Glossogobius aureus is a native species in the Philippines, Australia, Japan, Taiwan, and many other countries in Asia. In the Philippines, it is an important food fish as it is commonly caught in major lakes. In this study, a total of 307 specimens morphologically identified as G. aureus were sampled from nine major lakes in the Philippines and were sequenced for their mitochondrial cytochrome b (cyt b) gene. Two hundred sixty of the 307 cyt b sequences had sequence similarities of ≥ 99% with G. aureus reference sequence in GenBank, while the remaining 47  had sequence similarities of only 95% and were thus designated as Glossogobius cf. aureus and treated as a separate population. The sequences were then analyzed to examine the pattern of genetic diversity, relatedness, divergence, and demographic history among native and translocated populations of the species. Twenty-nine haplotypes were recovered, of which four haplotypes were shared among three to seven populations. Only one haplotype each was found in the native population in Lake Buhi and translocated population in Lake Paoay. Low haplotype and low nucleotide diversities were found for the populations in Laguna de Bay, Lanao, Bato, Buhi, Paoay, and Sebu lakes, which indicate founder event for the introduced populations in Lanao, Paoay, and Sebu lakes and recent genetic bottleneck for the native populations in Laguna de Bay, Bato, and Buhi. In contrast, high haplotype but low nucleotide diversities were found for the native populations of Taal, Naujan, and Buluan lakes, signifying a recent bottleneck followed by population expansion. Pairwise FST values showed generally large (FST = 0.168–0.249) to very large (FST = 0.302–1.000) genetic divergence between populations except between Laguna de Bay and Lake Bato, Laguna de Bay and Lake Buhi, and Lake Bato and Lake Buhi populations, which showed nonsignificant genetic differentiation. Lake Buluan and Lake Sebu populations showed moderate genetic differentiation (FST = 0.098). Neutrality tests showed significant negative Tajima’s D and Fu’s FS values only for the population from Laguna de Bay, which suggests that the population is undergoing expansion. These results are important for establishing scientifically sound strategies for effective conservation and sustainable exploitation of G. aureus in the Philippines.The golden flathead goby  Glossogobius aureus Akihito and Meguro 1975 (Gobiiformes: Gobiidae) is an amphidromous fish that is native to the Philippines, Taiwan, Japan, Thailand, Singapore, Australia, Malaysia, Cambodia, Papua New Guinea, Laos, and Indonesia [Glossogobius aureus belongs to the most speciose genus in the suborder Gobioidei (gobies) [G. aureus is the most widely distributed and the most abundant goby species in many lakes [Glossogobius aureus is a commercially important species. Unlike many species of gobies in which the adults are quite small, G. aureus can grow to a large size (up to 30 cm), and it has been found to have a high growth rate [G. aureus, are considered a delicacy in the Philippines. They are an important source of livelihood among residents surrounding many major lakes in the Philippines, such as in Laguna de Bay, Lake Lanao, Lake Taal, and Lake Naujan. Gobies are sold either as fresh or dried fish. In addition, the goby fry are a component of the ipon fishery [Ipon fishery was once reported to be an important source of livelihood among the fisherfolk of the northern coast of Luzon [Glossogobius sp. was one of the predominant fish species in Laguna de Bay in the 1950s [Glossogobius sp. has been observed by Mercene and Nasino [G. aureus) ranked as the 7th most abundant inland species caught by volume in the Philippines [th [The golden flathead goby ndonesia . Glossog(gobies) , 9, in wny lakes . Glossogwth rate , thus it fishery , 13, a cof Luzon . Goby frof Luzon . Glossoghe 1950s  and in Lhe 1950s . Over thd Nasino  in Lagund Nasino  in Lake lippines . HoweverG. aureus is widely distributed in East and Southeast Asia and serves as an important food fish [G. aureus in the Philippines. As an important food fish and a candidate species for aquaculture, conservation and management of this fish is of prime importance. There is a need to study the population genetics of this species to guide policymakers and concerned government agencies in the proper management and conservation of this important biological resource. High genetic diversity in a population would mean greater resilience to fluctuating environmental conditions [G. aureus in Laguna de Bay, Taal, Naujan, Buluan, Bato, and Buhi lakes. Additionally, low to high genetic diversity is expected among the translocated populations of G. aureus in Lake Lanao, Paoay, and Sebu, depending on the number of individuals introduced and how well the introduced populations were able to adapt to their new environments. Knowledge of the population genetic structure of G. aureus in the Philippines can serve as a basis for determining the number of independent management units (IMUs). The identification of distinct IMUs will enable fisheries managers to develop targeted conservation and management strategies based on the unique characteristics of each population.Although ood fish , the curood fish , scale sood fish , populatood fish , and repood fish . To datenditions  and greanditions . Thus, mnditions . On the nditions . Severalnditions –30. The nditions . These fcytochrome b (cyt b) gene, have been used in many studies to investigate the genetic diversity within and between populations of fishes and other animals. These mtDNA markers are often chosen for population genetic studies because they are easier to use and relatively cheaper compared to other molecular markers. In addition, mtDNA lacks repair mechanisms, resulting in an increased rate of evolution and making it useful in identifying major evolutionary lineages [cyt b gene, in particular, has been used in several fish population genetic studies. Sousa-Santos et al. [cyt b to determine the factors that affect the pattern and levels of genetic diversity in the different populations of native Portuguese cyprinids. Mitochondrial DNA cyt b was also used by Ju et al. [Rhinogobius giurinus in East Asia. Faulks et al. [cyt b to assess the genetic diversity and structure of the critically endangered endemic Edgbaston goby Chlamydogobius squamigenus from Great Artesian Basin springs at Edgbaston, Queensland, Australia. Their study aimed to assist management agencies in establishing relocated populations of C. squamigenus. Khosravi et al. [cyt b to molecularly identify Carassius species in Iran and investigate the origin and pathways of introduction of the non-native Carassius species. Genetic variation analysis was performed by Jose et al. [Carinotetraodon travancoricus using mtDNA cyt b. Abbas et al. [Elopichthys bambusa in Yangtze River using mtDNA cyt b. Recently, Gong et al. [cyt b to investigate the levels of genetic diversity and genetic structure in populations of the endemic cyprinid Schizopygopsis younghusbandi in the Yarlung Tsangpo River drainage, Tibetan Plateau. They also examined the correlation between the genetic structure of S. younghusbandi and landscape determinants such as longitude and elevation.Several mitochondrial DNA (mtDNA) markers, including lineages . The mtDs et al.  used mtDu et al.  to invess et al.  also usei et al.  used mtDe et al.  on the ts et al.  studied g et al.  used mtDG. aureus from nine major lakes in the Philippines. The genetic data from this study can be used in formulating possible conservation measures in the future and for selecting possible sources of broodstock for aquaculture.This study aimed to determine the genetic diversity, relatedness, and divergence among native and translocated populations of 2 and a maximum and average depth of 7.3 m and 2.8 m, respectively. It has an elevation of less than 2 m above sea level. The lake was originally part of Manila Bay before a blockage was formed by the movement of the West Marikina Valley Fault [Laguna de Bay  is located in Calabarzon region in Luzon Island, the largest island of the Philippines. It is bounded by Laguna, Rizal, and Metro Manila provinces. It is the largest lake in the Philippines. It has a surface area of 911–949 kmey Fault . Despitend largest lake in the Philippines. It has a total surface area of 357 km2, a maximum depth of 112 m, and an elevation of 702 m above sea level [Lake Lanao  is located in the province of Lanao del Sur in the northern part of Mindanao Island. It is the 2ea level . The lakrd largest lake in the Philippines. It has a surface area of 236.9 km2, an elevation of 2.5 m above sea level, and a mean and maximum depth of 90 m and 189 m, respectively [G. aureus in the lake were collected from San Nicolas and Talisay, Batangas.Lake Taal , formerly known as Bombon Lake, is also located in Calabarzon in Luzon Island, specifically in Batangas, a province bordering the southwest of Laguna province. It is the 3ectively . It is aectively . Historiectively , 44, butectively . The vioectively . This doectively . The oriectively , 46. Sin2 wide, making it the 5th largest lake in the Philippines [Lake Naujan  is situated in the northeastern part of Mindoro Island in the province of Oriental Mindoro. It is geographically shared by Naujan, Victoria, Socorro, and Pola municipalities. Mindoro is an isolated island surrounded by seawater. Lake Naujan is about 81.25 kmlippines . Lake Nalippines  and has lippines . It was lippines . The lak2 wide and considered the 6th largest lake in the Philippines [Lake Buluan  is found in the central part of Mindanao Island and bounded by the provinces of Sultan Kudarat and Maguindanao. It is about 130 km away from Lake Lanao. It is 61.34 kmlippines . It is alippines . It is alippines . The laklippines . Samples2, while Lake Buhi has an area of 17 km2; they rank as the 8th and 10th largest lakes in the Philippines, respectively [Lake Bato  and Lake Buhi  are both located in the Bicol peninsula in the southern part of Luzon. Lake Bato has an area of 28 kmectively . Both laectively . The disectively . During ectively  but not ectively , 49. Sam2 to 4.4 km2 and a depth that ranges from 5 m to 12.5 m [Lake Paoay  is situated in the province of Ilocos Norte in the northwestern part of Luzon. It has a surface area that ranges from 4 kmo 12.5 m . Lake Pao 12.5 m . It was o 12.5 m . Samples2 and an average and maximum depth of 5 m and 57 m, respectively [Lake Sebu  is located in the Municipality of Lake Sebu, South Cotabato, in the south-central region of Mindanao. It has a surface area of 3.54 kmectively . Lake Seectively . The samG. aureus were obtained from nine lakes (n = 30), Lake Lanao (n = 62), Lake Taal (n = 30), Lake Naujan (n = 30), Lake Buluan (n = 30), Lake Bato (n = 30), Lake Buhi (n = 30), Lake Paoay (n = 35), and Lake Sebu (n = 30). The map of the Philippines showing the sampling sites (http://qgis.osgeo.org [http://www.diva-gis.org/gdata) [A total of 307 individuals morphologically identified as ne lakes  in the Png sites  was drawsgeo.org . The basg/gdata) .Glossogobius species in each of the lakes was carried out through DNA barcoding and was published in a separate paper (see reference 10 for details). A small piece of muscle tissue behind the pectoral fins was obtained from the right side of each specimen, and the tissues were stored in separate tubes with absolute ethanol at -20°C. The fish were fixed and stored as voucher specimens in 10% formalin and deposited at the Institute of Biology, University of the Philippines, Diliman, Quezon City, Philippines.All fish samples were purchased from fish vendors and fishermen in each lake. This study did not require ethical approval because the used fish samples are commonly sold as food fish in the markets and were already dead when purchased. The specimens were identified morphologically based on Akihito and Meguro , Hammer ® Genomic DNA Purification Kit , InstaGene™ Matrix (BIO-RAD) or Vivantis GF-1 Tissue DNA Extraction Kit  following the manufacturers’ protocols. The extracts were stored at -20°C until further use. DNA yield was quantified using NanoDrop™ 2000/2000c spectrophotometer.Approximately 20 mg of tissue from each specimen was used for DNA extraction using Promega Wizardcyt b gene was amplified using the following primers that were designed with Primer3Plus [5’–AACCACCGTTGTTATTCAACTACA–3’) and reverse primer GCYTB2H (5’–AGGAAGTATCACTCTGGYTTRATATG– 3’). Polymerase Chain Reaction (PCR) was done in 25 μL reaction mixtures with 0.5 μL of 0.05 mM dNTP, 1.25 μL of each primer diluted to 0.01 mM, 2.5 μL of 10x PCR buffer, 0.125 μL of Taq polymerase (5u/ μL), 17.375 μ of ultrapure water and 2.0 μ of DNA template (5.0–25.0 ng/μL). The reactions were run using the following conditions: initial denaturation step of 95°C for 8 min, 35 cycles of 94°C for 30 s, 55°C for 35 s, and 72°C for 2 min, a final extension step of 72°C for 10 min, and 4°C for storage.The mtDNA mer3Plus : forward™ GeneJET Gel Extraction Kit . Purified PCR products were then sent to Macrogen, Inc.  for bidirectional DNA sequencing using Applied Biosystems™ 3730xl DNA analyzer.PCR products were analyzed by electrophoresis using 1% agarose gel with ethidium bromide. Approximately 800 bp-sized bands were excised from the gel and purified using QIAquick Gel Extraction Kit  or Thermo ScientificGlossogobius species or from contamination. The consensus sequences were submitted to GenBank and were assigned accession numbers MN306344–MN306498, MZ355635–MZ355664, OP086076–OP086097, OQ032698–OQ032789, and OQ551102–OQ551109. All the consensus sequences were aligned using ClustalW multiple alignment as implemented in BioEdit Sequence Alignment Editor version 7.2.6 [The consensus sequence of each specimen was assembled, edited, and trimmed using the pregap4 and gap4 tools in the Staden Package . Each coon 7.2.6 .h), nucleotide diversity (π), number of variable sites (S), and average number of nucleotide differences (k) were computed using DNA Sequence Polymorphism version 6.12.03 (DnaSP) [FST) and analysis of molecular variance (AMOVA) in Arlequin 3.5.1.5 [D [FS [Genetic diversity parameters such as haplotype diversity ( (DnaSP) . Populat 3.5.1.5 . To test5.1.5 [D  and Fu’s5 [D [FS  neutrali5 [D [FS , 67 were5 [D [FS . To visu5 [D [FS  was used5 [D [FS . Phyloge5 [D [FS . The Aka5 [D [FS  were useGlossogobius aureus had at least 99% sequence identity with G. aureus mtDNA cyt b in GenBank. These included all the specimens from Laguna de Bay (n = 30), Lake Taal (n = 30), Lake Naujan (n = 30), Lake Buluan (n = 30), Lake Bato (n = 30), Lake Buhi (n = 30), Lake Paoay (n = 35), Lake Sebu (n = 30) and 15 out of the 62 specimens from Lake Lanao. The remaining 47 specimens from Lake Lanao had the closest similarity to G. aureus mtDNA cyt b in GenBank but with percentage identities of only 95%. Hence, the 47 specimens of this group were designated as Glossogobius cf. aureus because they fit the morphological description of G. aureus, but they show genetic divergence from the other specimens based on mtDNA cyt b sequence. Nucleotide composition analysis of the 757-bp region of the mtDNA cyt b gene showed the following average frequencies: 31.89% for C, 27.68% for T, 24.15% for A, and 16.27% for G; showing obvious anti-G bias. A+T content (51.84%) was higher than C+G content (48.16%). Moreover, a total of 65 polymorphic sites (17 of which are singleton variable sites and 48 are parsimonious informative sites) defined the 29 haplotypes that were detected. Of the 29 haplotypes, a total of 25 (86.21%) were private . Only four (13.79%) haplotypes were shared among three to seven populations  analysis revealed that 260 of the 307 specimens morphologically identified as ulations , Fig 2. ulations . Nucleotulations .P-value = 0.000) genetic structure among the ten populations of G. aureus. The results showed that variation among populations (91.44%) was higher than variation within populations (8.56%). These values are supported by both chi-square test  and overall FST value (0.914). FST values were significant for all population pairs except between populations from Laguna de Bay and Lake Bato , Laguna de Bay and Lake Buhi , and Lake Bato and Lake Buhi  , large , and very large  . GeneticG. aureus specimens from Lanao, Taal, Naujan, Buluan, Paoay, and Sebu lakes. The second cluster (Cluster B) consists of G. aureus specimens from Laguna de Bay, Naujan and Buluan lakes; the third cluster (Cluster C) consists of specimens from all G. aureus populations except Lake Paoay, while the fourth cluster (Cluster D) is composed of all G. cf. aureus from Lake Lanao. All the specimens from Lake Paoay were grouped together in Cluster A, while all the specimens from Lake Bato and Lake Buhi were grouped together in Cluster C. This grouping is consistent with the results from maximum likelihood tree constructed using Tamura-Nei (TrN) with gamma distribution model of nucleotide substitution [The median-joining network analysis  separatetitution  Fig 3)..G. aureuD and Fu’s FS tests, including associated P-values, are shown in D and Fu’s FS tests. This suggests that the mutations in the mtDNA cyt b region are not neutral and that the population may be undergoing selection pressure in this gene region. The hypothesis of neutral evolution was rejected for Laguna de Bay population. On the other hand, populations from Lanao, Taal, Naujan, Buluan, Bato, Buhi, Paoay, and Sebu lakes had neutral mutations. The mismatch distribution plots of all populations except Buhi and Paoay populations are shown in r) [G. aureus populations from Lanao, Taal and Bato lakes.The results of Tajima’s wn in r)  under boGlossogobius, which they named as G. aureus [Glossogobius specimens from various lakes and other collection sites in the Philippines. They were the first ones to report the presence of G. aureus in Laguna de Bay, Taal, and Lanao lakes. Prior to 1975, only the other large-sized goby Glossogobius giuris was reported by Herre to be present in Laguna de Bay [Glossogobius giuris and G. aureus are morphologically similar and are commonly mistaken for each other [Glossogobius spp. in Laguna de Bay [Glossogobius species in these lakes are G. aureus. Based on the DNA barcoding studies [G. aureus is considered native to Laguna de Bay, Lake Taal, Lake Naujan, Lake Buluan, Lake Bato, and Lake Buhi. On the other hand, G. aureus is known to be an introduced species in Lake Lanao and Lake Paoay, as its presence was only detected in the 1960s in Lake Lanao [Glossogobius species in Lake Lanao revealed the presence of two lineages, G. aureus and G. cf. aureus, that are separated by a mean pairwise genetic distance of 3.7% based on mtDNA cytochrome c oxidase I (COI) gene [G. aureus and 22 G. cf. aureus) that were DNA barcoded [cyt b sequences of these 32 specimens generated in this study also formed two lineages that are concordant with the results obtained using COI sequences. The G. aureus population in Lake Sebu is also most likely introduced. Herre conducted extensive surveys of fishes in Mindanao lakes such as Lake Mainit, Lake Lanao, Lake Dapao, Lake Nunungan, and Lake Buluan in the 1920s [G. giuris in Lake Buluan [Glossogobius species in Lake Sebu. The only fish Herre mentioned as present in Lake Sebu is Chana striata [Glossogobius sp., G. celebius [G. circumspectus [G. aureus in Lake Sebu. In this study, we examined the pattern of genetic diversity, relatedness, and divergence among six native  and three translocated lacustrine populations  of G. aureus in the Philippines using the mtDNA cyt b.In 1975, Akihito and Meguro described a new species of a de Bay , 75, Taaa de Bay , 46, 75,a de Bay , 46, 75,a de Bay , 75, Bata de Bay , 75, anda de Bay , 75 lakech other . DNA bara de Bay , 77, Laka de Bay , 76, Laka de Bay , Lake Bua de Bay , Lake Baa de Bay  and Lakea de Bay  revealed studies , 76, 77  studies , 75, G. ke Lanao  and in 1ke Lanao . DNA barOI) gene . All thebarcoded  were alshe 1920s , 79, 80.e Buluan , a lake celebius  and G. cmspectus , in LakeG. aureus in nine Philippine lakes. High haplotype but low nucleotide diversities were observed in the native populations of G. aureus from Taal, Naujan, and Buluan lakes. High haplotype diversity and low nucleotide diversity can be attributed to a recent population expansion after a low effective population size caused by bottlenecks or founder events [Glossogobius aureus is an amphidromous fish that spends its planktonic larval stage in marine environment and ascends to freshwater habitat to mature and reproduce. The fact that the species inhabit ecosystems with high environmental heterogeneities may likely account for the high levels of haplotypic diversity observed. Since G. aureus are native to Taal, Naujan, and Buluan, and these lakes are large and still have a connection to the sea, these may explain why these populations were able to expand after a bottleneck event. These population expansions were also supported by the star-shaped haplotype networks detected for populations of G. aureus from Lake Taal and Lake Naujan  [Schizopygopsis younghusbandi  [Glossogobius callidus  [Schizopygopsis younghusbandi  [Values obtained for nucleotide and haplotype diversities can be used to infer the demographic history  of nativr events . Large er events , environr events , 86, bror events , 32 havee Naujan , unimoda= 0.36%) , river gto 0.5%) , and sch= 0.33%) .G. aureus, through the river. Laguna de Bay was then completely isolated; hence, the G. aureus population in the lake became landlocked as it had no opportunity to interact with other populations. This isolation prevented further gene flow between the population in Laguna de Bay and other populations.Both the haplotype and nucleotide diversities are considered low  for the Glossogobius aureus fry were possibly being caught indiscriminately along with another small goby species, the endemic sinarapan Mistichthys luzonensis. Sinarapan was recorded to have been overharvested from the 1960s to 1970s with the use of fine-meshed nets [Glossogobius aureus population in Lake Buhi is represented only by one haplotype, Hap2, even though the samples used in this study came from different batches collected at different times. The presence of only one haplotype might be due to a combination of founder event and genetic bottleneck. Lake Buhi is only three centuries old [G. aureus, from entering the lake, thus rendering the Lake Buhi’s G. aureus population landlocked. From 1975 to 1978, motorized push nets were being operated in Lake Buhi to increase the catch of sinarapan [sinarapan but also of the fry of other fish species. The use of motorized push nets did not only result in overharvesting of the fishes in Lake Buhi but also disturbed the lake’s aquatic vegetation that served as spawning grounds and hiding places for goby species from predators [Aulacoseira granulata was documented to be the dominant diatom species in Lake Buhi, indicating that the lake was under pollution stress [Both the haplotype and nucleotide diversities are also considered low  for the hed nets –96. Glosries old . It is pries old , 97, preinarapan . This fin stress . Since 1n stress .G. aureus in Lake Sebu also had both low haplotype and low nucleotide diversities. This can be attributed to founder effect because of the high likelihood that G. aureus has been introduced into Lake Sebu. Taxonomic study on Lake Sebu fishes is scanty. The presence of Glossogobius species in Lake Sebu was only recorded in 2015 [The population of  in 2015  and 2016 in 2015 . The low in 2015 , January in 2015  and Janu in 2015 . SpecimeG. aureus in Lake Paoay is also represented only by one haplotype (Hap5) and consequently, the haplotype and nucleotide diversity levels are zero. The presence of only one haplotype in Lake Paoay population might be due to the combination of founder event and lack of gene flow [G. aureus populations, possibly due to isolation. In the study of Santos et al. [Leiopotherapon plumbeus, it was found that the population from Lake Paoay was also the most distinct and the authors attributed this to isolation.Just like in Lake Buhi, the population of ene flow . The speene flow . Lake Paene flow . The pres et al.  on the mG. aureus in Lake Taal, Lake Naujan, and Lake Buluan than in introduced populations in Lake Lanao and Lake Paoay. Introduced populations tend to exhibit low diversity when only few individuals, carrying small fraction of the original DNA composition of the source population [Pangasius sp. [Channa striata [Silurus glanis [Hypophthalmichthys nobilis [Pangasius pangasius and low genetic diversity in introduced and hatchery-bred Thai and Vietnam striped catfish Pangasius hypophthalmus. Alam et al. [Channa striata populations and low diversity in introduced populations. Castagne et al. [Silurus glanis. However, in certain cases, introduced populations exhibit higher genetic diversity than native populations. In this study, the native population in Laguna de Bay and Bato had lower genetic diversity than the introduced populations in Lanao and Sebu lakes. Xing et al. [Protosalanx hyalocranius population had higher genetic diversity than the native populations in China. Similarly, some of the introduced populations of European catfish Silurus glanis demonstrate higher genetic diversity than their native counterparts [G. aureus into Lanao and Sebu lakes happened more than once. This is supported by the existence of two haplogroups (Cluster A and Cluster C) of G. aureus in the Lanao and Sebu populations and the G.cf. aureus in Lake Lanao. Additionally, the suitable new habitat and the species’ ability to successfully establish itself in its new environment can contribute to high genetic diversity in the introduced population. This ability can be facilitated by the high plasticity of their life-history traits, high tolerance to adverse environmental conditions and high reproductive success. These were the explanations suggested by Agdamar and Tarkan [Carassius gibelio in Turkey.This study revealed that genetic diversity is higher in native populations of pulation , are trapulation . Similarsius sp. , striped striata , Europeas glanis , and big nobilis . Ha et a nobilis  also foum et al.  also foue et al.  also obsg et al.  also fouterparts . Multiplterparts , 106. Sid Tarkan  for the th century [Pangasius hypophthalmus in Bangladesh [Myxocyprinus asiaticus in the Wanzhou reaches of upper Yangtze River [Hypophthalmichthys nobilis and silver carp Hypophthalmichthys molitrix populations in the Pearl River [G. aureus, the introduced populations have low genetic diversity probably because of founder effect, isolation, and lack of deliberate effort to introduce G. aureus individuals from other genetically diverse sources. All the introduction events were accidental [The practice of introducing economically important fish to new regions dates back to as early as the 17 century , with th century . However century . Prior kngladesh . When inze River . Conversrl River . These nrl River . In the cidental , 78 and FST values indicate pairwise genetic differentiation among all populations of G. aureus in this study except those from Laguna de Bay, Lake Bato, and Lake Buhi. Genetic divergence is expected among the different populations of G. aureus because they are landlocked freshwater gobies that thrive in lakes that are separated by physical barriers, such as terrestrial and aquatic barriers. Ward et al. [Nannoperca oxleyana populations that thrive in areas that are separated by physical barriers such as sand dunes. This genetic differentiation may also explain why majority of the haplotypes in this study were not shared among the populations. A high genetic differentiation was also detected in different populations of other Philippine freshwater fishes, such as cyprinid Barbodes tumba [Giuris laglaizei [The chi-square test for genetic differentiation, AMOVA, pairwise and overall d et al.  found a d et al.  in Oxleyes tumba  and the aglaizei , which aFST value observed among the G. aureus populations in Laguna de Bay, Lake Bato, and Lake Buhi suggests common ancestry in the past. This is supported by a shared haplotype (Hap2) among the three populations. In 1981, Laguna Lake Development Authority established lake-based hatchery and nursery facilities in Laguna de Bay [Glossogobius species as fish pens installed in Laguna de Bay served as breeding areas and refuges for other fishes, including Glossogobius [G. aureus populations in the two lakes was facilitated by stocking the lakes with tilapia seeds. Lake Sebu is represented by three haplotypes . Majority of the individuals (73%) belong to Hap5, which is shared with Lake Buluan population. Hap5 is also the most numerous (36.67%) among the five haplotypes in the Lake Buluan population.The nonsignificant a de Bay . Laguna a de Bay . It is psogobius . The modsogobius . It is pG. aureus.Both the MJN  and ML tG. aureus and G. cf. aureus, that are genetically highly differentiated  and are grouped into different clusters in the MJN , introduced milkfish larvae to Lake Lanao [COI sequences submitted to GenBank, Glossogobius cf. aureus were also detected in Taiwan , Pangasinan, Philippines , and San Jose, Antique, Philippines . The percentage identity between our G. cf. aureus and these three sequences ranged from 99.84% to 100%. Taiwan and Philippines are among the leading countries in milkfish production [G. cf. aureus.It is worth noting that specimens from Lake Lanao are divided into two lineages,  the MJN  and ML t the MJN . The pree et al. . It is pke Lanao . Based ooduction . The Phioduction , which ioduction , 118. Hooduction , and it G. aureus populations highlight the need for immediate conservation intervention. The best conservation option for populations of G. aureus that are carnivorous [G. aureus fishing during the peak spawning season must be implemented. Glossogobius aureus is reported to have a higher spawning activity during the months of August to October [The low levels of nucleotide diversity observed in all nivorous , geneticnivorous . Conservnivorous , and estnivorous . Regulat October .G. aureus in the Philippines exhibit low nucleotide diversity that ranges from 0.00% to 0.380%. These populations might benefit from augmenting or stocking individuals from closely related viable native or wild populations, preferably populations of higher genetic diversity [G. aureus, is being translocated; (3) systematic documentation of the stocking activity such as information on the origin, quantity, and genetic diversity information of the stock released to the recipient lake; and, (4) post-stocking monitoring and evaluation of the effect of the stocking activity on the genetic characteristic of the recipient population, as stocking activity can alter the genetic diversity of the recipient population [FST value between Laguna de Bay and Lake Bato populations is not significant, the two populations might benefit from reciprocal stocking. This will facilitate the transfer of unique haplotype from one lake to the other, thereby increasing the genetic diversity in each lake. Genetic diversity of Lake Buhi population can be increased if individuals from Laguna de Bay and Lake Bato are translocated into Lake Buhi, as the population of G. aureus in these three lakes are not genetically differentiated based on the results of this study.Introduced or non-native populations of iversity . This stiversity  or at leiversity . Howeverpulation . Since tcyt b in this study to investigate the pattern of genetic diversity, relatedness, and divergence among native and translocated populations of G. aureus in nine Philippine lakes. High haplotype and low nucleotide diversities were observed in the native populations of Taal, Naujan, and Buluan lakes. In contrast, low haplotype and low nucleotide diversities were observed for the native populations in Laguna de Bay, Bato, and Buhi lakes and introduced populations in Lanao, Paoay, and Sebu lakes. All the populations studied had low nucleotide diversity, indicating that these populations experienced bottleneck. Thus, this study calls for immediate conservation management intervention. Our results also showed genetic differentiation in majority of the populations. This indicates that each population must be managed separately, especially Taal, Lanao, Naujan, Buluan, Paoay and Sebu populations as these populations exhibit moderate to very high FST values. Since Lake Buhi and Lake Paoay populations are represented each by a single haplotype, individuals from other populations can be introduced into these lakes to increase their genetic diversity [G. aureus in other water bodies and other countries must also be studied to widen the possible sources of broodstock if the fish is to be used for aquaculture.We used mtDNA iversity . Populat


Apostichopus californicus, is one such exploited species whose management could benefit from further characterization of population structure. Using restriction site-associated DNA (RAD) sequencing, we developed 2075 single nucleotide polymorphisms (SNPs) to quantify genetic structure over a broad section of the species’ range along the North American west coast and within the Salish Sea, a region supporting the Washington State A. californicus fishery and developing aquaculture production of the species. We found evidence for population structure  = 0.0068) with limited dispersal driving two patterns of differentiation: isolation-by-distance and a latitudinal gradient of differentiation. Notably, we found detectable population differences among collection sites within the Salish Sea (pairwise FST = 0.001–0.006). Using FST outlier detection and gene-environment association, we identified 10.2% of total SNPs as putatively adaptive. Environmental variables  from the sea surface were more correlated with genetic variation than those same variables measured near the benthos, suggesting that selection on pelagic larvae may drive adaptive differentiation to a greater degree than selection on adults. Our results were consistent with previous estimates of and patterns in population structure for this species in other extents of the range. Additionally, we found that patterns of neutral and adaptive differentiation co-varied, suggesting that adaptive barriers may limit dispersal. Our study provides guidance to decision-makers regarding the designation of management units for A. californicus and adds to the growing body of literature identifying genetic population differentiation in marine species despite large, nominally connected populations.A growing body of evidence suggests that spatial population structure can develop in marine species despite large population sizes and high gene flow. Characterizing population structure is important for the effective management of exploited species, as it can be used to identify appropriate scales of management in fishery and aquaculture contexts. The California sea cucumber,  Over the last two decades, genetic studies have provided evidence that some marine species can develop spatial population structure despite very large population sizes and planktonic larval dispersal –3. The tEstimates of genetic population structure are also useful in informing best practices for aquaculture. Sourcing wild broodstock is a common practice in aquaculture production of species that are outplanted within their native range , 18. If Apostichopus californicus, supports wild fisheries and is a novel aquaculture species ; global genic differentiation test, p < 0.001), although clustering analyses with ADMIXTURE provided the strongest support for the model with a single underlying population .Global population differentiation was highly significant using all SNPs *x + 3.5*10−3) between pairwise linearized FST using all SNPs and in-water distance *x + 1.8*10−3) with a remarkably similar slope, but it was not significant in the southern samples *x + 3.2*10−3). Assuming an effective population density between 100 and 10,000 adults per km, this slope suggested a mean dispersal distance per generation of 2 to 20 km , with 8 of the 12 not statistically significant tests corresponding to collection site pairings with the small collection from Auke Bay, AK  and southern (Washington and Oregon) parts of the species range (FST = 0.005–0.015). Notably, pairwise differentiation between collection sites within Washington was broader in range and at times greater than that within Alaska, despite substantially smaller in-water distances .Of all 36 pairwise site comparisons using all SNPs , 24 reve Bay, AK . Only onFIS increased from North to South, while expected heterozygosity was similar across the range  SNPs as putatively adaptive  were identified as putatively adaptive. Each of these SNPs was correlated with an average of 2.5 environmental predictors and at least one SNP was correlated with each of the 29 environmental predictors (Table K in Using p = 0.063) and for the first axis , and the model using temperature and current velocity predictors (at sea surface or near-bottom) was significant at the full model level  and for the first axis . For the RDA using sea surface predictors, the first RDA axis explained 24.8% of the variance, and PC 2 had the greatest loading on the first RDA axis . We retained two RDA models (out of 8) for statistical significance: the model using sea surface predictors was marginally significant at the full model level  were only identified by Bayenv2  putatively adaptive SNPs, of which 41 (19.4% of 211 putatively adaptive SNPs) were detected with either Fig D in .Bayenv2, 15 SNPs matched to gene ontologies  developmental processes, 2) cell organization and biogenesis, 3) transport, 4) protein metabolism, and 5) stress response (Table N in Only 19 (9% of 211) putatively adaptive SNPs matched to gene ontologies, and 15 (79% of 19) matched to more than one gene ontology. Of the SNPs identified as putatively adaptive using tologies . Of thesΦCT /ΦSC) was higher using putatively adaptive SNPs for the State and NPC groupings, and higher using putatively neutral SNPs for the Victoria Sill and Admiralty Inlet groupings. Mantel tests for correlation among linearized FST and in-water distance were significant using putatively neutral SNPs *x + 1.7*10−3) and putatively adaptive SNPs  *x + 8.8*10−3), and not significant in samples from Oregon and Washington x + 1.6*10−2; R2 = 0.6757, p > 0.05, y = (1.5*10−6)*x + 9.5*10−4) or southern collection sites *x + 1.8*10−3). Correlation among linearized FST and in-water distance was strongest and the slope of the linear regression was greatest using putatively adaptive SNPs: the slope of the linear regression using putatively adaptive SNPs was nearly 7 and 20 times greater than the slopes using all SNPs and putatively neutral SNPs, respectively. Assuming a population density between 100 and 10,000 adults per km, we estimated mean dispersal distance to be between 3 and 34 km using putatively neutral SNPs and between 1 and 8 km using putatively adaptive SNPs  . Similarive SNPs .FST within the range observed in this study occurred in the long-term  with small effective population size (Ne = 500) and high migration (m ≥ 3%), with moderate effective population size  and moderate migration (1% ≥ m ≥ 3%), and with large effective population size  and small to moderate migration (0.1% ≥ m ≥ 1%). Most simulations that produced pairwise FST estimates within the range observed in this study were cases with moderate to large effective population size  and migration rates below 10%. Simulated pairwise FST remained below the lowest empirically observed pairwise FST in this study in the long-term  only when as effective population size was at least moderate  and migration rate was high (m ≥ 10%). Under realistic conditions , simulated pairwise FST was within the range of empirical pairwise FST when migration rates were 0.1–1.0%  and have undergone many  generations of drift after splitting from a single ancestral population. These estimates corresponded well to average dispersal distances per generation from isolation-by-distance patterns, which were on the scale of dozens of kilometers despite the long pelagic larval duration.In this study, we quantified patterns of population differentiation in A. californicus in British Colombia is due to limited dispersal across the bifurcation zone of the North Pacific Current. The North Pacific Current has also been identified as an oceanographic barrier to gene flow in the Bat Star, Patiria miniata [Sebastes helvomaculatus [A. californicus along the coast of British Columbia [Holothuria edulis [H. scabra [H. nobilis [Broad-scale population structure that we observed can be described by two patterns: differentiation across the bifurcation zone of the North Pacific Current and isolation-by-distance. Many species of marine invertebrate exhibit population differentiation along a latitudinal gradient, with the region between Alaska and Oregon as a known region of divergence , 88. Xue miniata , 89, andaculatus . In addiaculatus  detectedColumbia , but onlColumbia . Here, Ia edulis , H. scab. scabra , and H.  nobilis . Our res nobilis , 95, 96.FIS from north to south, a pattern that can also be observed in the data of Xuereb et al. [FIS values in the south could be an effect of selection against heterozygotes caused by high genetic load of deleterious mutations, as commonly observed in bivalves [FIS values in southern samples. Heterozygote deficiencies can also be caused by mixtures (Wahlund effect) of populations, cohorts or local demes [Additionally, we found a subtle pattern of decreasing observed heterozygosity, constant expected heterozygosity and increasing b et al. . In tempb et al. , oppositbivalves , but alsbivalves . Such sebivalves , thus exal demes –althoughal demes  in the sal demes , 103. Alal demes , and expNotably, we found that geographic distance was more strongly correlated to genetic distance among collection sites using putatively adaptive SNPs than using putatively neutral SNPs, though neither relationship was significant in the southern samples. The higher slope of the relationship resulted in lower estimates of mean dispersal estimates using putatively adaptive SNPs than putatively neutral SNPs. This finding suggests that selection gradients may reduce effective dispersal distances, leading to stronger signals in isolation-by-distance from neutral processes alone . One exckm (in-water distance) apart, consistent with earlier findings on differentiation as close as 60 km in A. californicus [km apart in the Tar-spot Sea Cucumber, Cucumaria pseudocurata [A. japonicus [FST in high latitude species [At finer scales, we found detectable differences among most collection site pairs and between collection sites close together as 110 fornicus  and 100 docurata . The samaponicus , and met species . AlternaFST outlier detection were identified using both methods and were also identified as putatively adaptive using at least one gene-environment association method. Using Bayenv2, we detected many SNPs that were not identified as putatively adaptive compared to redundancy analysis, consistent with expectations that Bayenv2 may produce more false positives compared to redundancy analysis [A. japonicus juveniles [A. japonicus larvae [Bayenv2 and the occurrence of temperature variables as top predictor variables is consistent with RDA results: the significant RDA models were those with environmental predictors at the sea surface and environmental predictors related to temperature and current velocity. Thus, we hypothesize that selection at early life history stages, when larvae are at the sea surface, drives adaptive differentiation in A. californicus, with salinity, nitrate, pH, and temperature as possible selection factors.We found evidence for putatively adaptive differentiation, corroborated by multiple lines of investigation. The majority of SNPs identified as putatively adaptive using analysis . The fivanalysis . Temperaanalysis  and haveuveniles  and grows larvae . The greWe note that we cannot exclude balanced polymorphism as an alternative explanation to observed putative adaptive differentiation: it is possible that selective mortality at early life stages leads to observed patterns of putative adaptive differentiation in adults (as were sampled here), but that genetic variation is maintained across time through high gene flow . As mentIn generating hypotheses about which environmental predictors are potential drivers of selection, it is prudent to be cautious and highlight 1) spatial auto-correlation of predictors, particularly in light of correlation between geographic distance and genetic distance using putatively adaptive SNPs as observed here, 2) correlation among environmental predictors and 3) the incomplete genome coverage of reduced representation approaches such as RAD sequencing. Almost all environmental predictor sets contained spatially auto-correlated variables, evidenced by high variance inflation factors for spatial variables and environmental predictor variables in partial RDAs . AdditioA. japonicus, high salinity conditions were correlated with downregulation of acetylcholinesterase, the enzyme that terminates signal transduction. Inhibition of this enzyme can lead to excessive stimulation of nerve and muscle tissue, ultimately leading to paralysis and even death in some species [A. californicus across environmental gradients or in common garden experiments with varying environmental treatments to build cases for which environmental factors shape adaptive differentiation and through which physiological pathways.For the environmental variables with the most correlated SNPs, and for such SNPs with associated biological processes, signal transduction appeared among the most common biological processes. Future research investigating the connections between genotype, phenotype, and observed patterns in adaptive differentiation may start with the potential connection between signal transduction pathways and salinity, nitrate, temperature, and pH. Others have identified signal transduction genes as part of an adaptive response to salinity adaptation and stress in sea cucumbers . In A. j species . TranscrA. californicus, though those implications may differ depending on the management aim. For example, marine protected areas for A. californicus may need to operate at smaller scales than expected from signals of genetic differentiation at broad scales, to maintain demographic connectivity and prevent overexploitation of local populations. Designation of management units for fisheries may need to occur at similar scales, because fishery management units are used to prevent overexploitation of local populations [Simulation results suggest limited dispersal among collection sites: if effective population sizes were at least 10,000 and separated at least 1,000 generations ago, genetic differentiation suggested migration rates of 0.1–1% . Here, oulations , largelyA. californicus, it is likely that IBD and oceanographic barriers such as the NPC shape population structure, such that variation may be gradual in many parts of the range at fine and broad scales, but that oceanographic barriers may create distinct boundaries at fine scales [km) may be a useful starting point for defining boundaries in delineation of management units, particularly for fishery management units. As populations are further subdivided for management based on genetic data, decision-makers must also consider the risk of reduced precision associated with the need to complete stock assessments for more stocks with less data [Gradual differentiation associated with the IBD pattern of population structure poses a unique challenge in determining the spatial scale of management units for fisheries and aquaculture. Without distinct boundaries between populations, it is difficult to delineate management units. However, in species with IBD, genetic variation can be preserved even if arbitrary boundaries are chosen . In A. ce scales . Assessiess data . From a S1 File(PDF)Click here for additional data file.


Chrysomela aeneicollis, an emerging natural model system for understanding how organisms respond to climate change. We used a genotype–environment association approach to identify genomic signatures of local adaptation to microclimate in populations from three montane regions with variable snowpack and a coastal region with no snow. We found that both winter-associated environmental variation and geographical distance contribute to overall genomic variation across the landscape. We identified non-synonymous variation in novel candidate loci associated with cytoskeletal function, ion transport and membrane stability, cellular processes associated with cold tolerance in other insects. These findings provide intriguing evidence that variation in snowpack imposes selective gradients in montane ecosystems.Organisms living in mountains contend with extreme climatic conditions, including short growing seasons and long winters with extensive snow cover. Anthropogenic climate change is driving unprecedented, rapid warming of montane regions across the globe, resulting in reduced winter snowpack. Loss of snow as a thermal buffer may have serious consequences for animals overwintering in soil, yet little is known about how variability in snowpack acts as a selective agent in montane ecosystems. Here, we examine genomic variation in California populations of the leaf beetle  For sLocal adaptation, which occurs when resident genotypes have a higher relative fitness in their local habitat than genotypes originating from other habitats, is an important mechanism by which genetic variation is maintained in heterogeneous environments –22. The Chrysomela aeneicollis, a well-described model species for understanding how climate change impacts montane ecosystems [In this study, we investigated relationships between microclimatic factors and genetic variation in the willow leaf beetle osystems ,33,34. Tosystems . During osystems . They ovosystems ,38.C. aeneicollis is found living on willows in cool, moist habitats separated by regions of arid or Mediterranean climates, resulting in highly fragmented distribution with little connectivity among populations [phosphoglucose isomerase, Pgi [Pgi and the mitochondrion reflect local adaptation [In western North America, ulations ,40. In Culations ,38,41,42ulations , suggestase, Pgi ,42,43. Pase, Pgi ,42,44,45aptation ,44,46, wHere, we address this gap by evaluating relationships between genomic variation and environmental conditions in locations where willow beetle populations occur in four distinct ecoregions of California . We quan. 2 (a)b) and that removed loci with low quality reads within populations .Illumina sequencing generated 5.06 billion paired-end reads from 175 individuals in 12 populations , of whic (b)The NicheMapR microclimate model simulated 24 variables for the 12 beetle populations that represent air, soil and snow conditions beetles experience throughout their life cycle . Simulated environmental variables demonstrated high sensitivity to the shade input parameter in the model , but relative multivariate environmental distances between populations were consistent between minimum and maximum shade conditions . Simulated microclimatic data under minimum shade conditions were more concordant with available empirical measurement based on RMSE , so downstream analyses were therefore conducted using simulated environmental variables under 10% shade. (c)b). Eastern Cascades and Coast Range (CR) ecoregions exhibited the greatest genomic divergence among populations, and population genomic variation in the SN and Central Basin ecoregions followed a latitudinal gradient . Individuals in the SN ecoregion show a strong pattern of genetic differentiation with latitude  than those for ‘mountain versus mountain' population pairs Partial redundancy analysis (pRDA)—The pRDA made it possible to identify specific genetic polymorphisms that were associated with environmental differences among populations. Among all California populations, the pRDA was globally significant  and maximum daily snowfall as significant environmental predictors of genomic variation . The first and second RDA axes also explained substantial proportions of genomic variation . Candidate SNPs were identified based on high correlation with temperature- and snow-related environmental variables (r > |0.65|) and z-score values of loadings of loci in ordination space . Based on these criteria, 107 SNPs were identified as candidate loci , and with MEM1 as the conditioning variable, the forward selection procedure identified only maximum daily snowfall as a significant predictor of genomic variation. Using the above candidate loci criteria, 116 SNPs were related to maximum snowfall .ate loci . Sixty-eLatent factor mixed model (LFMM)—The LFMM represents a second approach to identify SNPs related to environmental variability while accounting for overall population genetic structure. With all California populations, a LFMM was run with five estimated ancestry coefficients as latent factors to test single-locus relationships with annual air temperature range and maximum daily snowfall (ancestry coefficients shown in  (e)To reduce probability of false positive associations and narrow the search for candidate polymorphisms, we focused on SNPs identified by both pRDA and LFMM, and we assessed associations across coastal and montane populations and then again using only montane populations . In analyses with all populations, most SNPs correlated with annual air temperature range in pRDA were also identified using LFMM (67 of 70). A slightly lower proportion of SNPs associated with maximum daily snowfall based on pRDA were also identified by LFMM . In analyses with only montane populations, most SNPs correlated with maximum daily snowfall based on pRDA were also identified by LFMM (79 of 116), and 18 SNPs identified in the pRDA with only montane populations were also identified in analyses with coastal and montane populations .Analyses of all populations and only montane populations identified three non-synonymous SNPs associated with maximum daily snowfall found in genes coding for proteins involved in cell structure and movement (inverted formin-2 and microtubule-actin cross-linking factor). Analyses including all populations or only montane populations identified five non-synonymous SNPs associated with daily snowfall that are found in genes coding for proteins involved in ion transport or cellular membrane activity .. 3C. aeneicollis across California are differentiated across the nuclear genome, and we provide strong evidence that snow serves as a prominent selective gradient and driver of local adaptation across their geographical range. We show that both large-scale variation in snowfall across the California landscape and small-scale variation in snowfall within montane locations are associated with adaptive genetic variation. Specifically, we provide evidence that variation in maximum daily snowfall is linked to non-synonymous polymorphisms in genes associated with cytoskeletal motility, ion transport, and membrane structure and function, highlighting the potential role of adaptive protein modifications that could enhance insect cold tolerance in cold snowy regions.Detecting accurate signals of local adaptation in the genome requires linking observed genetic patterns to underlying selective features of the environment while accounting for associations imposed by neutral processes. Here, we demonstrate that populations of the willow leaf beetle  (a)C. aeneicollis living in different regions of California are genetically differentiated across loci in the nuclear genome. Three of four ecoregions sampled show substantial levels of genetic divergence among them, such that North Coast populations are distinct from those in the Eastern Cascades ecoregion, and both of those populations are distinct from populations in the SN and Great Basin  were strongly differentiated at mitochondrial and nuclear genetic markers, suggesting that geographical isolation among these regions predates recent fluctuations in the extent of glaciation over the past 50 000 years. Isolation of populations at mitochondrial loci was greater than nuclear genes, but there was overall agreement among loci that differentiation among geographical regions was substantial, which would contribute to conditions favouring local adaptation [The strong pattern of geographical differentiation of genomic variation across California was illustrated for the first time in the present study, but it is consistent with findings of Dellicour et al. . The earaptation . To date (b)C. aeneicollis indicate that the relationship between elevation and cold exposure in soil is strongly nonlinear, with cold exposure peaking at mid-elevation montane populations that are not buffered by persistent snow cover [Identifying climatic variables that act as drivers of spatially varying selection will be critical for predicting evolutionary responses to climate change and environmental disturbance . Among aow cover . Since sow cover ,50. ThisC. aeneicollis shows that air temperature varies between genetically differentiated populations and shows evidence of physiological adaptation to different thermal regimes [Prior work in SN populations of  regimes . However regimes ; thus, t (c)Genes containing non-synonymous SNPs associated with variation in snowfall encode proteins with functions related to ion binding, actin and cytoskeleton binding and organization, and membrane components; protein identifications were assigned with a high level of confidence, as all homologous proteins are present in other beetle species . These p (d)Chrysomela aeneicollis individuals have low levels of dispersal, with individuals often spending most of their life on a single host plant [In detecting genomic signatures of local adaptation, genotype–environment associations (GEAs) identified by various methods will depend strongly on demographic and sampling scenarios ,29,31,62st plant ,63.Nonetheless, correcting for population structure in pRDA can result in low power to detect true associations , and rec (e)A limitation of this study is sampling bias toward populations in the SN ecoregion relative to the other three eco-regions included in this study , which mN = 22 323 SNPs). While these thresholds did not alter overall estimates of population structure , candidate SNPs associated with environmental gradients in this study likely represent a subset of loci involved in local adaptation.Additionally, stringent SNP filter thresholds were applied to ensure quality genotypes within each population, resulting in a relatively modest set of polymorphisms a priori to 12 populations  designations . Beetle ulations  based on (b)C. aeneicollis reference genome [a). SNPs were removed if MAF across all individuals was less than 0.01 or if heterozygote frequencies deviated greatly from Hardy–Weinberg expectations (e.g. excess heterozygosity or inbreeding coefficient greater than ±0.5) . Finally, SNPs were retained if 70% of all samples and 70% of samples within each population showed a read depth between three and 30 and a genotype quality greater than 20  [Genomic DNA was extracted from individual beetles using NucleoMag Bacteria DNA Isolation kit , and whole-genome libraries were prepared following the plexWell library preparation protocol by the CCGP MiniCore. Paired-end sequencing (2 × 150 bp) was performed on an Illumina HiSeq4000 platform at UC Berkeley's QB3 Genomics Core Facility . Nextera adapter sequences and low-quality bases  were removed from each read using Trimmomatic v. 0.39 . Reads we genome  using the genome . Joint ge genome . Variante genome  (electroble S2c) . After fble S2c) ,76. Becable S2c) ,78, we a (c)NicheMapR [To obtain spatially explicit environmental variables representing local microclimate conditions across the life cycle, microclimate simulations were conducted for the 12 beetle populations using the biophysical modelling package icheMapR . The modicheMapR . The mid (d)K = 1–10) were tested using a sparse non-negative matrix factorization algorithm using the function ‘snmf' in the R package LEA v. 3.6.0 [K = 3–7 shown in electronic supplementary material, figure S11). The value of K that minimized cross-entropy and best explained genotypic data was five [K, and individual ancestry coefficients were extracted from the replicate with the lowest cross-entropy.Population structure from SNP genotypic data was assessed by estimating proportions of individual genomes originating from ancestral gene pools. A range of estimated ancestral gene pools . The ANCOVA tested whether the means of pairwise E v. 1.6 ,85. Pair v. 13.3 . Populat (e)vegan v. 2.6-2 [Signatures of local adaptation to climate were investigated using two GEA methods, pRDA ,87,88 anv. 2.6-2 . Outlierv. 2.6-2 ,75.a priori designated populations used previously in pRDA. The ‘lfmm' function in the LEA package was implemented using individual-level genotypic data (22 323 SNPs) as response matrix, forward-selected environmental variables used in pRDA as environmental predictors, and the best estimate of K  as number of latent factors. More detailed GEA methods are provided in electronic supplementary material, appendix 1.3.We then conducted a LFMM and documented overlap of detections with results from pRDA ,29,48,65 (f)Z-scoRE .


Following severe environmental change that reduces mean population fitness below replacement, populations must adapt to avoid eventual extinction, a process called evolutionary rescue. Models of evolutionary rescue demonstrate that initial size, genetic variation and degree of maladaptation influence population fates. However, many models feature populations that grow without negative density dependence or with constant genetic diversity despite precipitous population decline, assumptions likely to be violated in conservation settings. We examined the simultaneous influences of density-dependent growth and erosion of genetic diversity on populations adapting to novel environmental change using stochastic, individual-based simulations. Density dependence decreased the probability of rescue and increased the probability of extinction, especially in large and initially well-adapted populations that previously have been predicted to be at low risk. Increased extinction occurred shortly following environmental change, as populations under density dependence experienced more rapid decline and reached smaller sizes. Populations that experienced evolutionary rescue lost genetic diversity through drift and adaptation, particularly under density dependence. Populations that declined to extinction entered an extinction vortex, where small size increased drift, loss of genetic diversity and the fixation of maladaptive alleles, hindered adaptation and kept populations at small densities where they were vulnerable to extinction via demographic stochasticity. Novel or sudden environmental change can abruptly render a population poorly adapted to its habitat. When the environmental change is so severe that mean population fitness falls below the replacement rate, the population will go extinct if it does not adapt. This process of populations adapting to severe environmental change sufficiently to avoid extinction is called evolutionary rescue ,2.Gomulkiewicz & Holt  formalizModels examining rescue often rely on assumptions of density-independent growth , potentiAdditionally, models often assume that genetic variance in the population remains constant. This is present in several models e.g. ,9) relyi relyi9])Simultaneously incorporating realistic demographic and genetic processes such as negative density dependence and genetic erosion will improve the accuracy of predictions from models of evolutionary rescue for populations and species of conservation concern. Negative density dependence that is caused by intraspecific competition could constrain evolutionary rescue by accelerating population declines and slowing population recovery from reduced size . Some moModelling these demographic and genetic processes simultaneously is important, as they can interact and reinforce each other, creating a positive feedback loop of demographic and genetic decline. In the conservation biology literature, this positive feedback loop is called an ‘extinction vortex' . If popuHere, we relax the assumptions of density independence and constant genetic diversity to examine their simultaneous effects on evolutionary rescue and the extinction vortex, establishing a connection between these two major concepts in conservation biology. We do this by combining stochastic Ricker population growth and a genetic framework with a finite-locus quantitative trait in the same individual-based model. We simulate both rapid (15 generations) and longer (50 generations) timescales. We address two main objectives. First, we assess how negative density dependence interacts with initial conditions with respect to population size, additive genetic variance and degree of genetic maladaptation in the novel environment to influence trajectories of population size, probabilities of extinction and rescue, additive genetic variance and fitness over time. Second, we test for the presence of an extinction vortex and evaluate how negative density dependence influences this vortex.. 2 (a)We explored evolutionary rescue and extinction using stochastic simulations. This approach allowed us to incorporate several stochastic demographic and genetic processes that are important for rescue or extinction ,26. We dm biallelic diploid loci. Each allele-copy contributes a positive (1/(2√m)) or negative (−1/(2√m)) amount to the genotype of individual i, ig, which is the sum of allele-copies across all loci. From these allele frequencies, we estimate the additive genetic variance in the population, σa2, following )aptation  and thataptation  and inbraptation  are knowaptation .B) and increased extinctions b,c). Negative density dependence, rather than changing the demographic and genetic dynamics in the generations immediately preceding extinction, ‘widens' the opening to the vortex as populations are more likely to quickly decline to small size several generations before extinction (figure 5a). For managers of small, at-risk populations that are susceptible to sudden environmental change and extinction, intervention strategies such as translocation may be needed to pull populations out of the self-reinforcing demographic and genetic processes of the vortex [Challenging environments can draw populations into an extinction vortex . Althougfigure 5e vortex . (c)d and figure 3d). These ‘rapid rescues' appear to be a polygenic analogue of the phenomenon demonstrated in a haploid single-locus model [figure 3d). Another interpretation of this result is the fact that a sizeable proportion of small populations were rescued instantly owing to a ‘lucky’ initial genetic state Current definitions of rescue are difficult to operationalize in stochastic settings. Rescued is often understood to mean ‘not extinct', but this definition is lacking, as our analysis shows that even on relatively short timescales (50 generations in our simulations), populations can show sensible indications of ‘rescue' and still go extinct . Likewis (e)Our model makes several assumptions for simplicity and tractability that would be fruitful to explore in future work. First, our model shows that density dependence hinders adaptation through genetic erosion, but our model treats density dependence as independent of trait values and fitness. If, alternatively, density dependence acts differently on different trait values, then rates of adaptation will be affected by density dependence ,44. For  (f)a), leading to greater genetic erosion (A); in the event of future environmental change favouring another allele, its loss following the first environmental change event would hinder subsequent adaptation [Our simulations were on short timescales relevant to immediate or near-term conservation concern. However, we note that the reductions in population size or genetic diversity may be important on longer timescales, even after populations have been rescued. An effective population size on the order of hundreds is needed to reduce longer-term extinction risk due to accumulation of deleterious alleles –52. Popu3 erosion . Indeed, erosion . Furtheraptation .Here, we introduce a biologically realistic model for a more complete and applicable understanding of population rescue after environmental change, providing better prediction of potential outcomes and guidance for conservation and management as well as providing testable predictions for empirical validation. We demonstrate that negative density dependence impedes persistence in a novel environment, and that large and well-adapted populations previously predicted to be at minimal extinction risk ,3,24 do,


Argentina anserina, we measured ecological attributes  spanning a 1000 m elevation gradient, with high elevation populations at the range limit. We measured vegetative clonal potential in the greenhouse for populations spanning the gradient. We combined these data with a ddRAD-seq dataset to test the hypotheses that high elevation populations would exhibit ecological and genomic signatures of leading range edge populations. We found that population density on the landscape declined towards the high elevation limit, as is expected towards range edges. However, plant density was elevated within edge populations. In the greenhouse, high elevation plants exhibited stronger clonal potential than low elevation plants, likely explaining increased plant density in the field. Phylogeographic analysis supported more recent colonization of high elevation populations which were also more genetically isolated, had more extreme heterozygote excess and had smaller effective population size than low. Results support that colonization of high elevations was likely accompanied by increased asexuality, contributing to a decline in effective population size. Despite high plant density in leading edge populations, their small effective size, isolation and clonality could constrain adaptive potential.Climate change has influenced species distributions worldwide with upward elevational shifts observed in many systems. Leading range edge populations, like those at upper elevation limits, are crucial for climate change responses but can exhibit low genetic diversity due to founder effects, isolation, or limited outbreeding. These factors can hamper local adaptation at range limits. Using the widespread herb,  Climate change has elicited rapid geographic range shifts  often served as a source of genetic material for the rest of the range following historical glaciations  may vary with elevation if suitable habitat is unevenly distributed across elevation  is a self-incompatible perennial herb with the ability to spread clonally via above ground stolons . It is widespread in temperate regions in the Northern Hemisphere, where it spans wide elevation gradients in montane regions. We focused on 13 populations spanning >1000 m in Southwestern Colorado, with the highest elevation populations at the high elevational limit of the species range. We linked ecological attributes of populations (hereafter population attributes) that influence the genetic diversity and connectivity  with metrics of population genetic diversity to address the following questions and predictions:How do population density on the landscape, and plant distributions within populations change from lower elevation populations to the high elevation range edge? We predicted that population density on the landscape should decline towards the edge, and that high elevation populations will occupy smaller areas, and have lower plant density than lower elevation populations.Does the capacity for vegetative clonality exhibit an elevational cline? We predicted that if reproductive assurance is favored in leading edge populations, then vegetative clonality should increase with elevation.Are populations genetically structured by elevation, and are high elevation populations more genetically isolated? We predicted that high elevation populations should be less admixed, more recently colonized, and more genetically isolated than low.E, FIS, NE) covary with elevation, and directly with any population attributes? We predicted that high elevation populations should have lower NE and reduced HE due to recent founder events, and more extreme heterozygote excess (negative FIS) if they are more vegetatively clonal.Do metrics of within-population genetic diversity (HArgentina anserina (L.) Rydb. (Rosaceae) is a perennial herb, with a cosmopolitan global distribution in temperate environments. It reproduces both sexually via self-incompatible flowers . Specifically, we measured herbarium accession density within each 100 m elevational band within our sample region. We first downloaded all herbarium accessions of Argentina anserina from the SEINet database, an online portal for herbarium specimens throughout the Western USA. We identified all specimens in our sampling area within a bounding box of 1.5 degrees latitude (37.5 to 39.0) and one degree longitude (−107.5 to −106.5)  from SEINet with records of elevation. We again eliminated duplicate accessions prior to enumarting the number of non-focal taxa per 100 m elevational band. Finally, we downloaded a digital elevation model (DEM) raster of our sample area from the United States Geological Survey . Using the ‘raster’ package in R (Hijmans and van Etten 2) within each 100 m elevational band between 2200 and 3500 m, roughly the extent of our elevational sampling. We calculated a metric of standardized population density of A. anserina for each 100 m band as: A anserina accessions / total non-focal accessions / km2. We evaluated the relationship between standardized population density and elevation using a linear model in R (‘lm’) treating each elevation band quantitatively using its midpoint elevation.We used a collections-based approach to address whether the density of herbarium accessions of Question . Specifi.5) Fig. . To acco2) by walking straight-line transects and identifying population perimeters where plants were no longer present. We then ran two straight-line 50 m transects within each population, and within a 1x1m plot we scored the percent cover of A. anserina and the number of open flowers every 10 m (n = 10 plots per population). Plot locations were chosen randomly every 10 m by tossing a 1 × 1 m PVC-pipe square into the population.To assess how plant and flower distributions within populations changed over the elvation gradient (Question 1) we measured the following attributes in each population in Summer 2021: area of occupancy, plant density, plant patchiness, flower density and flower patchiness. Together, these estimate the census size of the population, the density and distribution of plants within populations, and the density and distribution of sexually reproductive units within populations. During peak flowering (late June- early July 2021) we estimated the area of occupancy  of A. anserina density across the 10 plots, and flower patchiness as the CV of flower number per plot. Populations with higher CVs for plant density and flower number among plots were more patchily distributed.We calculated population-level plant density as the average percent groundcover of +m from one another in the field in 2019, 2020, or 2021 and potted in a standard soil mix (3:1 Fafard to Turface). Each year, plants were subjected to a vernalization period in an environmental cold room set to 4.4 °C with total darkness for ~6 weeks once in the winter and summer. For 4 months in the Spring and 4 months in the fall, plants were kept in the greenhouse set to 15 °C. Plants were fertilized using slow-release Osmocote fertilizer  after each vernalization period. At the end of the plant growth cycle in Spring 2022, we measured the number of runners, the length of each runner, and the number of plantlets on each runner for 3–11 plants per population (mean = 7.2 + /−2.9 SD). In total we measured 280 runners across all plants. We calculated average runner length, average number of plantlets per runner, and plantlets per unit length of runner (cm) for each plant. From these metrics we generated population-level averages.To address whether clonal potential changed with elevation (Question 2), in 11 of the focal populations, we estimated metrics of vegetative clonal growth on plants in a common greenhouse environment. Plants from each population were collected 2N = 95) and extracted total genomic DNA using a modified cetyltrimethylammonium bromide extraction protocol . In each population, tissue from an individual was sampled at least every 2 m to reduce the likelihood of sampling ramets of the same genet. We selected 7–8 individuals per the 13 focal population (n = 2 (cstacks), p = 7, and r = 0.7 (populations). Different combinations of ustacks and populations parameters were tested before selecting parameters. Samples with a low number of reads retained  following ustacks were eliminated from the dataset. Two samples from each population with the highest number of reads were used to create the catalog and a single SNP per locus was obtained from the data (–write-single-snp function).To analyze the short read sequencing data, we used Stacks version 2.53  to reduce the numer of missing SNPs across individuals. However, 6 indiviuals with >30% missing SNPs were removed from the dataset due to convergence issues. Each individual removed was from a separate population, thus removal was not biased. With this pruned matrix, we modeled population genetic structure with 1 to 13 layers with 1000 iterations per MCMC chain. We measured the relative contribution of each additional layer to total covariance. Layer contributions ranged 4 to 77% with an average of 30% , we ran conStruct analysis , an outlier for FST among the lower elevation populations , and grouped genetically with high elevation populations in conStruct. This population is adjacent to a wastewater treatment plant, occurs on mounds of fill-dirt, and may be a very recent introduction. For these reasons, we excluded it from downstream analyses testing for elevational clines in population genetic parameters as well as ecological associations with genetic parameters.One low elevation population, MWL, was an outlier for FVisual assessment of conStruct figures suggested that individuals in high elevation populations were less admixed than those in low. To evaluate how the magnitude of admixture varied along the elevation gradient in a quantitatve manner, we identified the highest cluster assignment probability for each individual using 4 and 6 layers/clusters. For example, in a model with 4 clusters/layers, an individual that was highly admixed would have 25% ancestry from each cluster, and a highest membership of 25%. Conversely, an individual assigned to a single cluster would have 100% membership. We then calculated a ‘cluster uniformity’ index for each population as the average of the highest layer membership across individuals, with higher values indicating lower admixture. We modeled both the cluster uniformity index as a function of elevation with the best models (layers 4 and 6).Phylogeograhpic approaches are commonly used to evaluate the impacts of historical colonization on contemporary genetic structure of populations . We calculated pairwise vertical distances (m) between populations as the absolute difference in elevation and conducted Mantel’s test for isolation by vertical difference. We tested for isolation by topographic distance by estimating least-cost paths between populations based on topography. Because gene flow between populations via pollen or seed is likely inhibited not only by distance but topography, accounting for topography has the potential to provide a better metric of functional distance between populations. Using the DEM raster of our sampling area, we plotted each of the 12 populations and calculated paths between points using ‘topoDist’ , vertical distance (elevation), and topographic distance which incorporates both lateral and vertical distance. We first tested for isolation by lateral distance using a Mantel test between matrices of pairwise Fst’ Wang . From anE) using the heterozygote excess method in NeEstimator v. 2.1 . We calculated pairwise FST in GenoDive v.3.0 , we calculated the following population genetic diversity parameters in GenAlEx  as a function of elevation. We included the duration in years  that a population persisted in greenhouse conditions as a covariate because being pot-bound could impact on clonal potential. To obtain the direct effect of elevation on clonality metrics alone, we generated residual clonality metrics at the population from a model of each as a function of year in the greenhouse. We then regressed residual clonality metrics from these models as a function of elevation using linear models.E, FIS, and FST exhibited clinal variation with elevation by modeling each a function of elevation using linear regression. Finally, using the population-level phylogeny, we extracted divergence times estimated at each node, and modeled divergence time as a function of elevation and longitude with bootstrap node support as a weighting factor. We included longitude as a covariate in the model because preliminary graphical evaluations showed that divergence time was associated with longitude. While our sampling spanned a narrow longitudinal range, waterways in the Gunnison River Basin flow east-to-west, potentially structuring population genetic diversity of A. anserina which is a primarily wetland species. We estimated standardized regression parameters using the ‘lm.beta’ function for elevation and longitude such that their effects on divergence time were directly comparable.We then tested whether HE was directly predicted by population attributes measured in the field. To do so, we used stepwise regression starting with a global model predicting variation in HE using the following: area of occupancy, plant density, plant patchiness, flower density and flower patchiness. We used the ‘step’ function in R to iteratively remove terms until reaching a model from which AIC values no longer declined significantly with additional deletion of a single predictor. For the final model, we evaluated the variance inflation factor (VIF) using the ‘vif’ function. The final model’s VIFs ranged 1.3–2.5, indicating minimal effects of multicollinearity (Sheather Because variation in heterozygosity among populations was not explained by elevation (see results), we tested whether HSheather . We addiA. anserina on the landscape declined precipitously with elevation  and the length of runners  did not show a strong association with elevation.Accession density of ion Fig. . The majST = 0.13, SE = 0.012). Linear geographic distance between populations was a significant predictor of pairwise FST , while topographic distance was a weaker predictor of FST . The elevation difference between populations was the strongest predictor of FST   with both factors being important predictors   Fig. , C. Dive09) Fig. , and the09) Fig. , C.Fig. A, HO, and HE) are provided in Table E varied among populations over two-fold (0.05–0.13). The inbreeding coefficient (FIS) was consistently negative across populations, but exhibited wide variability from −0.14 to −0.78.Within population genetic diversity metrics  and higher elevation populations exhibiting higher isolation (FST > 0.15). These increases in isolation are accompanied by a reduction in the density of A. anserina accessions at high elevations, which we interpret as a reflection of lower population density at higher elevations because we controlled for elevational variation in collection bias. Our results are consistent with increased isolation at high elevation being driven greater distances between extant populations. Argentina anserina occupies the edges of ponds and streams, as well as wet meadows and roadsides, but does not commonly occur on steep slopes. A more rugged topography at higher elevation in the San Juan Mountains could limit suitable habitat for this species.The elevational cline in FGenetic isolation was better explained by vertical distance between populations than by lateral or topographical distance. This suggests that upward or downward elevational gene flow, either via pollen or seed, is likely constrained in this system. There was also more admixture among individuals in lower elevation populations based on conStruct analyses. When K = 6, even high elevation popualtions that are very geographically close to one another  were assigned to different clusters. This suggests that higher elevation populations are not only isolated from lower elevation populations, but from one another. Low connectivity of high elevation populations, especially from lower elevation populations that experience warmer conditions, has the potential to limit the introduction of novel genetic variants that could increase genetic diversity and contribute to enhanced performance under climate change  occur on wet gravelly edges of kettle ponds with reduced competition for space from other plant species. Thus, opportunity for clonal spread may be higher in high elevation populations.IS (extreme heterozygote excess) is a common feature of highly vegetatively clonal plant populations .The potential for increased clonality in field conditions at high elevation was reflected in the population genetic data as well. Strongly negative FE of only 1.4 individuals while two low elevation populations had an estimated NE of 15 or more. The continuous decline in NE with elevation is strongly suggestive of bottlenecks occurring as populations expanded from low to high elevation. Similar patterns of NE have been observed in other species during historical colonization  and those with higher flower density (characteristic of higher elevation) were associated with higher heterozygosity. Likewise, populations with more patchily distributed plants (low elevation) and those with higher plant density (high elevation) were linked with higher heterozygosity. Together, these correlates with heterozygosity were inconsistent with elevational patterns in population attributes, nullifying elevational trends in heterozygosity. If most heterozygosity in high elevation populations is harbored within individuals that largely propagate via vegetative clonality however, the generation of novel genotypes through sexual reproduction is unlikely.Argentina anserina in the Southern Colorado Rocky Mountains. Isolation of high elevation populations likely reflects limited dispersal across elevation bands as well as a reduction in the frequency of populations at high elevation. Increased vegetative clonality near the upper range limit likely contributes to reductions in effective population size, increased heterozygote excess, and increased plant density. While high elevation populations appear large due to high plant density, the fact that they are effectively small, highly clonal, and isolated suggests that they could have limited adaptive potential in response to altered environmental conditions which have been pronounced at high elevation populations in Western North America (Diaz and Eischeid Our study revealed a suite of ecological attributes that correlate with a decline in connectivity and effective population size towards the high elevation range limit of Supplementary material


Having higher adaptability against abiotic stress, whichis characterized in rural areas in developing countries, local farm animalgenetic resources (FAGRs) are increasingly precarious for random andunsystematic crossing with exotic breeds. In this study, 85microsatellite loci were utilized to assess genetic diversity among nativeAlgerian rabbits (NARs) sampled from an area of 753 km (from northto south) and 919 km (from east to west). Those distances covered25 significant geographical points in seven rural areas . Atotal of 558 alleles were observed in this study. The highest geneticdiversity was registered in the southern direction among NAR populations. Themean number of alleles per locus (MNa) and the inbreeding coefficient ( For a very long time, genetic diversity has allowed thousands of domesticated species to adapt to climate, disease, soilcharacteristics, sources of food, and topography .Genetic diversity within and between populations supports ecologicalfunctions and provides essential resources and services to humanity. Furthermore,diversity studies are very important in achieving the 2030 sustainable-development goals, with number 15 being to halt biodiversity loss.According to Food and Agriculture Organization  statistics, about 15 %–17 % of farm animal genetic resources (FAGRs) are classified as being on thebrink of extinction risk. In addition, more than 80 % of livestock breeds areunknown in the Middle Eastern region . Genetic diversity contributesto improving the herds of pastoralists and farmers that adapt livestockpopulations according to environmental conditions and changing demands.Several types of molecular markers were widely used in rabbit diversitystudies, e.g., random amplified polymorphic DNA (RAPD) according to Mohamed andAbdelfattah (2018); the polymerase chain reaction–restriction fragment lengthpolymorphism (PCR-RFLP) by Shevchenko and Kopylov (2015); simple sequencerepeat (SSR) microsatellites ; the mitochondrial DNA; and single-nucleotide polymorphisms (SNPs), which wereinvestigated by Ballan et al. (2022). Microsatellite markers are widely usedfor the naturally codominant, highly polymorphous, and Mendelian inherited.On the other hand, the disadvantages of microsatellites are the shadow appearance ofstutter bands, the null-allele presence , and too many alleles at certain loci thatwould demand a very high sample size for analysis . Inaddition, microsatellite markers sometimes contain mutation lengths thatcould produce identical-length variants when compromising thepopulation-level studies .In this regard, limited genetic diversity studies were carried out toinvestigate the NAR genetic situation. For this purpose, the current study isaimed at investigating the genetic diversity of NAR populations at 25different geographic locations belonging to seven rural regions using 85microsatellite markers.22.1In this study, an Algerian team conducted the survey over about 753 km (fromthe start point in the north to the end point in the south) and 919 km (fromthe start point in the east to the end point in the west). A total of 152tissue samples of NARs were collected from 25 points across seven Algerian ruralareas, as shown in Fig. 1, according to FAO conditions . The populationswere classified according to the following administrative divisions: ElTaref (24 samples), Mostaganem (20 samples), Sidi Bel Abbès (22samples), M'Sila (22 samples), Dar Chioukh (21 samples), Faidh El Botma (22samples), and Laghouat (21 samples), as shown in Fig. 2. The unrelatedanimals were sampled from the weaning rabbits and growing rabbits that wereprepared for market in the different backyards or from slaughtered rabbits in eachgeographic location. Tissue samples were stored in 90 % ethanol until DNAextraction was performed.2.2DNA was extracted from the tissue using DNA EasySpin kits  via the protocol recommended by the manufacturer.The DNA extraction was checked by agarose gel . Based on their annealing temperatures, 85 microsatellites wereamplified via 15 multiplexes (Table S1 in the Supplement). The multiplex contained 5 2.3The analysis of molecular variance (AMOVA) and of the estimated number of observedalleles per locus (Na), the mean number of observed alleles (MNa), the totalnumber of private alleles (Pa), and the observed and expected heterozygosity and Laghouat (4.482 and 23), respectively. A total of 558 alleles were recorded for 85 loci across the populations(Table S2), in which about 19 % of observed alleles were recorded asprivate alleles with the southern (Laghouat) and the western zones (Mostaganem and Sidi Bel Abbès), while the eastern zone (El Taref) was expressed as being far. The same concept is foundin the NJ tree .The STRUCTURE analysis and values of 3.3The results of AMOVA are summarized in Table 2. It is shown that the measuredgenetic differentiations of the total genetic variance among populations andindividuals were 3 % and 20 %. However, the variation ofwithin-population genetic diversity was 76 %.4As shown in Table 1, the mean values for MNa and Pa in NAR populations were4.277 and 15.14, respectively. This result is consistent with the findings of Bolet et al. (2000) and Alves et al. (2015) for European domestic rabbits; they stated mean values for MNa of 3.600 and 3.156, respectively. In addition, thisstudy result is nearly similar to the values recorded in north Africanregions such as Egypt and Tunisia. In this regard, in Egypt, Emam et al. (2017) found a high MNa that ranged from 4.316 to 6.000, while recordedvalues ranged between 5 and 15 for Pa. On the other hand, in Tunisia, Ben Larabi et al. (2014) reported low values of MNa that ranged from 3.000 to 4.370.Moreover, in this study, the Moreover, results in Table S2 showed that 57 % of loci were highlyformative in terms of PIC values. The high percentage of formative PIC was also observed inseveral studies . On the other hand, 89 % of the loci were significant in terms of HWE atthree significance levels  allowed us to classify NAR populations into four major groupsaccording to geographical groups . The geographical integration was found between the middlepopulations . It is due to thegeographical proximity of those areas (less than 150 km). On the otherhand, the overlap between the middle and southern populations could beexplained by the distance between the Laghouat population (in the south) and theFaidh El Botma population (the last city in the middle zone), which is less than110 km. In addition, the distance between the western points (Mostaganem andSidi Bel Abbès) is less than 130 km. In contrast, the eastern population was expressed as being far separated from the western (919 km), middle (635 km), and southern (753 km) populations. The geographical isolation was found for wildrabbits . In addition, the rabbit population classification according to thegeographical zones was reported in wild rabbits  and localrabbit breeds . The same results were recorded in the NJ tree.According to the analysis of STRUCTURE , more populations wereclustered together in the middle and west . Emam et al. (2016)reported that more populations clustered together in the Egyptian localbreeds, while other populations were separated in each cluster (in the eastand south), which confirmed the results in Figs. 3 and 4. The mostlikely values of The results of AMOVA (Table 2) showed a high value of genetic variationamong individuals (20 %), which is a strong indicator for permittingflexibility and the survival of a population in the face of changingenvironmental circumstances . On the other hand,the among-population variation (3 %) is a strong indicator ofthe closing inbreeding system and could be the direct result of theinfrequent gene flow that increased the chance of recombination . In contrast,El-Aksher et al. (2016) in Egypt and Adeolu et al. (2021) in Nigeria found random breeding systems with lower values among populations (1 %) andindividuals (4 %) in commercial rabbit lines.5The current study is the first study dependent on using a large number ofmicrosatellite loci to understand the genetic diversity of native Algerianrabbit populations. According to the discovered results, high diversity wasrecorded in the south. Also, a high degree ofgeographical distribution  was noticed in the results. Generally, thecurrent study records a high internal-breeding factor, although the sampleswere collected randomly. This is a strong indicator that native Algerianrabbits are in an endangered situation. This study could be used as a guidefor rabbit genetic improvement and conservation strategies in Algeria. Theresults of this study could open up areas for cooperation among northAfrican countries by studying the genetic diversity of native rabbits. Inthe same context, cooperation could be included to design maintenanceprograms (genetic improvement and conservation) for native rabbit breeds,which could facilitate sustainable rural development plans in this importantregion. It will meet goal 15  of the United Nations'sustainable development agenda for 2030.10.5194/aab-66-207-2023-supplementThe supplement related to this article is available online at: https://doi.org/10.5194/aab-66-207-2023-supplement.