Astrangia poculata is a temperate scleractinian coral that exists in facultative symbiosis with the dinoflagellate alga Breviolum psygmophilum across a range spanning the Gulf of Mexico to Cape Cod, Massachusetts. Our previous work on metabolic thermal performance of Virginia (VA) and Rhode Island (RI) populations of A. poculata revealed physiological signatures of cold (RI) and warm (VA) adaptation of these populations to their respective local thermal environments. Here, we used whole-transcriptome sequencing (mRNA-Seq) to evaluate genetic differences and identify potential loci involved in the adaptive signature of VA and RI populations. Sequencing data from 40 A. poculata individuals, including 10 colonies from each population and symbiotic state , yielded a total of 1,808 host-associated and 59 algal symbiont-associated single nucleotide polymorphisms (SNPs) post filtration. Fst outlier analysis identified 66 putative high outlier SNPs in the coral host and 4 in the algal symbiont. Differentiation of VA and RI populations in the coral host was driven by putatively adaptive loci, not neutral divergence . In contrast, we found evidence of neutral population differentiation in B. psygmophilum . Several putatively adaptive host loci occur on genes previously associated with the coral stress response. In the symbiont, three of four putatively adaptive loci are associated with photosystem proteins. The opposing pattern of neutral differentiation in B. psygmophilum, but not the A. poculata host, reflects the contrasting dynamics of coral host and algal symbiont population connectivity, dispersal, and gene by environment interactions. Population connectivity in marine systems is shaped by a complex mixture of factors, including oceanographic currents , planktoIn scleractinian corals, the holobiont consists of the coral host, algal symbiont  or without (aposymbiotic or white) B. psygmophilum across its range  of respiration was elevated in VA corals compared to RI , corresponding with warmer in situ temperatures in the more southern population and suggestive of a signature of adaptation of VA and RI corals to their native thermal habitat.Here, we consider patterns of neutral and adaptive differentiation in the northern star coral, mophilum . Astrangts range . Therefots range  demonstrA. poculata is a gonochoristic broadcast spawner that releases its gametes into the water column between early August and September  both by population (VA vs. RI) and by symbiotic state (brown vs. white) using SNPs derived from high-throughput mRNA-Seq data. Leveraging our previous work on physiological adaptation in these populations, we explore the relative strengths of neutral and adaptive divergence in this broadcast spawning, facultatively symbiotic, and extraordinarily thermally resilient species across the northern half of its range.In the current study, we characterized population structure of the Astrangia poculata colonies were collected each from Virginia (VA) and Rhode Island (RI), USA (populations will be referred to as VA-brown (n = 10), VA-white (n = 10), RI-brown (n = 10), and RI-white (n = 10)). All brown and white VA colonies were collected from the wreck of the J.B. Eskridge  on September 15, 2017 at 20 m depth  and salinity of approximately 35 ppt for a recovery period before fragmentation . Corals were fragmented over 2 days  using a high-speed cut off tool  fitted with a diamond tip circular blade  to consider population structure.Following both collections, all de sensu , then afTo compare long-term temperature trends across the two collection sites, sea surface temperature (SST) data was downloaded from the NOAA 1/4° daily Optimum Interpolation Sea Surface Temperature dataset  and deionized (DI) water. Temperature was maintained using a temperature controller  in combination with an in-line water chiller  and 1,500 W immersion heater . The holding aquarium was equipped with a filter sock for mechanical filtration, protein skimmer for removal of organic material, and powerheads  to maintain flow. All experimental corals were fed three times a week with freshly hatched Artemia sp. nauplii and maintained under approximately 200 μmol photons m−2s−1 of light supplied by 165 W LED aquarium lights .Similar to ® (code assembled by D. Barshis) connected to the same style heater and chiller as the holding aquarium . As in the holding aquarium, artificial seawater was mixed to 35 ppt and each aquarium was equipped with a filter sock and protein skimmer. In the heat and cold aquaria, temperature was manipulated using a custom-programmed Arduinoum sensu . Tempera−1, and the target temperatures of 22 °C (heat) and 14 °C (cold) were reached at 10:30. The corals were held at their respective target temperatures for another hour, and the experiment therefore ended at 11:30 . All corals were given 30 min of acclimation in the dark, after which the lights were turned on and a 1 h hold at 18 °C began. At 09:30, temperature was increased (heat aquarium) and decreased (cold aquarium) at a rate of 4 °C hat 11:30 .2 were taken from each coral fragment and preserved in an RNAlater-like solution . The light levels used here were based on an estimation of minimum saturating irradiance from our previously published photosynthesis vs. irradiance curve  according to manufacturer’s instructions. The purified RNA was eluted in 50 μL of DNase/RNase-free water and immediately stored at −80 °C for future library preparation. The concentration of all RNA extractions was assessed using a Qubit 2.0 Fluorometer  and diluted to 0.1–1 μg total RNA before mRNA-Seq library preparation.RNA was extracted from all coral fragments between November 1 and 13, 2017 using Trizol . For each extraction, coral tissue samples were thawed, crushed with a sterile razor blade, combined with Trizol and sterile 0.5 mm zirconia/silica beads  and bead beat on high for 2 min to break open cells. Following a 5-min incubation at room temperature, samples were centrifuged for 10 min at 12,000×N = 96) were prepped using the Illumina TruSeq mRNA prep kit  and half-sized reaction volumes. The 96 libraries consisted of 7 genotypes per population , with each genotype represented across all temperature treatments (7 genotypes × 4 populations × 3 temperature treatments = 84 libraries). These 84 libraries were intended for use in differential gene expression analysis. An additional 12 libraries, intended to increase the sample size for SNP analyses, were prepared from 3 additional genotypes per population (3 genotypes × 4 populations = 12 libraries), all of which were in the 18 °C control treatment.Libraries  and KAPA Library Quantification Kit for Illumina platforms . Any libraries that did not successfully amplify after the first attempt were prepped again using the same method listed above, except 25 μL of un-diluted RNA was used in the Illumina TruSeq mRNA prep kit. The 96 libraries were sequenced on 6 lanes of an Illumina HiSeq4000 (16 libraries per lane) at the University of California Berkeley Vincent J. Coates Genomics Sequencing Laboratory, which yielded single-end 50 base pair (bp) reads. All RNA sequences have been deposited in NCBI BioProject under accession number github.com/hannahaichelman/Astrangia_PopGen). Raw sequences were processed using the adapter trimming/quality filtering functions of the Fastx-Toolkit to remove adapter sequence contamination and reads with a quality score less than 33. All sequences were then used as input for de novo transcriptome assembly using Trinity . “Good hits” to these rRNA databases were defined as matching at least 78% of the read over at least 100 bp, and once identified were removed from the reference assembly. A total of 1,273 matches to the LSU database and 688 matches to the SSU database were removed from the reference assembly.Detailed descriptions for all data analyses can be found on the electronic notebook associated with this publication (n = 10 datasets), (2) all available Symbiodiniaceae data , (3) aposymbiotic cnidarian data only , and (4) cultured Symbiodiniaceae data only . These four databases contained the same sequencing data used by Once rRNA contamination was removed from the reference assembly, it was filtered to include only sequences greater than 500 bp in length. Host and symbiont contigs in the reference assembly were differentiated and assigned as described previously . Brieflye-value cutoff of 1e−4 using a custom annotation script  to 28 fastq files. These concatenated files, along with the 12 libraries originally intended for population genetic analyses, yielded a total of 40 libraries that were used for all downstream analyses. It should be noted that the dataset presented here had relatively low read counts, resulting from an error in the library preparation that led to sequencing of rRNA in addition to mRNA. The primary result of this error was a larger than usual percentage of sequence yield going to rRNA, which resulted in a lower read count on an individual basis. To combat this, we have used conservative cut-off values at every step of the data analysis pipeline to address these issues and account for missing data.p-value cutoff for SNPs = 0.01). This fourth filter resulted in no SNPs being removed from the symbiont file, but 11 SNPs being removed from the coral file. The filtered vcf files were converted to genepop format using a custom python script (written by D. Barshis). The coral host analysis was conducted considering the data as four populations . The symbiont data only included brown hosts and was therefore analyzed as two populations (VA-B and RI-B). Additionally, to look for consistent loci driving patterns of differentiation between brown and white phenotypes within VA and RI, the coral host analysis was also conducted as two populations for each origin separately (VA-B and VA-W separately from RI-B and RI-W).The quality filtered reads were mapped to the de novo holobiont transcriptome using the “very-sensitive” method of Bowtie2.2.4 . Single Potential clones in the coral host data were assessed using the R package poppr v2.8.5; . The filNeutral and outlier SNPs were detected using BayPass3.04  under thFollowing BayPass outlier detection, the coral and symbiont vcf files were each filtered to create separate files for neutral and high outlier SNPs. These separate vcf files were then randomly filtered to include only one SNP per contig using the Filter_one_random_snp_per_contig.sh script from the dDocent pipeline . This waAdegenet (Pairwise differentiation (Fst) calculations were conducted in GenoDive v3.04  using thAdegenet .K) was assessed using the DeltaK method (STRUCTURE (v2.6), with a 100,000 burn-in period and 500,000 MCMC runs after burn-in, was used to determine the number of distinct genetic groups in the coral host . The optK method , which wK method  and plotK method  using thK method .A gene ontology enrichment analysis was conducted to determine if genes containing outlier SNPs were enriched for any particular GO categories. Each gene in the reference transcriptome received a binary indicator of whether it contained an outlier SNP or not, and a GO enrichment analysis based on Fisher’s exact test was used to determine if these outlier SNPs were enriched for any GO categories. Results were plotted as a dendrogram , which iA. poculata mapped reads/genet ranged from 9,340,966 to 167,484,549, and mapping efficiencies ranged from 45.96% to 54.10%  as well as removing one putative clone (RI-B), a total of 34 individuals remained in the coral analysis . For the symbiont analysis, two individuals with a high percentage of missing data (n = 2 VA-B) were removed, leaving 38 individuals in the symbiont analysis .The total counts of o 54.10% . The unfo 54.10% . After rp = 0.269); however, there was significant population structure at high outlier loci . At high outlier loci, all pairwise Fst values were significant .For the symbiont analysis, BayPass analyses identified 52 neutral and 4 high outlier SNPs, and further filtering for one SNP per contig left 20 neutral loci and 4 high outlier loci that were used in population differentiation analyses . In contThe coral host Fst results are reflected in the STRUCTURE analysis, with a pattern of admixture in all samples at neutral loci in the coral host, but two distinct clusters separating VA and RI individuals at high outlier loci . The optFor the coral host analysis, at neutral loci there were no clear patterns for one population having higher observed (Ho) or expected (Hs) heterozygosity than another, with Ho ranging between 0.217 (RI-B) and 0.283 (RI-W) and Hs ranging between 0.224 (RI-B) and 0.267 (VA-W) . At highn = 13) were manually inspected to look for agreement with previous studies on coral adaptation to distinct temperature environments  CP43 reaction center protein, photosystem I (PSI) P700 chlorophyl When analyzing the data as two populations separately for VA and RI to look for consistent high outlier SNPs that could be driving differentiation between brown and white morphs, 11 high outlier SNPs were identified as shared between VA and RI , which iGene Ontology enrichment analyses showed that the coral host outlier SNPs detected in this study were enriched for 10 GO terms in the “molecular function” category and seven GO terms in the “cellular components” category. The significant cellular components terms consisted of several ribosome-associated terms, including large ribosomal subunit, small ribosomal subunit, ribosome, and ribosomal subunit. The significant molecular function terms consisted of several ribosome-associated terms, including structural constituent of ribosome and rRNA binding, as well as several terms related to coral stress response, including ferric iron binding and oxidoreductase .Astrangia poculata. Between Virginia and Rhode Island, the coral host exhibited neutral panmixia, but adaptive divergence. While there was no significant population differentiation at putatively neutral loci in the host, there was evidence of neutral divergence in the algal symbiont. Astrangia poculata is a gonochoric broadcast spawning species that horizontally transmits its symbionts, a life history strategy that likely lends to the contrasting patterns of genetic differentiation in the host and symbiont observed here. Marine broadcast spawning species have extended pelagic larval durations (PLDs) of up to 244 days , it is not unreasonable to expect that A. poculata larvae can remain in the water column for extended periods of time, facilitating larval connectivity across VA and RI.Here, we find evidence of contrasting patterns of population structure between members of the holobiont in the temperate scleractinian coral 244 days , facilit244 days . CombineOrbicella franksi enables export of larvae from the Flower Garden Banks to more distant Caribbean reefs, including Broward and Palm Beach, Florida approximately 1,500 Km away . However, this overall connectivity was complicated by regional patterns of genetic structure, including an east-west genetic barrier at Mona Passage and differentiation across only 470 Km along the Mesoamerican Barrier Reef System  associated with A. hyacinthus and A. digifiera across Micronesia occurs across smaller scales compared to the coral animal (Durusdinium glynni population structure sampled from Pocillopora (type 1) across the Eastern Tropical Pacific. In contrast to the host, which exhibited genetic connectivity over 3,400 Km  affect each symbiotic partner  within the Virginian Province to be connected and from an ancestral gene pool that diverged from populations south of Cape Hatteras in the Carolinian and Caribbean provinces. In contrast to panmixia within the Virginian Province, as we also observed in the A. poculata host, Gemma gemma; no planktonic larval phase) around New Jersey, separating southern populations of Maryland, Virginia, and North Carolina from northern populations of Maine, Massachusetts, and Connecticut. Such divisions in population structure between what are called the Upper and Lower Virginian provinces (at ~39°N latitude) correspond with a gradient in average SST and with population structure of several other species, including amphipods (Ampithoe longimana), killifish (Fundulus heteroclitus), polychaetes (Marenzellaria viridis), and copepods  across the stark environmental differences between Maryland in the south and Nova Scotia in the north by sequencing the mitochondrial cytochrome oxidase I gene. The authors suggest that high levels of dispersal and gene flow, facilitated by a 3 week planktonic larval phase, likely contribute to the observed pattern  differentially induced heat shock protein 70 (HSP70) expression as a function of thermal history. Namely, shallow-water corals that historically experienced warmer temperatures more strongly induced expression of HSP70 upon heat stress, and microsatellite loci showed that corals from the three depths were genetically differentiated across this steep environmental gradient . However it is possible that, similar to B. psygmophilum symbionts that A. poculata larvae acquire upon settling in their respective environments. Additionally, in the facultatively symbiotic coral Oculina patagonica, Gradients in environmental parameters such as temperature also play an important role in local adaptation  and geneironment . In contA. poculata host connectivity at neutral loci despite evidence for adaptive divergence at several high outlier sites.It is important to distinguish between neutral and adaptive loci when considering population divergence. Namely, loci can still be under selection even in the face of extensive gene flow, and therefore populations can appear homogeneous at neutral loci but still exhibit local adaptation . AdaptivAcropora palmata after 2 days of thermal stress  CP43 reaction center protein , photosystem I (PSI) P700 chlorophyl a apoprotein A2 , and PSII protein D1 . Species of Symbiodiniaceae have been previously shown to be differentiated by mutations on genes related to photosystem machinery. Durusdinium glynnii was distinguished from other symbionts within the genus Durusdinium as well as from symbionts of other genera by a non-synonymous mutation that effected a change on the D1 protein of photosystem II (psbA gene), a highly conserved region in dinoflagellates  or considering cell size differences , but not in symbiont physiology . Sampling for RNA occurred between 11:30 and 13:10.Temperature was recorded using Hobo PendantClick here for additional data file.10.7717/peerj.10201/supp-3Supplemental Information 3Height on the y-axis is based on Hamming’s distance, or the number of allelic differences between individuals. The box indicates the two putative clones that had relatively few allelic differences, and RI_B_06_merged was removed for all downstream analyses.Click here for additional data file.10.7717/peerj.10201/supp-4Supplemental Information 4Vertical columns represent individual samples within a population and the associated probability of assignment to K = 2 genetic clusters (light blue and dark blue). The four site abbreviations are: VA-B (brown/symbiotic Virginia), VA-W (white/aposymbiotic Virginia), RI-B (brown/symbiotic Rhode Island), and RI-W (white/aposymbiotic Rhode Island).Click here for additional data file.10.7717/peerj.10201/supp-5Supplemental Information 5Dendrograms indicate gene sharing between GO categories. The numbers listed with each GO term correspond to the number of genes in the module over the total number of genes assigned to each category in the entire SNP dataset.Click here for additional data file.10.7717/peerj.10201/supp-6Supplemental Information 6The sequences were added to one of three databases (‘Database’): 1) Dirty Coral = all available cnidarian data, 2) Clean Coral = aposymbiotic cnidarian data only, 3) Clean Sym = cultured Symbiodiniaceae data only. ‘Reference’ refers to the species the sequencing data was obtained from and ‘Source’ refers to the publication or website associated with the data.Click here for additional data file.10.7717/peerj.10201/supp-7Supplemental Information 7A, n = 279), high outlier coral SNPs , neutral symbiont SNPs  and high outlier symbiont SNPs . Observed heterozygosity (Ho) is the observed frequency of heterozygotes within a subpopulation, values can range from 0  to 1 . Heterozygosity within populations  is the expected frequency of heterozygotes within subpopulations (assuming Hardy-Weinberg equilibrium) and includes a correction for sampling bias from sampling a limited number of individuals per population. Values were calculated in Genodive using default parameters.Data is included for the putatively neutral coral SNPs  and Rhode Island (RI) analyses used to look for signatures of genetic differentiation by symbiotic state. ‘Contig Name’ and ‘Contig Length’ refers to the name and length of the contig containing the SNP(s) in the reference transcriptome. The description of the contigs was determined by blasting to the NCBI nr database. The E-value and percent match describe the match between the reference transcriptome contig and the nr database match. ‘No Description’ and ‘No sig hit’ refer to contigs that did not match an annotation.Click here for additional data file.


ST estimates were higher in the populations sampled in 2017, showing a larger genetic distance between them. Considerable differences exist between the populations of the same breeds, which can most likely be attributed to genetic drift. STRUCTURE results have shown a clear separation between the Hungarian populations, which is in agreement with the principal coordinate analysis. The most likely clustering was found at K = 6, classifying the populations of the same breed as one group. No considerable allele loss was found in the Hungarian indigenous chicken breeds after 15 years of conservation. In general terms, after 15 years, the level of inbreeding within the populations was, in fact, higher, although this could be effectively reduced through the use of an improved mating system. Consequently, the breed management applied in the case of Hungarian local chicken breeds was found to be effective at adequately conserving their genetic variability.Investigation into the genetic diversity of certain endangered native breeds of domestic animals has been in common practice for several decades. The primary objective of these investigations has been to reveal the exceptional genetic value of such breeds, both for their conservation and also to gain insight into their current genetic status, as they have been undergoing a progressive decrease in population size and general diversity; this has been compounded by the general lack of an optimal breeding scheme. In this study, we have investigated changes in the genetic diversity of six Hungarian local chicken breeds based on 29 microsatellite loci over a period of 15 years. In terms of the basic diversity measures, populations sampled in 2017 generally exhibited a lower heterozygosity and mean number of alleles and thus, experienced a higher degree of inbreeding. Although the effective population size increased, the estimates of populations sampled over different periods indicated comparatively low values, suggesting overall lower genetic variance. Pairwise F Over the last several decades, exploration has been under way regarding the genetic diversity of endangered domestic animal breeds. The loss of genetic variability, together with the limited number of commercial hybrids, both suggest that genetic diversity in poultry is one of the most endangered genetic resources –3. ThereMaintaining an effective population size (Ne) plays an important role in conservation genetics, as it is a determining factor in the development of the population in regard to inbreeding and genetic drift. Minimising loss of genetic variability is one of the main concerns of conservation breeding programmes and can be achieved through the maximisation of the effective population size, and hence, decrease the occurrence of genetic drift. Estimation of the effective size of a population and the monitoring of its changes over time are important to accurately assess the effectiveness of the breed management, particularly in small populations, where the allele frequencies change more easily over time. In the absence of selection, any change in allele frequency could solely be the result of drift and thus indicates the effective size of the population .Although high-throughput SNP techniques have been more preferred for a while , microsaHaving a long history in this area, one of the primary activities of the National Centre for Biodiversity and Gene Conservation  has been the preservation of local Hungarian poultry species. The aim of this study was to investigate the extent of divergence which has developed in the genetic diversity of six Hungarian indigenous chicken breeds over a period of 15 years. We trust that our results will provide useful information regarding small populations which have been managed for the purpose of maintaining their genetic variation in gene banks for gene conservation as well as future breeding programmes.Animals were kept and maintained according to general animal welfare prescriptions of the Hungarian Animal Protection Law . Permission to undertake experimental animal research at the National Centre for Biodiversity and Gene Conservation was granted by the National Food Chain Safety Office, Animal Health and Animal Welfare Directorate, Budapest (Permission no. PE/EA/2485-6/2016). All experimental methods described herein were approved by the Institutional Ethics Review Board.Six native Hungarian chicken breeds were included in this study: the Yellow (HY), the Speckled (HS) and the White Hungarian (HW), as well as the Speckled (TNNS), the White (TNNW) and the Black Transylvanian Naked Neck (TNNB). A major breeding program has resulted in good dual-purpose, medium-sized Hungarian chicken breeds which have been propagated not only for their sizable egg production  but also for their excellent meat quality . In the case of the HY, HS and HW breeds, the body weight of the hens is 2.0–2.3 kg, while that of the roosters is typically 2.5–3.0 kg. The Transylvanian Naked Neck chickens are characterized by their featherless necks and only slight plumage on the top of their heads. These breeds are extraordinarily resistant and known for their good winter-laying ability; however, their body weight  and egg production  are slightly lower when compared to the feathered Hungarian breeds . 1 ml of5'-CAGGACCAGGCTACCGTG-3') was placed before the 5’ end of the forward primers , and the second, mainly by their inbreeding coefficients and observed heterozygosity [PCA2 = (0.279* MNA)–(0.395* HO)–(0.067* HE) + (0.645*FIS)]. Two principal components were extracted from the Hungarian native chicken populations with eigenvalues of 2.376 for the first principal component (PCA1), and 1.321 for the second (PCA2); these two principal components explained 92.4% of the total variance. Based on the cluster analysis of the individual principal scores of diversity the TNNB2002 and TNNB2017 stocks as well as the populations of the HW and TNNW breeds sampled in 2017 formed a cluster separate from the others, showing a higher level of inbreeding  of the summary diversity statistics was performed to collapse several parameters into two principal components . The firbreeding .IT, FST and FIS) of the Hungarian chicken populations are shown in IT) amounted to 0.241 ± 0.022. Its estimate was higher in those populations (0.298 ± 0.025) sampled 15 years later. The genetic differentiation (FST) of the set from 2002 was slightly lower than the corresponding value of the populations sampled in 2017. Estimate of within (FIS) population variation was significantly lower in the stocks sampled in 2002 than those taken 15 years later. The results of AMOVA indicated the same trends (Wright’s fixation indices (average Fe trends .ST estimates between the Hungarian populations sampled in 2002 varied between 0.15 (HW2002–TNNW2002) and 0.28 (HY2002–TNNB2002), while in case of the populations sampled after a period of 15 years the range was from 0.21  to 0.30 (HW2017–HY2017). Values of the two populations of the same breed sampled at different points in time varied between 0.03 (HY2002–HY2017) and 0.08  which are relatively low but still indicate a differentiation between them (Pairwise Feen them .2002 and HY2017 stocks formed a cluster and remained together until K = 12. The Transylvanian Naked Neck breeds (except for the TNNW) grouped with the HS stocks, which are slightly overlapping with the HY breed. At K = 4, the two TNNB populations split to form a separate cluster. The HS stocks are still overlapping with the HY and TNNB breeds, but at K = 5 they showed a clear population structure. The clustering of highest probability was obtained at K = 6 (N = 100) based on Evanno’s method, where the HW and the TNNW breeds separated from each other and this state remained until K = 12. From K = 6 to K = 9 the clustering was the same, however, the HW2002 showed a slightly-mixed population structure from K = 7. At K = 10 the two populations of the TNNS breed formed separate clusters, while at K = 11 the TNNB2002 and TNNB2017 stocks split off as individual groups. K = 11 and K = 12 showed identical solutions, indicating that a maximum of eleven clusters illustrate the data.The results of the STRUCTURE analysis are given in IS estimates were not significantly different from zero, suggesting that the populations sampled in 2002 were close to the Hardy-Weinberg equilibrium state. After 15 years of conservation, heterozygosity estimates differed significantly in most of the breeds, consequently, the inbreeding coefficient (FIS) increased in all breeds with the exception of the HS. This is in agreement with the principal component analysis based on diversity estimates  indicated that heterozygote deficiency was lower in the Hungarian populations sampled in 2002 and it was almost fully explained by between-population differentiation ; however, they were well managed in terms of maintaining the genetic variation found 15 years prior. The FIS value of the Hungarian populations sampled in 2017 is even coherent with that of the Polish local breeds investigated [IS, ST, The overall population inbreeding coefficient , which is remarkably close to the pairwise FST estimate (0.15) of two distinct breeds (HW2002–TNNW2002) sampled in the same year. This suggests that the diversity of TNNB and TNNS populations has changed more than that of the others. This concurs with the STRUCTURE results.The pairwise F in 2017 . The popST estimate . Generally, the level of inbreeding appears to be higher after 15 years of conservation, but it is capable of being reduced through the use of a sire-rotation scheme different from that being used at present. The allele frequency of small populations may be subject to fluctuations by leaps, therefore, the allele set itself has proven to be a more stable indicator, as it changes only when the gene pool of the populations changes significantly, and this is not the case here.The breed management which has been applied to these populations, i.e. maximising the genetic variance based on phenotypic traits and sire rotation used between families, has sufficiently demonstrated its suitability for adequately conserving genetic variability with the addition of some improvements. Thus, we hereby suggest the implementation of a controlled random male rotation scheme in place of the currently constant pattern of rooster exchange between families. Introducing new individuals into the breeding system from time to time may also help reduce the inbreeding level. Increasing the effective population size with either the use of a higher number of roosters in smaller families, or through the application of rooster exchange during periods of egg collection for pedigree, is recommended to conserve the genetic variance more effectively, however this practice does have its financial constraints.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.


Magnolia kobus consists of two morphologically different varieties, kobus and borealis. The latter variety is characterized by larger leaves than the former. For the conservation of M. kobus genetic resources in natural forests, the relationships between morphological and genetic variation should be clarified. We investigated variations in nuclear microsatellites, chloroplast DNA (cpDNA) sequences and leaf morphological traits in 23 populations of M. kobus over the range of species. Two genetically divergent lineages, northern and southern were detected and their geographical boundary was estimated to be at 39°N. The northern lineage consisted of two genetic clusters and a single cpDNA haplotype, while the southern one had multiple genetic clusters and cpDNA haplotypes. The northern lineage showed significantly lower genetic diversity than the southern. Approximate Bayesian computation indicated that the northern and southern lineages had experienced, respectively, population expansion and long-term stable population size. The divergence time between the two lineages was estimated to be 565,000 years ago and no signature of migration between the two lineages after divergence was detected. Ecological niche modeling showed that the potential distribution area in northern Japan at the last glacial maximum was very small. It is thus considered that the two lineages have experienced different population histories over several glacial-inter-glacial cycles. Individuals of populations in the central to northern part of Honshu on the Sea of Japan side and in Hokkaido had large leaf width and area. These leaf characteristics corresponded with those of variety borealis. However, the delimitation of the northern and southern lineages detected by genetic markers (39°N) was not congruent with that detected by leaf morphologies (36°N). It is therefore suggested that variety borealis is not supported genetically and the northern and southern lineages should be considered separately when identifying conservation units based not on morphology but on genetic markers.Species delimitations by morphological and by genetic markers are not always congruent. The online version of this article (10.1007/s10265-019-01134-6) contains supplementary material, which is available to authorized users. Fagus crenata, Hiraoka and Tomaru Cryptomeria japonica, Tsumura et al. Kalopanax septemlobus, Sakaguchi et al. Quercus aliena, San Jose-Maldia et al. Magnolia salicifolia, Tamaki et al. Fagus crenata have indicated that variations in leaf morphology and physiology are based not on phenotypic plasticity but on the genetic make-up of individuals , whereas those of leaf width do not . Thus, the leaf width could become a key to distinguish the two varieties. However, Callaway  to clarify the genetic diversity and structure of M. kobus natural populations over the range of the species; (2) to assess the existence of variety borealis based on leaf morphological and genetic traits and (3) to infer how best to conserve genetic resources of M. kobus.In this study we investigated genetic variation in nuclear microsatellites and chloroplast DNA sequences, and leaf morphological traits, in Magnolia kobus populations, which cover its entire distribution range  the leaves collected were dried using silica gel and stored at room temperature prior to DNA extraction. We could not collect enough leaves for morphological measurement in this population, so the leaves were used only in genetic analysis.We sampled 10–20 leaves per individual for DNA extraction and measurement of leaf morphology from 23 M. obovata, M6D8  method (Murray and Thompson A), average gene diversity within population (HS), gene diversity in the total population (HT) and Weir and Cockerham’s FST were calculated. Hedrick’s standardized GST  is an intercept and the other βs are regression coefficients. σs are standard deviations and assumed to take a value greater than zero. Non-informative priors were used for all parameters. Stan via rstan package version 2.18.2 of R was used to estimate posterior distributions of parameters  and populations 10, 11 and 17 were thus removed from this analysis. To reduce the computational cost and uncertainty of parameter estimation, the parameters of the demographic model were sequentially inferred  approach was applied. We used only pure populations without admixture from the recessive lineage at NCUR). The unit of NCUR was the number of diploid individuals. (2) the population growth model (PGM) assumed that the current effective population size shrank exponentially towards the past with the rate G , i.e. there was exponential growth from past to present. PGM had two structural parameters, NCUR and G. The unit of the time parameter was generations. (3) The size reduction model (SRM) assumed that the effective population size changed at time T and had three structural parameters NCUR, T and relative ancestral effective population size . Prior distributions of parameters of the three models are shown in Table S1. We simulated nSSR and cpDNA sequence data simultaneously under these three models by fastsimcoal2 version 2.5.2.21  was used as a mutation model for nSSRs  and the geometric parameter (PGSM). PGSM ranges from 0 to 1 and represents the proportion of mutations that change allele sizes by more than one step; a value of zero means a strict stepwise mutation model. We simulated 13 independent loci. The prior distribution for the mean value of μ among 13 loci was drawn from a log-uniform distribution from 10−5 to 10−3 and each locus value of μ was randomly drawn from a gamma distribution with shape and rate parameters. The prior distribution of the shape parameter was drawn from a uniform distribution from 0.5 to 5 and the rate parameter was calculated by shape/the mean value of μ. The prior distribution of the mean value of PGSM among the 13 loci was drawn from a uniform distribution from 0 to 1 and each locus value of PGSM was randomly drawn from a beta distribution with a and b parameters. The values of a and b were calculated by 0.5 + 199 × the mean value of PGSM and a × /the mean value of PGSM, respectively, according to Excoffier et al.  Thr et al. . For cpDion Muse .Fig. 2Co5 simulations were conducted and posterior distributions of parameters were estimated using neural network regression implemented in abc package version 2.1 in R with the logit transformation option . When running simulations, NmNS and NmSN were divided by NN and NS, respectively, then the migration rates calculated were passed to fastsimcoal2. In angiosperms, the migration rate measured in the nuclear genome reflects both pollen and seed dispersals, whereas that in the chloroplast genome reflects only seed dispersal because the chloroplast genome is generally maternally-transmitted. When simulating cpDNA sequences, we thus multiplied migration rates by a coefficient β, which ranges from 0 to 1, in order to take account of the reduction in the migration rate for the chloroplast genome. The prior distribution of β was drawn from a uniform distribution from 0 to 1. (3) and (4) IMM models with one way migration from the northern to the southern lineage (IMMNS) and from the southern to the northern lineage (IMMSN) were also defined. For the mutation parameters of the four population divergence models, the same settings as for population size change analysis were used. All prior distributions for parameters are listed in Table S1. Model choice and parameter estimation of the best model were conducted in the same way as population size change analysis except for the number of simulations (1.5 × 106) and tolerance value  when parameter estimation was carried out.Taking into account the results of the population size change models, four different population divergence models were constructed Fig. b. (1) ThTo confirm that the model fits the observed data, posterior predictive simulations using 1,000 randomly drawn posterior samples were conducted for both population size change and population divergence analyses  and last inter-glacial  with the maximum entropy method implemented in Maxent version 3.3.3 k  GBIF Occurrence Download http://doi.org/10.15468/dl.ifurvo]. All records from GBIF were carefully checked against satellite images on Google Maps (http://maps.google.com) and ambiguous or erroneous location data were removed. A current distribution model was constructed with six bioclimatic variables, annual mean temperature (bio1), mean temperature of warmest quarter (bio10), mean temperature of coldest quarter (bio11), annual precipitation (bio12), precipitation in warmest quarter (bio18) and precipitation in coldest quarter (bio19), at a resolution of 2.5 arc-minutes, as used in a study of Magnolia salicifolia  ranged from 12 to 37 with an average value of 24.6 and the average gene diversity within populations (HS) ranged from 0.388 to 0.911 with an average value of 0.762  based on four individuals ranged from 3.34 to 5.30 with an average value of 4.48 and expected heterozygosity (HE) ranged from 0.580 to 0.859 with an average value of 0.764 (Table FIS) ranged from 0.008 to 0.340 within populations and its value over all populations was 0.097. Twelve of the 23 populations showed significant deviation from Hardy–Weinberg equilibrium.Among the 13 nuclear microsatellite loci across 23 populations, the number of alleles  than for cluster 2 (0.001). Populations 10, 11 and 17 showed more than 5% genetic admixture of the recessive cluster. At K = 14, although populations 1–10 were dominated by the mixture of two clusters, most of the remaining 13 populations were dominated by one of the other 12 clusters .The log probability of data in each  14 Fig. a. Howeve= 2 Fig. b. We thents Fig. c. The dins Figs. c. AccordAR and HE were higher in the southern lineage than in the northern lineage with the exceptions of populations 20 and 23 . Populations 20 and 23 were located apart from the other populations on the axes. . PopulatR2 = 0.400 and P < 0.001) and in both the northern and the southern lineages . However, the strength of IBD in the northern lineage was less than that in the southern one.Significant isolation by distance (IBD) patterns were detected over all 23 populations .A total of 3,932 bp-length of aligned cpDNA sequences was obtained. Four substitutions were detected within the species and three haplotypes  were determined  estimated by SHAPE which had more than 5% contribution to the overall variance (Table S3). PC1 reflected relative leaf width, for which populations in central to northern Honshu Island and Hokkaido Island had large values . All explanatory variables  were significant in the Bayesian linear mixed effect models of PC1 and leaf area  and the standard neutral model (SNM) were selected as the best models for, respectively, the northern and the southern lineages, with high posterior probabilities of 0.973 and 0.837, and low classification error rates of 0.193 and 0.192 Table . All posNN and NS) were 73,700  and 120,300 , respectively (Table TDIV) was 11,300  generations ago.Using the information obtained from population size change analyses, we constructed four population divergence models with different migration patterns. Model choice among the models was carried out and the isolation model (ISM) was selected as the best model with posterior probability 0.842 Table . AlthougPosterior predictive checking of the best models for both population size change and population divergence analyses showed good fits of the estimated models to the observed data .M. kobus  being 0.987 ± 0.002. The predicted distributions projected onto the current climate were a good fit to the species’ range except for Shikoku Island, where the species was predicted to be present currently as well as in LGM and LIG, but has no current populations of us Figs. , 6. The P > 0.6), the distribution areas with high probability north of 36°N were much smaller in CCSM than in MIROC. In Jeju, Honshu north of 41°N and Hokkaido, no potential distribution areas with P > 0.3 were detected. The predicted potential distribution areas in LIG and present were very similar.Although under both LGM climate scenarios, several potential distribution areas in coastal regions south of 36°N for both Sea of Japan and Pacific Ocean sides were predicted with high probability  and in Hokkaido during the LGM were very low. These results suggest that genetic variation in the northern lineage has been affected by severe genetic drift during one or more past glacial periods. The significant genetic differentiation between the northern and southern lineages, and the lower level of genetic diversity in the northern lineage, that we observed in M. kobus are common trends among temperate tree species that are widely distributed along the Japanese archipelago  on both Sea of Japan and Pacific Ocean sides in southern Japan. Although most of the populations of the southern lineage showed high genetic diversity and low FST values between each cluster and the ancestral population, two populations (20 and 23) did not. In the case of population 23 (Jeju), this population might have been affected by a severe historical isolation effect, as is suggested by the low probability of occurrence and the discontinuity with surrounding populations during both glacial and inter-glacial periods that were predicted by ecological niche modeling. Populations of Rhododendron weyrichii, which is a shrub growing in the warm temperate zone from Jeju to southern parts of Honshu Islands, on Jeju Island also showed long-term low effective population size and severe isolation as predicted by ABC and ecological niche modeling, respectively  and several areas with high probabilities of potential distribution during the LGM  years ago. As we have made two major assumptions, of mutation rate and generation time, when estimating the divergence time, and since the estimated value has some degree of uncertainty denoted by the HPD, we should be cautious about its interpretation  in LGM near the coast from 39° to 40°N on the Sea of Japan side, where the tail of the current distribution of the northern lineage is located, and it is considered to have been the glacial refugium for the northern lineage  of divergence time estimated by the isolation model was 11,300  generations ago. To convert the divergence time into years ago, we must assume a generation time for Callaway . As far Callaway  reportedM. kobus var. borealis . Because variety borealis recognized based on morphological characters comprises not only the northern lineage but also the southern one, we can conclude that it is not supported genetically and systematically.Populations from central Honshu on the Sea of Japan side to Hokkaido  had wide leaves and large area. Populations with these leaf morphological traits corresponded well to s Ohashi . Althougborealis appeared across both the northern and southern lineages. However, ABC analysis detected no significant historical migration between lineages after divergence. Population admixture was only detected in populations 11 and 17 for the southern lineage, but these populations showed leaf morphology of variety kobus. Therefore, it is unlikely that introgression has contributed to shape the morphology of variety borealis in some southern populations. We detected significant effects of environments on relative leaf width (PC1), the position of the maximum leaf width (PC2) and leaf area. Moreover, we also detected significant effects of population history on relative leaf width and leaf area. These results suggest that variation in the position of the maximum leaf width has been shaped by only natural selection, while variations in relative leaf width and leaf area have been shaped by both natural selection and population demographic history. However, it is possible that variations in these leaf morphological traits affected by natural selection are expressed through phenotypic plasticity and functional gene products , only one cpDNA haplotype and single variety, and the level of isolation by distance was relatively low, seed and/or seedling transfer may be permitted within the area north of 39°N, although the information about genetic differentiation of adaptive traits would be ideally needed to determine the range of seed and/or seedling transfer. On the other hand, since the southern lineage contained multiple genetic clusters, most populations showed clear genetic structure dominated by a single distinct genetic cluster in nSSR, composition of cpDNA haplotypes were different among populations and there were multiple varieties, it is obvious that seed/seedling transfer should be restricted even within the southern lineage and the optimal seed source would be the nearest natural population. In the southern lineage (< 39°N), for example, prefectural level conservation units may be practicable Below is the link to the electronic supplementary material.


Halyomorpha halys (Stål) is a globally invasive species that harbours the primary bacterial symbiont ‘Candidatus Pantoea carbekii’. In this work, P. carbekii was used as another genetic marker to investigate the biodiversity and biogeographical patterns of this important pest, in native and newly invaded areas, especially in Italy. The correlation between the genetic structure of the symbiont and that of its host was studied through the analyses of one bacterial and one host marker, the putative pseudogene ΔybgF and the mitochondrial gene COI, respectively. As a result, five new P. carbekii haplotypes were identified, and an association pattern between host-symbiont haplotypes was observed. Host species showed higher haplotype diversity than symbiont, which can be expected in a long term host-symbiont association. Populations from the north-eastern Italy showed the highest values of genetic diversity for both markers, highlighting that this particular Italian area could be the result of multiple ongoing introductions. Moreover, some of the symbiont-host haplotypes observed were shared only by populations from north-eastern Italy and native areas, especially Japan, suggesting further introductions from this native country to Italy. Overall, our findings improve the understanding of the potential origin of multiple accidental introductions of H. halys in Italy.The brown marmorated stink bug  P-symbionts are transmitted vertically, from mother to offspring, and may accurately mirror the history of their insect hosts. This ultimately helps, in the case of invasive host species by shedding light on their invasion pathways4. Bacterial symbionts can be used as reliable markers, showing even higher resolution than their hosts6.Symbiotic bacteria, especially primary or obligatory symbionts (p-symbionts) have the potential to be used as genetic markers to investigate host biodiversity and biogeographical patterns9, and consequently shape defining new control strategies.During the last century, biological invasions by exotic pests into new areas, generally facilitated by increased international trade and human movement, have caused costly economic damages in agriculture and forestry. Any new venue of information regarding the invasion pathways of a pest may help understanding its colonizing capacity, adaptability and behaviorHalyomorpha halys (Stål) (Hemiptera: Pentatomidae), harbours the primary, vertically transmitted symbiont ‘Candidatus Pantoea carbekii’ (hereafter referred to as Pantoea carbekii)10. Compared to free-living bacteria of the gammaprotobacteria, P. carbekii genome has experienced a consistent reduction, like other p-symbionts, but still provides its host with essential nutrients, vitamins, cofactors and protection of the most vulnerable stages of early development 11. Pantoea carbekii is highly stress tolerant, especially once secreted to cover the eggs, by its unique biofilm-formation properties, securing host offspring survival11.The highly invasive brown marmorated stink bug Halyomorpha halys is native to Southeast Asia but has recently invaded North America and Europe causing substantial damage to agricultural crops and creating nuisance to residents in rural and urban areas14. In Europe, since its first occurrence in Switzerland in 200415, it has rapidly spread to almost other 17 European countries33, as well as to Russia, Abkhazia and Georgia35. Based on the newly reported distribution points and recent models of expansion, its dispersal will likely continue in the coming years37.H. halys across the continents using genetic markers. Although most studies have focused on the use of the mitochondrial Cytochrome Oxidase I (COI) and Cytochrome Oxidase II (COII) genes42, one laboratory has attempted to resolve the genetic structure and origin considering some pseudogenes of the symbiont, P. carbekii, as genetic markers (e.g. ΔybgF)3. However, the bacterial marker did not explicitly incorporate geographic information among populations probably due to the limited sampling, especially in Europe3.Several efforts have been made to identify the origin and movements of H. halys from native  and newly invaded areas  was investigated utilizing the putative pseudogene ΔybgF. Special attention was given to invasive populations of north-east Italy, where high levels of genetic variability of the insect host have been recently reported43. The resulting data from the ΔybgF (bacterium marker) analysis was combined with the genetic structure of their insect hosts, for which the mitochondrial gene COI was used. Both native and newly introduced populations were studied to elucidate the pathways of expansion of the pest across the invaded area.In this work, the genetic structure of the primary symbiont of populations of P. carbekii in GenBank NCBI11. After alignment of the sequences obtained from this study, a total of six variable sites, and three parsimony-informative sites were identified.A total of 194  high quality sequences of ΔybgF were obtained. For quality assessment of the sequences, the chromatograms were analysed, and low quality regions found at the beginning and end of each one were trimmed, resulting in high quality sequences 206 bp long. All sequences showed high identity (> 99% sequence similarity) with the available sequences of Calculation of population diversity indices was limited to those populations represented by more than five samples. The haplotype diversity (H) of the p-symbiont per population ranged from 0 for KGJ (South Korea), and IFVM and IFVU , to 0.778 in IVeMo . Similarly, nucleotide diversity (π) ranged from 0% in KGJ (South Korea), IFVM and IFVU  and 0.66% in IVeMo  Table .Table 1DP1 = 100 (38%) and NP2 = 138 (52%), respectively. They were followed in numbers by haplotype P3 (N = 22), haplotype P4 (N = 2), and the remaining haplotypes, P5, P6, and P7, with one sequence each. Haplotype P1 was the most frequent among populations from the native area (China) and from the newly invaded areas , revealed seven different bacterium haplotypes. Five haplotypes of them  have been retrieved for the first time in this study Fig. . HaplotyCOI gene was obtained for all 219 specimens  representing the 22 populations of H. halys object of this study. The rarefaction curve reached an asymptote at 0% difference among sequences  plus many rare haplotypes. Haplotypes H1 and H3 included samples from all countries of the invaded areas , and few samples from the native areas in China  and two newly invaded populations (Switzerland and France) rejected the null hypothesis of neutrality, suggesting a past population expansion of H. halys in these areas . The NODF value, significantly lower than expected, did not exhibit a nested pattern  confirming the specificity between symbiont and host haplotypes.A bipartite interaction matrix between the haplotypes of the host and the symbiont was built considering only the samples in which the sequence of both the symbiont and the host were obtained Fig. . The netP. carbekii haplotype, P1, was more often associated with H1 host haplotype. This association was observed both in samples collected in China and in all the new invaded regions considered in this work, which were twelve populations from Italy, three from USA and one from Hungary  coming from native  and newly invaded areas ; notably, in the majority of these associations (11 out of 16), the host haplotypes were exclusively associated with the symbiont haplotype. Host haplotype H3 was most frequently associated with haplotype P2, and it was present in samples from China and several populations from the newly invaded areas: six populations from Italy, two populations from USA, and one from Hungary  were exclusively associated to one symbiont haplotype, and only five host haplotypes showed associations with more than one. Host haplotype H53 was mainly associated with symbiont haplotype P3, except for 2 samples out of 15, which resulted to be associated with haplotype P2. Similarly, host haplotype H3 was mostly associated with haplotype P2, and only 2 samples were associated with haplotype P1. In general, H1 harboured p-symbiont haplotype P1, fewer samples (N = 11) carried P2, and only one H1 sample was associated with haplotype P7. Host haplotypes H62 and H8 were associated with P2 and P5, and with P2 and P1, respectively  was the finding of a higher haplotype diversity in the host species, H. halys than in the symbiont.This study, based on populations of 44. Likely, populations of north-east Italy could be the result of multiple, still ongoing, introductions from either different parts of the native areas and even other invaded regions.It is assumed that increased genetic variability of invasive species in the invaded areas is associated with multiple introductionsH. halys. The north-eastern Italian populations showed symbiont-host haplotypes associations that differed from the rest of north Italian populations, which support the presence of multiple H. halys invasions from different origins. Haplotypes P1 and P2, already detected by Otero-Bravo & Sabree3, included the highest number of sequences: 100 (38%) and 138 (52%) of the dataset including both sequences of this study and NCBI, respectively. The topology of the host network matched with the one obtained by Cesari et al.39 and Valentin et al.41 that was characterized by the presence of two frequent and spread haplotypes (H1 and H3) and many rare haplotypes.The whole dataset allowed to observe some dispersion patterns of the invasive pest 45, the documented interception of H. halys in Europe, being all haplotype H1, suggests that European populations with haplotypes P1-H1, could have arrived from USA instead of coming directly from China46. The presence of the host haplotype H1 in Italy has been considered the result of an introduction event (from China or USA) different from the one reported for the first time in 2004 in Europe, Switzerland46. Indeed, the first populations found in Switzerland hosted the p-symbiont haplotype P23, supporting that the P1-H1 association found in samples collected in Italy might have resulted from a different introduction event either from China or USA.The p-symbiont haplotype P1, strongly associated with H1, was the most spread haplotype across the invaded areas including Europe and USA and it was found in all the Chinese populations object of this study. The symbiont-host association P1-H1 seems the most successful combination for invasiveness, or the most widespread combination. Its presence in the newly invaded areas may be attributed to a single or few introductions of H1 carrying P1, probably from China, and from there it could have spread across the globe. However, we cannot exclude that this widespread association may be the result of multiple introductions in different parts of the world by many samples sharing the same Chinese combination of haplotypes (P1-H1), possibly due to the commercial trades. Overall, it could be due to a single small initial invasive population with haplotypes P1-H1 or multiple invasions from the same source. After a shipment from USA to Germany46. Active dispersal involves movement of the entire organism through its own ability  while in passive dispersal, an organism uses different mechanisms (e.g. wind and human-mediated transport) to exploit new habitats48. Other symbiont-host associations, like P2-H52, P2-H54, including samples from both China (Shandong province) and north-west Italy, support the hypothesis that they came from China, in particular north-eastern areas of the native country. Moreover, host samples with haplotype H23 harbouring the p-symbiont haplotype P2 found in a number of populations from north-east Italy, ITAR, IVeL and IVeN, and Japan, highlight their possible recent arrival in Italy from Japan. In Oregon, the symbiont haplotype P2 was associated with two novel host haplotypes, H67 and H79, as well as with host haplotypes H56 and H23, which included also samples from Japan and Italy. The results pointed out the possibility that new introductions of H. halys from native or even newly invaded areas can take place continuously. In recent studies it was observed that after years of low genetic variability in USA populations42, new H. halys haplotypes have been found, suggesting that new invasion events have recently occurred in USA42.P-symbiont haplotype P2, was found in association with the highest number of host haplotypes, and more frequently linked to the host haplotype H3. The association P2-H3 was detected in samples collected within the native areas in China, and in the early invaded areas of Switzerland, Italy (mostly north-western areas), USA, and Hungary. The presence of P2-H3 in Italy could have resulted from its spread from China to Switzerland and, subsequently, from there to the North of Italy, by active and passive movementH. halys from Japan to Italy. While haplotype P4 was strictly associated to host haplotype H40 from China and north-eastern Italy, strengthening the possibility of multiple introductions from China to Italy.P-symbiont haplotype P3 was the most frequent among the five new symbiont haplotypes reported for the first time in this study. It was mostly associated with host haplotypes H53 occurring in populations of north-eastern Italy and Japan; adding to further introductions of 42 shed new light on the dispersion patterns of H. halys into the new invaded regions of north-east Italy. It is likely that (1) one or more introduction events of P1-H1 from China (and/or USA) to north Italy have taken place, (2) that one or more introduction events of P2-H3 came first from China to Switzerland, and, subsequently, to north-west Italy and, (3) that one or more introduction events of P3-H53 and P2-H23 came directly from Japan to north-east Italy.These findings, combined with the information provided by other authorsH. halys retrieved in samples collected in north-eastern Italy, could be explained by a large national and international commercial trade in this area. This can be supported by the presence of two of the largest and busiest Italian ports importing high volume of solid commodities49. The ability of H. halys to travel, joining commercial or domestic transport and with any type of goods, being often undetected during phytosanitary checks facilitates its accidental introductions into new territories52. Moreover, a study conducted by Zhu et al.40 proposes that H. halys populations from northern China possess high dispersal ability and/or adaptability. The former characteristics, together with the available suitable climate space in the invaded areas, suggest that the pest may expand northward to higher latitudes40.The complex network route of introduction and the high diversity of H. halys, which showed the existence of 25 haplotypes, interacting with only seven P. carbekii (p-symbiont) haplotypes can be expected from a long term host-symbiont association.The high genetic diversity of the host, 53. Studies on molecular evolution rates of these pentatomid symbionts have revealed the existence of reduced genomes (due considerable gene losses), coupled with high AT composition and accelerated evolution, a characteristic of stable host-symbiont associations, and not observed in frequently promiscuous symbionts 53. A phylogeny carried out on pentatomid symbionts, showed that the stable lineages are conserved and host species specific53. This same study found that P. carbekii has an accelerated molecular evolution, being one of the few uncultivable symbionts within the stink bugs clade. This pattern is generally observed and expected from endosymbionts, or intracellular bacterial symbionts which have reached a stable, old host association living in the protected intracellular environment54. P-symbionts have well established ancient associations that are also explained by the provision of bacteriomic or mycetomic organs by their host55. Halyomorpha halys, as all other pentatomid bugs, provides a unique bacteriomic structure, consisting of midgut caecal crypts in the distal end of the gut10. The physiological processes involving this organ include the selective uptake of the unique bacterial symbiont species, being the organ able to discriminate from non-symbiotic bacterial species56. This is a feature of long-standing, ancient host-symbiont associations.The stink bug family (Pentatomidae) carry several lineages of gut symbionts standing at different evolutionary stages54. Instead, p-symbionts seem to be exposed to selective pressure over time, removing deleterious mutations with reduction in base substitutions; this is dominating in older or ancient associations, slowing down the molecular evolutionary rates of the symbionts54. As H. halys is a ~ 160 million years old bug57, the association with Pantoea might have been established since the emergence of the clade, evolving for as long as the polyphagous host exists. Results from this work and previous research3 suggest that P. carbekii is under strong selective pressure, consequently keeping a low diversity (haplotypes). Pantoea carbekii stability makes possible the generalist polyphagous habits of H. halys. This host can rely on a stable p-symbiont that secures colonization, by providing nutrients, vitamins, and crucially securing survival of vulnerable developmental stages, regardless of the type of plant species and/or new environments invaded.According to the Muller’s ratchet hypothesis, p-symbionts  increasingly acquire deleterious mutations, leading to sharp extinction; but this is not happeningHalyomorpha halys is highly flexible, rapidly adapting to new and varied environments. The pest has high genetic diversity, observed in the multitude of haplotypes revealed. This genetic plasticity is not new, it has been observed in populations from the native and original areas, likely central China40, and seems to be shaped since the milder Pleistocene climate of East Asia. Latest research modelling the Pleistocene history of H. halys, estimated that the rapid expansions that took place in the past have occurred towards the Last Glacial Maximum. The same study has predicted future expansions toward higher latitudes, as it is already happening, in US and Europe40.This study improves the basic knowledge of the symbiotic relationship and provides new insights about potential origin of accidental introductions of the exotic species in Italy. Considering the impact that free trade exhibits on the rapid expansion of alien pests, a specific strategic plan might be developed in order to reduce the invasion risk by this pest. The results herein provide a framework for future researches that could help to optimize specific monitoring programs of material trade between national and international authorities.H. halys were collected during three years (2017–2019) in six countries; three representing their native ranges  and three new invaded areas . Extensive sampling was conducted in Italy encompassing the northern regions where the pest is mostly present . In total, 22 localities (populations) were sampled  harbouring the symbionts was extracted, transferred to Eppendorf tubes and kept in 96% ethanol at − 20 °C for further analysis.3 was selected to analyse the genetic variability of the p-symbiont. Same samples were used both for amplifying P. carbekii pseudogene ΔybgF, and H. halys mitochondrial cytochrome oxidase I (COI) gene.Extraction of DNA from each individual sample was performed using the Qiagen DNeasy Blood & Tissue Kit  following the manufacturer’s instructions. The putative pseudogene ΔybgF previously used by Otero-Bravo & Sabree3 were used in order to amplify the putative pseudogene ΔybgF in 20 μl reactions . Thermal cycling conditions were 3 min at 95 °C followed by 30 cycles of 95 °C for 30 s, 50 °C for 30 s, and 72 °C for 30 s with a final extension of 72 °C for 2 min.Primers dYbg-F and dYbg-RH. halys was studied using a region of the mitochondrial DNA corresponding to a fragment of the Cytochrome C Oxidase subunit I (COI) which was amplified using the universal primer pairs LCO-1490/HCO-2198, following procedures as in Folmer et al.59.Genetic diversity of the host https://www.geneious.com)60 and trimmed before further analysis. Low quality sequences were not included in the analysis.PCR products were checked via electrophoresis on 1.0% agarose gels stained with SYBR Safe nucleic acid stain (Invitrogen), purified using Exonuclease and Antarctic Phosphatase  and sequenced at the BMR Genomics Service . For the sequences obtained, the chromatograms were quality checked in Geneious prime 2020.1.2  to assess the identity of the sequences. In order to estimate the adequacy of sampling a rarefaction curve of the COI data set was generated using DOTUR 1.5362 considering the number of OTUs obtained at the 0% level of sequence divergence. If rarefaction curves reach an asymptote it is considered that most of the actual diversity have been sampled. Haplotype and nucleotide diversity of symbiont and host markers (each population), were calculated with Arlequin 3.563 using a Kimura 2-parameters model. The symbiont haplotype names in this study follow the nomenclature given by Otero-Bravo & Sabree3.Sequences were edited and aligned using MEGA X64 and TCS 1.2165.Sequences of the same markers available in the NCBI database were retrieved and added to this study datasets, in order to build haplotype networks for both the symbiont and the host. Statistical parsimony haplotype networks were inferred using the software PopART 1.7H. halys was inferred within each country, using the COI dataset, through the Tajima’s D and Fu’s Fs test and mismatch distributions of the pairwise genetic differences using Arlequin 3.5. The raggedness index (r) was used to quantify the smoothness of mismatch distribution and the sudden expansion model was tested through the analysis of the sum of square deviations (SSD) representing the modality of the distribution, obtaining the corresponding P values with a parametric bootstrap approach .The demographic history of e.g. symbiont-host association) show high H2′ values, whereas networks composed of not specialized associations show H2′ value of zero67. Moreover, the Nestedness Overlap and Decreasing Fill (NODF) index was used to detect and quantify the level of nestedness. Increasing values of NODF show increasing nestedness in the network68. Graph and metrics were generated by the bipartite package69 in R 3.0.1 software (R Development Core Team—https://www.r-project.org).In addition, associations among symbiont-host haplotypes were represented in a bipartite network. The specialization index (H2′) was used to determine the overall network specialization. Networks composed of specialized associations (Supplementary Information


Phaethon lepturus) in the Atlantic, Indian, and Pacific Oceans. Our primary aim was to identify appropriate conservation units for this little known species. Three morphometric characters were also examined in the subspecies. We found a clear pattern of population structuring with four genetic groups. The most ancient and the most isolated group was in the northwestern Atlantic Ocean. The South Atlantic populations and South Mozambique Channel population on Europa were genetically isolated and may have had a common ancestor. Birds from the Indo‐Pacific region showed unclear and weak genetic differentiation. This structuring was most well defined from nuclear and mtDNA markers but was less well resolved by morphological data. The validity of classifying white‐tailed tropicbirds into six distinct subspecies is discussed in light of our new findings. From a conservation standpoint our results highlight that the three most threatened conservation units for this species are the two subspecies of the tropical North and South Atlantic Oceans and that of Europa Island in the Indian Ocean.Investigations of the genetic structure of populations over the entire range of a species yield valuable information about connectivity among populations. Seabirds are an intriguing taxon in this regard because they move extensively when not breeding, facilitating intermixing of populations, but breed consistently on the same isolated islands, restricting gene flow among populations. The degree of genetic structuring of populations varies extensively among seabird species but they have been understudied in their tropical ranges. Here, we address this across a broad spatial scale by using microsatellite and mitochondrial data to explore the population connectivity of 13 breeding populations representing the six subspecies of the white‐tailed tropicbird ( Phaethon lepturus) over its entire range were analyzed in order to investigate population connectivity and relatedness. Our results are an example of how thorough genetic analyses can reveal critical information about subspecies delimitation and conservation priorities.Morphology and population genetic structure of the white‐tailed tropicbird ( The three small subspecies breed in the Pacific (P. l. dorotheae), Indian , and south Atlantic Oceans (P. l. ascensionis). The species has golden or apricot plumage morphs in some populations in which birds have white parts of the body plumage that are tinged with these shades. Populations of P. l. fulvus and P. l. europae comprise large fractions of apricot and golden birds, respectively, whereas other subspecies contain mainly white individuals  win2.3Total DNA was extracted from blood or tissue samples using the QIAmp Blood and Tissue kit (Qiagen). Genotyping was conducted for 10 polymorphic microsatellite loci , unbiased expected heterozygosity estimated according to Nei (HE), and Wright's F‐statistics FIS according to the method of Weir and Cockerham , aockerham  were calockerham –2004. Deh; Nei, π; Tajima, To test whether patterns of genetic variation in the mtDNA sequences deviated from neutral expectation, Ewens–Watterson test Ewens,  was perf2.64 simulation replicates. The L‐shaped method illustrates the frequency of rare alleles in the populations; an L‐shape graph indicates that the population is in mutation–drift equilibrium  was performed on microsatellite allele identity and mtDNA to determine how genetic diversity was distributed within and among subspecies and populations. Statistical significance was determined in ARLEQUIN 3.5.1.3 . For choosing the optimal number of clusters, three criteria were used; (1) the log likelihood given K (L(K); Pritchard et al., K; Evanno, Regnaut, & Goudet, L(K). The first two were calculated using STRUCTURE HARVESTER online Web server  to assess the optimal number of genetic clusters; the K with the lowest BIC value is the optimum  in all populations, (b) after removing the much‐differentiated Bermuda population, and (c) after removing the three differentiated genetic groups on Bermuda, Ascension/Fernando de Noronha and Europa.2.8P. lepturus.A statistical parsimony haplotype network was obtained from mtDNA sequences  using the pegas package 0.11 in R Paradis, . The hapP. lepturus and six P. rubricauda. The model of DNA substitution was determined using jModelTest 2  using the Tracer program 1.7.1  runs of 5 × 102.9FST matrix of genetic distances to calculate a new matrix based on Slatkin (FST/(1−FST). Differentiation in morphometric measurements among 10 populations was estimated using pairwise Mahalanobis D2, calculated with the HDMD package in R  had significantly longer tarsi than birds in the other populations  were consistently smaller for all three measured traits compared with the remainder , tarsus , and bill  lengths, respectively; Table P. l. catesbyi, P. l. europae, and the others . Pairwise permutation MANOVAs showed that P. l. catesbyi and P. l. europae were significantly bigger and smaller, respectively, compared to all other populations .Based on three morphometric measurements, birds of the subspecies ns Table . Birds o3.2p < .05) but no significant linkage disequilibrium was observed among any of the loci after the Benjamini and Yekutieli (α = 0.011), suggesting that all loci were independent. The mean allelic richness, based on minimum sample size of 11 individuals, ranged from 3.5 to 6.8 alleles per locus and was relatively similar among populations, except for the less variable Atlantic Ocean and Mozambique Channel populations (Table Ps > 0.05).No null alleles, large‐allele dropout nor stutter peaks were detected for the 10 microsatellite loci. The percentage of missing data was 0.81%. Linkage disequilibrium among loci was detected for three of the 45 loci pairs (ekutieli  correctiPs > 0.09) and Tajima's D  tests within all populations were not significant, providing no evidence of deviations from selective neutrality.Eighteen different mtDNA COI haplotypes were obtained from 872 bp COI sequences of 123 individuals sampled across 13 populations Table . HaplotyPs < 0.05; Appendix The L‐shaped allele frequency distribution graphs and M‐ratio statistics were not consistent with recent bottleneck events in any population. All populations had normal L‐shaped distributions and M‐ratio statistics well above 0.68. However, heterozygosity excess was detected in Ascension, Europa, Reunion and Mauritius colonies (3.3Ps < 0.001). With mtDNA, 73% of the variance was attributed to differences among subspecies, 13% to among populations and 14% to among individuals .The AMOVA using microsatellite allele identity indicated that 65% of microsatellite variation was attributed to differences among the delineated subspecies, 10% among the populations and 25% among individuals . Based on FST values, three populations were clearly distinguishable: (1) Bermuda, (2) Ascension, and (3) Europa, suggesting a strong isolation of these three populations , although statistically significant genetic structuring for two‐thirds of the pairwise comparisons and suggested isolation of the Reunion population  at K = 5 and optimal K using median value of L(K) at K = 5; Figure K varied from three (Evanno's procedure) to eight clusters (mean and median L(K) values; Figure K, analyses clearly distinguished three clusters corresponding to North and South Atlantic regions (the Bermuda population forming one and the Ascension and Fernando de Noronha populations the second) and the Europa population in the Mozambique Channel  were admixed.Clustering of microsatellite genotypes using STRUCTURE analysis showed that the best‐supported model contained three to eight genetic clusters, depending on the optimal K method used Figure  and suggl Figure . All thel Figure . WithoutK = 5–8 for all populations, K = 4–6 for all populations except Bermuda, and K = 2–5 at the Indo‐Pacific Ocean scale except Europa  was the most isolated with 13 nucleotide changes distinguishing it from the Ascension population and three private haplotypes. The Europa population was related only to the Ascension/Fernando de Noronha populations (four substitutions between them) and the Ascension/Fernando de Noronha populations were separated from the Indo‐Pacific populations by four substitutions. The Europa and Ascension‐Fernando de Noronha populations had private haplotypes . Pairwise ΦST values among populations confirmed these findings with all comparisons among these three populations and the remainder yielding ΦST values > 0.81 which were significantly different from 0 (Appendix P. l. fulvus) contained 92% of individuals with a private haplotype (VI). All comparisons among populations in the Indo‐Pacific region except Europa yielded ΦST values < 0.77 (and <0.66 if the Christmas Island population was excluded); half of them were not significantly different from 0  The first clustered the Bermuda population (P. l. catesbyi), (2) the second was divided into two well‐supported phylogroups consisting of the Ascension/Fernando de Noronha (P. l. ascensionis) populations and the Europa population (P. l. europae), and (3) the third included all of the remaining individuals of Indio‐Pacific Oceans . Morphometric distances estimated for the same 10 populations were weakly correlated with nuclear genetic distances  but not with mtDNA genetic distances .Genetic distances based on nuclear microsatellites and mtDNA sequences estimated for 10 populations showed significant relationships with geographic distances  were the smallest and constituted a highly differentiated genetic cluster. The subspecies P. l. ascensionis from the South Atlantic populations is morphologically similar to the Indo‐Pacific populations but genetically isolated. Birds from the remaining populations of the Indo‐Pacific region were morphologically similar and showed weak genetic differentiation. These results are discussed below in terms of subspecies validity and appropriate units for conservation strategies.The present study has provided data on genetic and morphological differences among putative white‐tailed tropicbird subspecies. Patterns of variation in white‐tailed tropicbirds revealed significant differences at different geographic scales and suggested disparities in connectivity among populations. This structuring was broadly in agreement in morphological, nuclear, and mtDNA marker analyses. Our results showed clear morphological and genetic differentiation in some subspecies such as 4.1P. l. catesbyi, the smaller subspecies P. l. europae and the other subspecies that were intermediate in size  and small subspecies . This difference in findings of these two studies may be partly explained by Le Corre and Jouventin  and P. l. europae (South Mozambique Channel) were each isolated and may have a common ancestor. Birds from the remaining subspecies  of the Indo‐Pacific region showed no clear and weak genetic differentiation but sample sizes were generally low.Microsatellites and mtDNA analyses suggested complex structuring among white‐tailed tropicbirds subspecies. P. l. ascensionis) during chick‐rearing from Fernando de Noronha archipelago and showed that long foraging trips reached an average distance from the breeding colony of 105 ± 48 km. This reinforces the hypothesis of a lack of connectivity between northern and southern populations of the Atlantic Ocean. Indeed, birds from Bermuda are genetically and morphologically distinct from birds of Ascension and Fernando de Noronha, suggesting that gene flow does not occur between the southernmost and northernmost populations of the Atlantic Ocean. Bermuda and Ascension Islands are separated by 39° of latitude , and so it is likely that latitudinal distance plays a major role in their genetic isolation. There is a pressing need to investigate the genetic structure of birds along the southeast–northwest gradient of the tropical Atlantic (including Great and Lesser Antilles), to identify the spatial limits of northern and southern genetic clusters.Birds from Bermuda are the most divergent and isolated genetic group, as suggested by microsatellite clustering Figure , DAPC F, the mtDP. l. ascensionis and P. l. europae could have a common ancestor that probably diverged from the ancestor of the Indo‐Pacific region about 290 kya  demonstrates that the Indonesian archipelago is not a physical barrier to seabird dispersion, suggesting that gene flow occurs among populations in the two ocean systems. An alternative scenario that would explain this result would be from only recent isolation of a previously panmictic population. An ongoing regional study of migration strategies of 10 seabird species of the western Indian Ocean has shown that some species, such as Lesser frigatebirds (Fregata ariel), sooty terns (Onychoprion fuscatus), and red‐tailed tropicbirds (Phaethon rubricauda), occasionally fly over the Timor Sea and the Indonesian straits to reach the Pacific Ocean  was genetically indistinguishable from populations of the Indian Ocean (except birds in the Europa population). The lack of nuclear and mtDNA genetic differentiation between Indo‐Pacific subspecies . Lee and Walsh‐McGehee . This subspecies is particularly at risk on the Brazilian islands where populations are small, threatened by introduced predators and exhibiting low genetic diversity and distinctiveness between the tiny yet discrete populations at Abrolhos and Fernando de Noronha , the subspecies P. l. lepturus appears not to be at risk currently. They include hundreds of populations, and tens of thousands of breeding pairs. Furthermore, different populations seemed to be genetically connected, implying a functional metapopulation of birds , endangered (P. l. ascensionis) or critically endangered (P. l. europae), especially under the enduring threats from invasive animal species. They warrant targeted and urgent scientific attention leading to conservation actions.Defining appropriate conservation units is the most important contribution of population genetics research to conservation. Our study suggests five conservation units for white‐tailed tropicbirds based on our genetic data: (1) Bermuda ; (2) Ascension/Fernando de Noronha ; (3) Europa; (4) Christmas Island; and (5) the other Indo‐Pacific colonies. The first three conservation units are clearly vulnerable ; Formal analysis ; Funding acquisition ; Investigation ; Methodology ; Project administration ; Software ; Supervision ; Validation ; Writing‐original draft ; Writing‐review & editing . Matthieu Le Corre: Conceptualization ; Funding acquisition ; Project administration ; Resources ; Supervision ; Writing‐original draft ; Writing‐review & editing . S. James Reynolds: Methodology ; Resources ; Validation ; Writing‐original draft ; Writing‐review & editing . Colin Wearn: Resources . Janos C. Hennicke: Funding acquisition (supporting); Resources . James C. Russell: Resources ; Writing‐original draft. Yann Gomard: Investigation . Hélène Magalon: Formal analysis; Writing‐original draft; Writing‐review & editing. Patrick Pinet: Resources . Pauline Gélin: Investigation. François‐Xavier Couzi: Resources . Etienne Bemanaja: Resources . Vikash Tatayah: Resources . Bacar Ousseni: Resources . Gérard Rocamora: Resources ; Writing‐review & editing. Patrick Talbot: Resources . Nirmal Shah: Resources . Leandro Bugoni: Funding acquisition (supporting); Resources ; Writing‐review & editing . Denis Da Silva: Investigation. Audrey Jaeger: Formal analysis ; Resources ; Validation ; Writing‐original draft ; Writing‐review & editing .Appendix S1Click here for additional data file.