post-fire increase in genetic diversity was also reported; e.g. the mountain brushtail possum Trichosurus cunnunghami43, and the Gran Canaria chaffinch Fringilla teydea polatzeki18. A combination of life history traits , and habitat characteristics  could have contributed to increase genetic diversity.The response of Podarcis guadarramae is a highly saxicolous lizard and it is associated with rocky habitats44. Likewise, its preference for these habitats with a naturally high number of crevices and burrows, where the lizard could take refuge during the fire events, may allow a considerable level of survival among animals (see a similar result for the saxicolous gecko Tarentola mauritanica34), allowing the retention of a large fraction of the pre-fire genetic diversity. This fact is in line with the lack of evidence for severe bottlenecks in burnt populations.Our results suggest that refuges are preponderant both in the survival rate of wall lizards as well as for colonizers. IS in recently burnt localities, which may be in line with Wahlund effects caused by recent migrants from different gene pools45. We acknowledge that this hypothesis cannot be corroborated by the absence of pre-fire genetic data nor movement data. However, the negative correlation between genetic diversity and the time since the last fire was indirect evidence of the mobility/migration lizard pattern in burnt populations. Indeed, long-unburnt localities showed reduced genetic diversity of lizard populations likely by vegetation re-growth that reduces lizard mobility and isolates small populations in small rocky spots. The incorporation of knowledge of both direct, in particular the demographic and mobility processes13, and indirect effects (shift in habitat structure) of fire is important to understand the underlying processes behind the patterns of genetic diversity found11.Moreover, given that fire clears vegetation and exposes previously unsuitable rocky outcrops, unoccupied habitats represent a new ecological opportunity for wall lizards. This not only supports an increase in the local effective population size, but also allows the dispersal of individuals between metapopulations, and potentially, recruitment and settlement of migrants from outside the affected area. Both mobility and migration can positively increase genetic diversity at the population level. Our results shown an increase in FP. guadarramae populations. This result configures a classical metapopulation scenario, especially when individuals show low dispersal ability, like the lizard case46, and matches findings from closely related species (P. bocagei and P. carbonelli) also inhabiting western Iberia, in which, similarly low or slightly higher levels of population differentiation were reported across their distribution areas, yet with a clear correspondence to geography .The observed general increments of genetic differentiation according to geographic distance (isolation by distance) and not to fire history, suggested limited gene flow among all 49 that are driven by habitat structure changes and are a product of species life history . Thus, as general rule, the open-habitat dwelling species are favoured by post-fire habitat conditions, then, while the ecological succession is recovering, forest-species increase in abundance whereas open-habitat species tend to decrease . In our study areas (i.e. locations), these metapopulations could be spatially arranged according to the distribution of rocky and agricultural  areas given its saxicolous ecology28.Reptiles show strong succession responses to fireP. guadarramae, all the population trends suggest that this lizard benefits from recurrent fires. Microhabitat preference and post-fire dispersal seem to be traits that help this lizard to resist fire and persist afterwards. Mobility is an attribute that makes fauna a challenging group to examine their responses to fire12. However, data on this trait needs to be integrated with other biological traits to capture all relevant information in predicting how reptile populations respond to recurrent disturbances50. Integrative approaches are necessary to forecast biodiversity patterns in future scenarios of shifts in fire regime.Due to their particular life-history traits , reptiles are important organisms to examine in their responses to fire at different complementary approach levels, i. e. demographic, genetic, ecologic. Given the saxicolous preferences of Podarcis (Squamata: Lacertidae) are among the most conspicuous, abundant and widely distributed reptiles in Europe and North Africa. Podarcis guadarramae occurs in northern Portugal, north-western Spain, and Central Iberian Mountains in Spain  diurnal lacertid lizard28 with Atlantic ecological affinities53. Its depressed body shape and flattened skull facilitate entrance into narrow, irregular crevices. Individuals aggregate around favourable open areas with rock crevices, artificial walls, and isolated big blocks33.Wall lizards of the genus 56. Fire frequency and intensity have increased remarkably since the 1960s58, due to a combination of socioeconomic ; plantations of fire-prone tree species such as eucalypt and pines  and environmental factors . Coincident with P. guadarramae distribution, the northern part of the country has the highest fire frequency and size  due to the dominance of fire-prone tree species  and shrubs .Portugal is currently one of the European regions with the highest percentage of burnt land62 with a transitional climate from Atlantic (NW) to Mediterranean (NE). Average annual precipitation ranges from approximately 1200 mm at the coast to 2800 mm at the upper elevations in the interior, mostly falling between October and April and with 0–2 rainless months. The average annual temperature is 14.5 °C, mean maximum and minimum temperatures are respectively 25 °C in July and 4 °C in January63. Native forests have become very fragmented, and the characteristic species are Quercus robur, Q. pyrenaica, Acer pseudoplatanus, Pinus pinaster, Pyrus cordata, and Ilex aquifolium. The most common type of vegetation are fire-prone scrub communities composed by genus Erica, Calluna, Ulex and Cytisus64.The sampling area  was located in the transition between the Mediterranean and Eurosiberian biogeographic zones close to the Atlantic coastP. guadarramae, we selected ten locations based on a digital national cartography of burnt areas from 1975 to 2013 . These locations followed a gradient of recurrent fires and time-since-fire periods. Burnt locations had 5.8 fires on average (range 2–8 fires) during the period 1975–2013  from the Portuguese Government that provided the permit for sampling lizards in northern Portugal (number 578/2014/CAPT).P. guadarramae lizards using the EasySpin Genomic DNA Tissue Kit  following the manufacturer’s instructions. Samples were genotyped at nine microsatellite loci chosen based on further multiplex optimization and reports of scoring errors by Ribeiro65; Supplementary material Table et al.66. PCR products were then separated by size on an automatic sequencer ABI 3130xl Genetic Analyser  with size standard GS500 LIZ . Allele scoring was performed with GeneMapper v4.1  and checked manually.DNA was extracted from tail muscle of 201 loci. No individual or locus was excluded based on missing data. We used MICRO-CHECKER v2.2.367 to test for the presence of null alleles or scoring errors. We used ARLEQUIN v3.568 to test for departure from Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium (LD). Bonferroni corrections were applied due to multiple tests69. With the final data set, we performed three types of analyses: i) population genetics; ii) population differentiation; and iii) bottleneck tests.The final data set included 201 samples genotyped for nine microsatellite E) and observed (HO) heterozygosities, mean number of alleles (NA), and number of private alleles (PA) for each population. We also calculated allelic richness  and inbreeding coefficient FIS71 with FSTAT v2.9.3.272.Using Arlequin, we calculated allele frequencies, expected  between each population pair, and between burnt and unburnt populations as a whole were explored using a permutation approach with 999 permutations. These analyses were performed with Python 2.7 scripts (available from the authors upon request).Differences in genetic diversity indexes  and fire history variables  by using simple linear regression in STATISTICA v8.073. Due to the independence of each fire event, these analyses disregard the paired sampling design and focus on an overall comparison of fire-history and genetic diversity among all ten populations sampled.We further explored the relationship between genetic diversity indices  grouping by pairs of location in order to test the null hypothesis that genetic variation was not associated with spatial structure. Both analyses were performed in ARLEQUIN using 10,000 permutations.To test for genetic differentiation among all populations pairs, pairwise Fhttp://biodiversityinformatics.amnh.org/open_source/gdmg). We tested for isolation by distance by regressing log transformed geographic distances (in km) against Slatkin’s linearized FST (FST/(1 − FST)). Statistical significance was examined by Mantel test with 999 permutations in PASSaGE74. A Principal Coordinate Analysis (PCoA) was computed in GenAlex 6.576 to visualize patterns of genetic differentiation among populations.Geographic distances between populations were calculated from the pairwise Euclidean distance according to the latitude and longitude of each location with Geographic Distance Matrix Generator, version 1.2.3  M- ratio, and a test for heterozygosity excess developed by Cornuet & Luikart77. This test is based on the principle that, when populations experience a sudden decrease in population size, the number of alleles decreases faster than heterozygosity. Hence, a transient excess in heterozygosity can be detected in bottlenecked populations when compared to mutation-drift equilibrium expectations. We used the software BOTTLENECK v. 1.280 to evaluate such heterozygosity excess. Because the expected patterns of variation vary extensively depending on the mutation model assumed, the test of the robustness of the inferences was performed under the infinite allele model , the strict stepwise mutation model , and under a wide range of two-phase mutation model  parameters. BOTTLENECK provided several tools to compare hypothesis; we chose the Wilcoxon’s test, since it has been shown to be the most powerful, especially for data sets with a small number of loci such as in our case80.Bottlenecks (such as those expected to derive from the occurrence of wildfires) can be detected by taking into account patterns of microsatellite allele size distribution and/or frequency datak is expected to decrease faster than allele range r. Values of M = k/r lower than expected given equilibrium expectations may thus be indicative of a bottleneck. We used the R package strataG84 to perform the calculation of M and the script “Critical_M” (obtained at https://swfsc.noaa.gov/textblock.aspx?Division=FED&id=3298) was used to determine the “Critical M” (Mc) value. This program calculates the M-ratio from simulated data sets under equilibrium for a given set of relevant parameter values and returns the value representing the fifth percentile of this distribution. We calculated Mc based on the sample size and the number of loci specific to our study. We also used a range of reasonable parameter values for the pre-bottleneck population mutation rate (from 5 to 20) and for the microsatellite mutation model parameters, including both the strict SMM as well as allowing for larger size mutations in varying proportions .The M-ratio test quantifies gaps in the distribution of microsatellite allele sizes. Under a bottleneck, the number of alleles Both types of tests rely up to a great extent on the mutation model assumed for microsatellite loci. Except in the case of the IAM, the mutation models typically used for microsatellite data do not tolerate the existence of alleles resulting from insertions and deletions in the flanking regions. An observation of allele sizes in our case revealed the existence of a few alleles not compatible with strict repeat-number mutations. Therefore, to accommodate our data to the models under which these hypotheses were tested, we performed the analyses described above in a reduced data set resulting from the removal of the loci that, in each population, deviated from the expected repeat number pattern. This resulted in data sets containing six loci in all localities, except Santo Tirso – burnt, where five loci were retained.Supplementary informationDataset 1


Xenophysogobio boulengeri and X. nudicorpa are the only two species within the genus Xenophysogobio , and both are endemic to the upper reaches of the Yangtze River. In recent years, due to human activities, the natural resources available to both species have declined sharply. Sympatric species with overlapping niches inevitably compete for their habitats, and genetic structure and diversity can reflect population history and their potential for adaptation to changing environments, which is useful for management decisions.X. boulengeri and X. nudicorpa. Microsatellite DNA data, jointly with traditional summary statistics including FST and Fis, were used to assess the population genetic structure by structure analysis. The mtDNA sequences were then used to examine these patterns through time to detect demographic history.In the present study, microsatellite DNA and mitochondrial DNA (mtDNA) markers were used to investigate the patterns of population genetic structure for Xenophysogobio boulengeri and X. nudicorpa exhibited high levels of genetic diversity in Yangtze River populations, except for two populations of X. nudicorpa in the Jinsha River, which were low in mtDNA diversity. X. boulengeri showed genetic homogeneity among populations, whereas X. nudicorpa appeared to have significant geographic genetic divergence. Both species experienced a late-Pleistocene sudden population expansion in Yangtze River populations, but not in the Jinsha River populations of X. nudicorpa.X. boulengeri populations might result from similar population expansion events and environment features. The geographic genetic subdivision for X. nudicorpa between the Jinsha and Yangtze Rivers might be caused by the geographic isolation in the middle Pliocene, as well as climate and environmental heterogeneity.The genetic homogeneity of  The Yangtze River is the longest river in China and the third longest river in the world. It originates from the Qinghai-Tibetan Plateau and follows a sinuous easterly route before emptying into the East China Sea at Shanghai city. The section above Yichang city in the Huhei province (the Three Gorges Dam site) is generally regarded as the upper reaches and is characterized by mountains, raging torrents and a high altitude compared with the middle and lower reaches. The section above Yibin (YB) city in the Sichuan Province, known as the Jinsha River , is the Gobiobotia and Xenophysogobio, in this subfamily with about 17 species of which Xenophysogobio represent the primitive species -Panzhihua (PZH) section  is a group of small freshwater fish, distributed across East Asia, including Korea and China . There a species . Xenophyze River . They shg rivers , while X section  Fig. 1)Gobioboti section , which i section . For insucturing . For exaudicorpa . Previou habitat . Habitat habitat . To the  studies . The com studies .X. boulengeri and X. nudicorpa. Simple sequence repeat (SSR) data, jointly with traditional summary statistics including FST and Fis, were used to assess the population genetic structure by Structure analysis. The Cytochrome b , control region  sequences were then used to examine these patterns through time to detect demographic history. We aimed to: (i) compare genetic diversity and population genetic structures between X. boulengeri and X. nudicorpa; (ii) compare the occurrence of recent population expansions between the two species in order to assess demographic history; and (iii) clarify spatial genetic sub-structuring as a good base for improving Xenophysogobio stock management.In the present study, microsatellite DNA and mitochondrial DNA (mtDNA) markers were used to investigate the patterns of population genetic structure for X. boulengeri and X. nudicorpa specimens was conducted in strict accordance with Animal Experimental Ethical Inspection of Laboratory Animal Centre, Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences (ID Number: FRE0006).All handling of X. boulengeri individuals from five sites, Jiangjin (JJ), YB, Shuifu (SF), Yongshan (YS), and Qianwei (QW), and 126 X. nudicorpa individuals from four sites, JJ, YB, QJ and PZH, were collected between 2011 and 2018  in X. nudicorpa were used, according to previously reported protocols (b gene was amplified by polymerase chain reaction (PCR) using primers L14724: (5′-GACTTGAAAAACCACCGTTG-3′) and H15915 (5′-CTCCGATCTCCGGATTACAAG-3′) (Genomic DNA was isolated by proteinase n method . Nine porotocols . The finrotocols . The CytCAAG-3′) , and CR CAAG-3′) . The PCRLatitude, longitude and altitude were measured by a hand-held sub meter GPS .X. nudicorpa by SPSS 17.0. The analysis of molecular variance (AMOVA) and pairwise FST based microsatellite data were assessed in Arlequin version 3.1.1 , and Group 2 represented the Yangtze River populations (JJ and YB). The global FST was also calculated between Group 1 and Group 2. Correlations between geographical distances (ln km) and genetic distances (FST/(1-FST)) based on microsatellite data were tested in GENALEX version 6.5 (Fis) and allelic richness were obtained by FSTAT version 2.9.3  and expected heterozygosity (He) were all detected by POPGENE version 1.32 . Duncan’on 3.1.1 . Benjamision 6.5 . Geograpsion 6.5 . POPGENEon 2.9.3  and the on 2.9.3 .K method was used to determine the appropriate value of K ] and the Delta K statistic for a given K. The results were summarized on the online platform Structure Harvester (http://taylor0.biology.ucla.edu/struct_harvest/). Genetic relationships among populations were also examined by applying discriminant analysis of principal components (DAPC)   using ths (DAPC) . For mics (DAPC)  was usedh) and nucleotide (π) diversity, the number of haplotypes and the haplotype frequencies were obtained using the software Dnasp version 5.10  and genetic distances (FST/(1-FST)) were tested using a MANTEL matrix correlation test in GENALEX version 6.5  were obtained for Cyt b and CR sequences using the Bayesian information criterion in IQ-TREE . Bayesian inference was performed to reconstruct the phylogenetic tree among the Cyt b and CR haplotypes in X. boulengeri and X. nudicorpa in MRBAYES version 3.1.2 (Hemibarbus barbus (AB070241) and Hemibarbus labeo (DQ347953) or Gnathopogon elongates (AB218687) and Gobiocypris rarus (NC_018099) based on fossils from the Late Miocene , which occurred before this time  (P). Default settings were used for the prior range for maximum intraspecific divergence , corrected distances (JC69) and minimum slope increase (X) of 1.5. Although there is still a lack of consensus of how to interpret discordant ABGD results  . The fir results , previoucurately .D  to 0.825 (SF) and from 0.852 (JJ) to 0.879 (SF), respectively,  to 0.613 (JJ) and from 0.469 (PZH) to 0.705 (YB), respectively, . Overall, haplotype and nucleotide diversities were high for X. boulengeri , but lower for X. nudicorpa   for Cyt 0.00348) . Within X. boulengeri individuals and 126 X. nudicorpa individuals were distributed into 43 (784 bp fragments) and 23 haplotypes (854 bp fragments), respectively, . The CR sequences had 39 and 18 variable sites for X. boulengeri and X. nudicorpa, respectively. The overall Ts/Tv ratio was 7.908 in X. boulengeri and 1.273 in X. nudicorpa. The global haplotype and nucleotide diversities were similar for both X. boulengeri  and X. nudicorpa    . Comparelations) .X. boulengeri populations appeared similar in genetic diversity. However, there was a difference in genetic diversity among X. nudicorpa populations. Duncan’s test shown that the genetic diversity in Jinsha river populations were significantly lower than that in Yangtze River populations  deviated from HWE (X. boulengeri (0.061–0.102) and JJ and YB populations of X. nudicorpa (0.121 and 0.174), except the values were negative in QJ and PZH populations of X. nudicorpa  displayed a departure in ulengeri . For X. from HWE . Estimatudicorpa .X. boulengeri  , with highly significant FST values   . High ge 0.0001)   and Yangtze River (JJ and YB) populations ) for all the three markers dataset in X. boulengeri. For X. nudicorpa, a significant correlation was detected in the SSR  and Cyt b dataset , but not in CR   and genetic distances (= 0.160)  and S5.X. boulengeri and X. nudicorpa, the Delta K values were highest  when K = 2  in network topology. There were five mutational steps between clades   .X. nudicorpa, the results of haplotype network showed two clades based on Cyt b (Clade A and Clade B) and CR (Clade C and Clade D) respectively. Clade A and Clade C encompassed individuals from four populations with the most frequent haplotype usually being present in the Jinsha River populations (PZH and QJ), whereas Clade B and Clade D individuals were found only in JJ and YB  (D and Fu’s Fs) for the Jinsha River and Yangtze River X. nudicorpa populations were calculated separately. The values of Tajima’s D and Fu’s Fs were significant and negative in Yangtze River populations (except Tajima’s D for the CR dataset), but not significant in Jinsha River populations.Neutrality tests were used to detect population expansions using Cyt dicorpa) , which iX. boulengeri and X. nudicorpa based on the Cyt b and CR datasets. For X. boulengeri, a smooth unimodal mismatch distribution was observed, which was compatible with a single expansion  of X. nudicorpa was similar to X. boulengeri in JJ and YB populations  might have been caused by the founder effect or genetic drift  (X. boulengeri (X. boulengeri was congruent with genetic structure reports in other taxa from these regions (For low (Nm) , which mlow (Nm) . In addilow (Nm) , may havulengeri . The gen regions .X. nudicorpa, a significant correlation between genetic and geographic distance in Mantel’s test was observed using the Cyt b and SSR dataset, although the result was not supported by the CR datasets. Hence, population genetic differentiation cannot be explained by the isolation-by-distance model using the present data. The lack of strong evidence in the CR  accumulation of mutational steps between the two clades of X. nudicorpa, corresponding to either side of the dams, need a longer period of time than that lapsed since dam construction (2007 for the Xiluodu Dam and 2008 for the Xiangjiaba Dam). Thus, the current genetic patterns of the fish are likely to be derived from historical events. The Jinsha River was insulated in the middle Pliocene  was mostly due to excess of homozygosity. The micro-checker detected excess of homozygosity for most HWD tests  and S9. tructure . If the tructure . The phyX. boulengeri, and two separate MUs within X. nudicorpa including Jinsha River Unit and Yangtze River Unit.The concept of management unit (MU) is useful for identifying and prioritizing conservation units within a species. MUs are recognized as populations with significant divergence of allele frequencies at mitochondrial or nuclear loci, regardless of the phylogenetic distinctiveness of the alleles . Accordib and CR provide evidence of a recent population expansion in X. boulengeri and X. nudicorpa derived from the Yangtze River. However, the population expansion times obtained by Cyt b and the CR gene were not coincident. The interval might have been caused by the substitution rates of the markers we used. The average substitution rates used in the present study were those of vertebrate mtDNA (0.65% for Cyt b and 3.6% for CR). However, variation of the CR was lower than that of Cyt b in this study, against the common property of vertebrate mtDNA, and therefore the time deduced from the CR may be underestimated. For both X. boulengeri and the Yangtze River populations of X. nudicorpa, the beginnings of the expansion took place in a similar period in the Late Pleistocene. X. boulengeri has undergone a greater long-term expansion period than Yangtze River X. nudicorpa populations. Population expansion for both species has provided evidence that the main stream in the upper reaches of the Yangtze River could have been a refuge for X. boulengeri and X. nudicorpa in the Late Pleistocene. Similar population expansion events might contribute to the similar genetic structure pattern found in X. boulengeri and X. nudicorpa  Xenophysogobio nudicorpa of four populations.Delta K as a function of the K values according to 10 run outputs. (a) Click here for additional data file.10.7717/peerj.7393/supp-2Supplemental Information 2Xenophysogobio boulengeri based on nine microsatellite loci and (b) Xenophysogobio nudicorpa based on nine microsatellite loci.(a) Click here for additional data file.10.7717/peerj.7393/supp-3Supplemental Information 3a-b) Xenophysogobio boulengeri based on Cyt b and CR, (c-d) Xenophysogobio nudicorpa based on Cyt b and CR. Numbers represented nodal supports inferred. The supported value was only displayed among main clades. Xenophysogobio boulengeri from all populations based on CR; (c-e) Xenophysogobio nudicorpa from all populations, Jinsha River and Yangtze River based on Cyt b, (f-h) Xenophysogobio nudicorpa from all populations, Jinsha River and Yangtze River based on CR. XB and XN are the abbreviation of Xenophysogobio boulengeri and Xenophysogobio nudicorpa respectively.(a) Click here for additional data file.10.7717/peerj.7393/supp-5Supplemental Information 5p < 0.05) are denoted with a*.Statistically significant estimations .Genetic distance has been calculated by the formula: Click here for additional data file.10.7717/peerj.7393/supp-9Supplemental Information 9FST/(1-FST).Genetic distance has been calculated by the formula: Click here for additional data file.10.7717/peerj.7393/supp-10Supplemental Information 10Click here for additional data file.10.7717/peerj.7393/supp-11Supplemental Information 11Click here for additional data file.10.7717/peerj.7393/supp-12Supplemental Information 12Yes indicates the presence of an allele, No indicates the absence of an allele,–indicates insuffient sample.Click here for additional data file.10.7717/peerj.7393/supp-13Supplemental Information 13Yes indicates the presence of an allele, No indicates the absence of an allele,–indicates insuffient sample.Click here for additional data file.10.7717/peerj.7393/supp-14Supplemental Information 14Xenophysogobio boulengeri from five populations based 9 microsatellite loci..Raw data of Click here for additional data file.10.7717/peerj.7393/supp-15Supplemental Information 15Xenophysogobio nudicorpa from four populations based on 9 microsatellite loci.Raw data of Click here for additional data file.10.7717/peerj.7393/supp-16Supplemental Information 16Xenophysogobio boulengeri from five populations based CR data.Raw data of Click here for additional data file.10.7717/peerj.7393/supp-17Supplemental Information 17Xenophysogobio boulengeri from five populations based Cyt b data.Raw data of Click here for additional data file.10.7717/peerj.7393/supp-18Supplemental Information 18Xenophysogobio nudicorpa from four populations based CR data.Raw data of Click here for additional data file.10.7717/peerj.7393/supp-19Supplemental Information 19Xenophysogobio nudicorpa from four populations based Cyt b data.Raw data of Click here for additional data file.