Juglans regia L.) and sweet chestnut (Castanea sativa Mill.) have been economically and culturally important crops for millennia; there, in ancient times, they were invested with symbolic and religious significance. In this study, we detected a partial geographic congruence between the ethno‐linguistic repartition of human communities, the distribution of major cognitive sets of word‐related terms, and the inferred genetic clusters of common walnut and sweet chestnut populations across Eurasia. Our data indicated that isolation by distance processes, landscape heterogeneity and cultural boundaries might have promoted simultaneously human language diversification and walnut/chestnut differentiation across the same geographic macro‐regions. Hotspots of common walnut and sweet chestnut genetic diversity were associated with areas of linguistic enrichment in the Himalayas, Trans‐Caucasus, and Pyrenees Mountains, where common walnuts and sweet chestnuts had sustained ties to human culture since the Early Bronze Age. Our multidisciplinary approach supported the indirect and direct role of humans in shaping walnut and chestnut diversity across Eurasia from the EBA  until the first evidence of active selection and clonal propagation by grafting of both species. Our findings highlighted the benefit of an efficient integration of the relevant cultural factors in the classical genome (G) × environmental (E) model and the urgency of a systematic application of the biocultural diversity concept in the reconstruction of the evolutionary history of tree species.A biocultural diversity approach integrates plant biology and germplasm dispersal processes with human cultural diversity. An increasing number of studies have identified cultural factors and ethnolinguistic barriers as the main drivers of the genetic diversity in crop plants. Little is known about how anthropogenic processes have affected the evolution of tree crops over the entire time scale of their interaction with humans. In Asia and the Mediterranean, common walnut ( Juglans regia L.) and sweet chestnut (Castanea sativa Mill.). In this study, we evaluated the impact of isolation by distance processes, landscape heterogeneity, and cultural boundaries among human communities on shaping genetic resources of walnut and chestnut across Eurasia. Our findings highlighted the benefit of an efficient integration of the relevant cultural factors in the classical genome (G) × environmental (E) model and the urgency of a systematic application of the biocultural diversity concept in the reconstruction of evolutionary history of tree speciesLittle is known about how anthropogenic processes have affected the evolution of tree species with a long‐time‐scale history of human utilization such as common walnut ( These factors are interrelated because plants, especially crops, coevolved with humans in a complex socio‐ecological system at scales from the global to the local  and sweet chestnut (Castanea sativa Mill.).A few pioneering studies shed light on the cultural forces affecting plant diversity and seed dispersal routes at a continental scale. A strong geographic coincidence between ethnolinguistic boundaries, used as a proxy for human cultural differences, and the population genetic structure of plants has been detected for sorghum and pearl millet in Africa  and 3 Asian  countries . If the etymological reconstruction was available, the proto‐word for walnut and chestnut forms, conventionally denoted with an asterisk (*) at the start of the word, was reported in the Dené‐Sino‐Caucasian , and Afro‐Asiatic and Eurasiatic  Super‐Phylum. Despite the lack of written records, the etymological analysis of later attested words provided the opportunity to trace back through successive intermediate steps to the common ancestral word form in the ancestral reconstructed proto‐language. We considered linguistic terms as “inherited” when they were inherited from a proto‐language through nodes of phylogenic tree of more recent descendant languages following a vertical transmission. We classified linguistic terms as “loanwords” when words were borrowed from different language families and adopted in other languages by horizontal transmission. The identification of loanwords was further corroborated by archeological and historical data. We referred to a “cognate set” when the words have a common etymological origin sharing the same proto‐word. However, the cognates progressively changed their form and sometimes meaning over the course of the time , but in most cases they have similar sounds , we computed pairwise genetic differentiation with Jost's coefficient .To explore the genetic relationships among 91 common walnut populations and 73 sweet chestnut populations in the native range  was performed as implemented in Arlequin version 3.11 software  versus human linguistic diversity (dLAN) with geographic distance as a covariate (dGEO) was used to test whether any statistical significance inferred by the AMOVA was a result of isolation by distance (IBD) ] pairwise values against the corresponding natural logarithm of geographic distances. Subsequently, we performed a partial Mantel test to calculate the partial correlation between linearized dGEN values and human linguistic diversity after controlling for straight‐line geographic distance. Considering that partial Mantel tests showed inflated type‐1 error rate in the presence of spatial autocorrelation  parameter using the rarefaction method as implemented in the HP‐Rare  for all 91 common walnut and 73 sweet chestnut populations across Eurasia. For each sampling site, we calculated the number of common walnut and sweet chestnut linguistic forms recorded from published sources and from the LWED in terms of inherited terms, loanwords and open compound words. The nonparametric Kruskal–Wallis test was applied to compute statistical differences in the within‐population genetic variation Rs among classes of word‐term richness. Pairwise comparisons among word‐term classes for Rs values were performed based on post hoc Dunn's test.The level of genetic diversity was estimated for each common walnut and sweet chestnut population by computing the allelic richness (2.4.3K), we derived two synthetic maps representing the genetic structure of common walnut and sweet chestnut in Eurasia. In addition, the absolute number of migrants exchanged between the inferred genetic clusters per generation (2Nm) was calculated using Arlequin version 3.11 software. The spatial distribution of walnut/ chestnut word‐terms and their proto‐words was compared to the inferred genetic clusters of J. regia and C. sativa populations across Eurasia. Chi‐squared tests were conducted to compute statistical differences in the distributions of the major cognate sets that referred to walnut and chestnut linguistic terms among the inferred genetic clusters.The geographic coincidences between genetic structure of walnut and chestnut populations and the distribution of the major cognitive sets referred to their respective word forms were examined. As reported in Pollegioni et al.  and Matt33.1p < .0001). . An increase of mean pairwise dGEN was associated with an increase of mean dLAN among human communities in both species, varying from dGEN = 0.086 ± 0.003  and dGEN = 0.374 ± 0.01 (chestnut) for the category dLAN = 0 (same language) to dGEN = 0.393 ± 0.003  and dGEN = 0.693 ± 0.001 (chestnut) for the category dLAN = 4 (different phyla)  and sweet chestnut . Simple Mantel tests revealed that human linguistic diversity was also positively correlated with straight‐line geographic distances among common walnut  and sweet chestnut  sampling sites. Thus, the observed relationship between dGEN and dLAN matrices might have occurred in J. regia  and C. sativa  as a result of a common spatial component . The MRM analysis indicated that the effects of geographic distance and human linguistic diversity on genetic tree divergence were significantly positive among common walnut populations  and sweet chestnut populations  (Table p = .0002) and 39.7% (p = .0002) of common walnut and sweet chestnut genetic differentiation, respectively  among J. regia and C. sativa populations and linguistic distance (dLAN) among human communities living in the Eurasian sampling sites  Fig. . FurtherJ. regia (79.77%) and C. sativa (76.65%) individuals, while 11.75% and 7.93% was due to differences among language phylum areas, respectively (p < .0001). The remaining molecular variance, 8.49% for common walnut and 15.42% for sweet chestnut, was distributed among populations within language phylum groups. A multivariate population graph displayed a nonrandom pattern of association between the language phyla and the genetic differentiation inferred in common walnut and sweet chestnut populations across Eurasia  parameter for each common walnut and sweet chestnut population based on SSR markers. By comparing these datasets, we detected statistically significant differences between genetic allelic richness (Rs) within J. regia and C. sativa populations, and the associated number of the related inherited terms, loanwords, and open compound words . The nonparametric post hoc Dunn's tests revealed that geographic sites with low Rs values were often associated with singletons (single word‐terms) in both species  and doon, and two in the ancient isolated Burushaski language from Dené‐Sino‐Caucasian super‐phylum, khakhā́jo and tili , large kernel in Proto‐Basque (*kankano) and small stone, grain and egg in Proto‐North Caucasian phylum (*ḳV̆rḳV̆(‐nV)). The adjacent Northern Pamir ridges of Tajikistan included one Persian walnut‐word gôz  and the loanword cormacz borrowed in the last century from Russian within the Indo‐European phylum , the Svan gak’  or Georgian ḳaḳali  belonging to Proto‐Karvelian *ḳaḳ‐al‐ , and ni‐gosi/goz‐ borrowed into the Georgian from the Old Persian language. The ancient root *ʔwǟrƛ̣_V  served as the primary equivalent for the meaning “walnut” also in the Proto‐Basque (*hur̄), while, conversely, forms from the Proto‐Sino‐Tibetan word *HwV́rƛ̣V  with similar sound but different meaning were found in the Sinic (Old Chinese *lit = fruit) and Tibetic (Tibetan li = apple) groups , the Turkic loanword koz (950–650 BP), and the current open compound word ceviz ağacı. It is worth mentioning that the eastern Himalayas of Western China showed high walnut genetic diversity and one autochthonous Sino‐Tibetan name of walnut star‐ka, and two loanwords, the Sino‐Tibetan Chinese hú táo  and the Indo‐European kara  , the Georgian c̣abli  from the ancient proto‐Kartvelian *ć̣ab‐ , and the Russian kaštán within the Indo‐European phylum  , the Turkic loanword kestane (950–650 BP), and the current open compound word bayat fıkra. In the Balkans, Greek sampling sites shared with Bulgarian areas the Indo‐European inherited proto‐word *kastAno‐ , and displayed two open compound words dios balanos and karua Euboikè. Finally, the Aquitania site located in the Pyrénées Atlantiques (France) was characterized by a progressive accumulation of two chestnut terms. The Indo‐European French form châtaigne belonging from Latin castanea  joined the Basque koskol from the Proto‐Basque relict form *kVl . The etymology of *kVl belonged to a heterogenous core group ranging from the Proto‐Eurasiatic *bVrq̇wV (edible fruit) to Proto‐North‐Caucasian q̇wăɫV   Figure . Sites i) Figure .3.3J. regia and C. sativa populations across Eurasia. STRUCTURE analysis of the 91 common walnut and 73 sweet chestnut populations based on SSR markers indicated four  and three (Kchestnut = 3) as the most appropriate number of genetic clusters, respectively  to 12.06  migrants per generation  of the inferred cluster 1 , Iran, Kashmir‐western Himalayas (Pakistan), and Tibet‐eastern Himalayas, except one population (Karankul) in Uzbekistan  and Akkadian  to current Aramaic (egoz), Syriac (gauza), and Arabic (jauz) , Northern Uzbek (jɔŋɣɔq), and Uyghur (jaŋɣaq). The Central‐Asian distribution of *ńaŋo term overlapped with the inferred genetic clusters 2 and 3 and admixed populations between cluster 2 and 3, including all common walnut populations from, Kyrgyzstan, northern China, and Uzbekistan. Apart from ten populations sampled in Hungary in which the loanword dió was borrowed from the Turkic language (409 BP) and Crete (Greece), the remaining 35 European common walnut populations included in the genetic cluster 4  displayed a co‐dispersal with the related Euro‐Asiatic *ŋuńV > PIE *khneu, mainly restricted to Italic (*xnut), Celtic (knūs), and Germanic languages (*xnut) . Finally, the inherited terms from the PIE *kar borrowed from the Proto‐Euro‐Asiatic *KVrV were co‐distributed with the five easternmost Balkan populations showing admixed profiles between cluster 1 and cluster 4. Although its geographic distribution was wide across all Indo‐European languages, the proto‐form *kar referred to the common walnut in the Anatolian‐Hittite , Old Hellenic ((k)árüo‐n, , Latin , Albanian (arrë), Greek (karydiá), and in all Balto‐Slavic languages with *reîš‐a‐ and *orēxъ as a Baltic and Slavic proto‐form , Albanian gështenjë, Proto‐Balto‐Slavic *késten, Germanic kastan(i)e, Celtic castan, and Latin castanea . The overlay approach suggested *kastAno‐term penetrance in the Hungarian language via Turkic‐elite dominance scenario  and through South‐Western Asia in the Turkish (kestane) and Arabic (kastanāʾ) territories. A small ancient cognate class of the Dene‐Sino‐Caucasian super‐phylum, *q̇wăɫV́, was restricted to two Basque chestnut populations of Western Europe included in cluster 3 , evolved in “tree” and/or “tar” in the Anatolian‐Hittite (taru), Germanic (*tri(w)u) and Balto‐Slavic languages , “wood” in Albanian (dru), “hard” in the Italic (dūrus), “oak” in Old Hellenic (drũs), Celtic  but also “chestnut” in the ancient Anatolian languages (dōru) . A fourth cognite set *blwt’ was detected in the Levant and Iran with a semantic shift from oak to chestnut and vice versa  3,00 BP. A 44.1In this study, spatial analysis showed that the inferred barriers to gene flow among tree populations coincided with large differences in human language. Genetic differentiation computed between common walnut and sweet chestnut populations and the divergence in current human language phylogeny were both positively correlated with geographic distances between sampled sites. Language and genetic tree data are therefore not statistically independent, exhibiting significant spatial autocorrelation. As postulated by Gavin et al. , biologiTopography is not the sole determinant of spatial patterns of diversity, however; a large body of research characterizes human migrations and socio‐cultural factors such as political complexity and social connectedness as additive drivers behind the diversification of languages or their geographic homogenization over the time  for which parallel demic and cultural expansion is assumed across Eurasia Fort, . Small s4.2Rs) in samples from (a) the Himalayas and Pamir Ridge of Central‐South Asia , (b) Pyrenees Mountains of Western Europe (for sweet chestnut), and (c) Trans‐Caucasus and Anatolia‐Balkan circuit for (both species) in the Eastern Mediterranean. The Himalayas, Trans‐Caucasus, and Pyrenees Mountains are considered glacial refugia and reservoirs of high levels of genetic diversity for many plant species, including common walnut and sweet chestnut  and the species persisted through the beginning of the Holocene  in the Himalayas, in the western Kunlun mountains of the Tibetan Plateau, in the Pamir mountains of southern Tajikistan and in the Tien Shan mountains of Northeast China , Kartvelian  to Indo‐European  word‐terms. Similarly, both species have been closely associated with human activities in these primary centers of fruit tree diversity since the Neolithic or Early Bronze Age onward.At the global scale, ecoregions of high biological diversity often coincide with hotspots of indigenous‐linguistic diversity  at Kanispur and Burzahom sites of Kashmir Valley, , and three Indo‐European  walnut word‐terms were highlighted. There is evidence of the religious importance of common walnut in Kashmir from the 1st millennium BC onward; walnut became the essential element of several rituals in the Shaivism religion of Kashmir , hazelnut (Corylus), common walnut, and sweet chestnut were stored in ceramic vessels as offerings for votive/ritual purpose in the geographically adjacent settlements of the Kura‐Araxes culture in the Southern Caucasus , the subsequent Indo‐European speakers of the Early Kurgan settlements in Georgia showed similar habits using animals, cereals, hazelnuts, figs (Ficus carica L.), walnuts (Indo‐European proto‐form *kar and *a‐/an‐gōza), and chestnuts (Indo‐European proto‐form *kastAno‐ and *blwt') as major components of funeral gifts  and Ceramic  Neolithic cultures of Central Turkey Siddiq, , both spC. sativa were uncovered in the Neolithic site of Kobeaga II cave in the Spanish Basque Country  and cluster 4 (Western Europe) walnut germplasm. The genetic modeling analysis of 91 walnut populations suggested that walnut genotypes from Anatolia hybridized with autochthonous Balkan trees in southeastern Europe over several generations starting from the EBA   along with seeds of non‐native fruit species such as pine (Pinus pinea) and pomegranate (Punica granata) from the Late Bronze Age shipwreck at Ulu Burun, Turkey , revealed the existence of a complex, sophisticated maritime trade network dominated by the proto‐Phoenicians more than three millennia ago  in several Roman vessels .It is also true that we cannot exclude the adoption of Haldane, . The arckástanon and Armenian kast (castanea) and the subsequent wide spread of the Indo‐European proto‐form *kastAno‐ attested the growing importance of nondomesticated chestnut for fruit, fodder and timber production during the Greek and Roman Empire. Even so, sweet chestnut was mainly consumed by the low social classes and was subsidiary to common walnut  in fact derived from a compound meaning, wealh = foreign + hnutu − nut. The genetic homogeneity of sweet chestnut (cluster 3) and common walnut (cluster 4) populations inferred across Western and part of Eastern Europe confirmed the main expansion of these species throughout Europe occurred after 2,500 BP, as a result of Roman fruit cultivation and their commercial activities . Although the language root of a‐/an‐gōza has been classified as Indo‐Iranian, its origin is uncertain. In the Afro‐Asiatic phylum, this cognate set is present as loanwords from the Persian language such as Syriac gauza and Arabic jauz but also as inherited terms from unknown Afro‐Asiatic proto‐form in three extinct Semitic languages, the Aramaic egoz , the Mesopotamian Akkadian gūzu , and Levantin Ugaritic 'rgz   and chestnut/ oaks in Aramaic , Akkadian , and Assyrian . The adoption of non‐Indo‐European proto‐words for common walnut and sweet chestnut by Iranian tribes cannot be excluded. Recently, cuneiform scripts provided tangible evidence of Mesopotamian‐Assyrian royal gardens and buildings for ex situ conservation of fruit trees such as almond, date, olive, pomegranate, fig, grapevine pear, and walnut  and the geographic dispersal of the third cognate set of common walnut, the Old Persian proto‐word i et al. , walnut ut Bowe, . In partut Bowe,  in Euras5In this study, we detected a partial geographic congruence between ethno‐linguistic repartition of human communities, the distribution of major cognitive sets, and the inferred genetic clusters of common walnut and sweet chestnut populations across Eurasia. Our data indicated that IBD processes, landscape heterogeneity, and cultural boundaries might have promoted both human language diversification and walnut/chestnut differentiation across the same geographic macro‐regions. In particular, we found three hotspots of common walnut and sweet chestnut genetic diversity associated to high linguistic‐related form richness: Himalayas, Trans‐Caucasus, and Pyrenees Mountains. In agreement with genetic and linguistic data, the archeological evidence suggested a long‐term interaction between walnuts, chestnuts, and people living in these macro‐areas. An early integration of common walnut and sweet chestnut cultivation in the Trans‐Caucasian agricultural landscape of EBA cultures was postulated. Our multidisciplinary approach supported the indirect and direct role of humans in shaping the walnut and chestnut diversity across Eurasia, in particular during Persian Empire and Greek–Roman colonization, until the first evidence of active selection and clonal propagation by grafting of both species.i) an efficient integration of the relevant cultural factors in the classical genome (G) × environmental (E) model, and (ii) a systematic application of the biocultural diversity concept in the reconstruction of evolutionary history of tree species.We are aware that our reconstruction of walnut and chestnut history will be affected by adjustments and revisions based on the availability of linguistic databases and theoretical developments in linguistics. As explained by Moran, Grossman, and Verkerk , variabi6The authors declare no conflicts of interest.Paola Pollegioni: Conceptualization (lead); Data curation (lead); Formal analysis (lead); Funding acquisition ; Investigation ; Methodology (lead); Project administration ; Supervision ; Validation (lead); Visualization ; Writing‐original draft (lead); Writing‐review & editing (lead). Stefano Del Lungo: Conceptualization ; Data curation (supporting); Investigation (supporting); Methodology ; Supervision ; Validation (supporting); Writing‐review & editing (supporting). Ruth Müller: Conceptualization (supporting); Formal analysis (supporting); Project administration (supporting); Supervision (supporting); Writing‐original draft (supporting); Writing‐review & editing (supporting). Keith Woeste: Conceptualization (supporting); Funding acquisition (supporting); Methodology (supporting); Project administration (supporting); Supervision ; Writing‐review & editing (supporting). Francesca Chiocchini: Data curation (supporting); Methodology (supporting); Software (supporting); Visualization ; Writing‐review & editing (supporting). Jo Clark: Project administration (supporting); Resources (supporting); Validation (supporting); Writing‐review & editing (supporting). Gabriel Hemery: Project administration (supporting); Resources (supporting); Validation (supporting); Writing‐review & editing (supporting). Sergio Mapelli: Project administration (supporting); Resources (supporting); Validation (supporting); Writing‐review & editing (supporting). Fiorella Villani: Conceptualization (supporting); Formal analysis (supporting); Resources (supporting); Validation (supporting); Writing‐review & editing (supporting). Maria Emilia Malvolti: Conceptualization ; Data curation (supporting); Funding acquisition (supporting); Project administration (supporting); Resources (supporting); Supervision (supporting); Validation (supporting); Writing‐review & editing (supporting). Claudia Mattioni: Conceptualization ; Data curation ; Formal analysis (supporting); Investigation ; Resources (supporting); Supervision ; Validation ; Writing‐original draft ; Writing‐review & editing .Supplementary MaterialClick here for additional data file.


Helianthus verticillatus (whorled sunflower). This endangered, wild relative of the common sunflower, Helianthus annuus, is endemic to four locations in Alabama, Georgia, and Tennessee, United States. Despite its endangered status, there is no recovery plan for H. verticillatus, and knowledge related to its basic plant biology and importance in ecosystem services is mostly unknown. In this study, we utilized 14 microsatellite loci to investigate fine-scale population structure and genetic diversity of H. verticillatus individuals found on two sampling sites within the Georgia population. Our results indicated moderate genetic diversity and the presence of two distinct genetic clusters. Analyses of molecular variance indicated that the majority of variance was individually based, thus confirming high genetic differentiation and limited gene flow between H. verticillatus collection sites. The evidence of a population bottleneck in these sites suggests a recent reduction in population size that could be explained by habitat loss and population fragmentation. Also, high levels of linkage disequilibrium were detected, putatively suggesting clonal reproduction among these individuals. Our study provides a better understanding of fine-scale genetic diversity and spatial distribution of H. verticillatus populations in Georgia. Our results can underpin an original recovery plan for H. verticillatus that could be utilized for the conservation of this endangered species and to promote its persistence in the wild.Evaluating species diversity and patterns of population genetic variation is an essential aspect of conservation biology to determine appropriate management strategies and preserve the biodiversity of native plants. Habitat fragmentation and potential habitat loss are often an outcome of a reduction in naturally occurring wildfires and controlled prescribed burning, as seen in  Helianthus verticillatus , commonly known as the whorled sunflower, is a herbaceous perennial endemic to four locations in the southeast United States   . After tl (1898) . Soon afl (1898) .Helianthus verticillatus is morphologically similar to three other Helianthus spp., Helianthus angustifolius (narrow-leaved or swamp sunflower), Helianthus divaricatus (woodland sunflower), and Helianthus microcephalus , with shared traits of rounded hairy leaves and small flower heads  and H. angustifolius (n = 17) , overlapping habitats, and H. grosseserratus displayed verticillate leaf patterns in “exceptions” (This species was first speculated to be a hybrid of two other tifolius . However(n = 17) . Due to eptions” .H. verticillatus were investigated using nuclear and chloroplast DNA microsatellite loci , McNairy and Madison Counties, and two near the Georgia (GA)-Alabama (AL) border , roughly 3.5 km apart .As a result of these studies and documented habitat loss due to agricultural expansion and timber harvest , in 2014km apart . In Madich Creek . The AL ch Creek . In addiH. verticillatus populations, our knowledge concerning the basic biology, census data, and a lack of a well-defined conservation plan remain a major problem for preservation of this species  and Echinacea tennesseensis (Tennessee Purple Coneflower)   could beHelianthus verticillatus leaf samples (n = 206) were collected from two sites located near Cave Spring, GA, which is managed and maintained by The Nature Conservancy. Within this location, there are only two discrete areas where endangered H. verticillatus was growing. We collected samples from both sites. Site one consisted of five contiguous 1 × 1 m quadrants of H. verticillatus. All stems were counted in each quadrant and five leaves per stem were collected at random from no more than 30% of the individual stems. Site one yielded 74 samples, 27 from the first quadrant, 17 from the second, 5 from the third, 11 from the fourth, and 14 from the fifth quadrant  to avoid collecting genetically identical individuals, per previous studies, and remove potential bias caused by clonality from this portion of the study (n = 206) were placed in plastic bags containing silica gel at a ∼10:1 ratio  and stored on ice at the time of sampling to prevent degradation  with sterile 13-mm zirconia/silica beads , and submerged into liquid nitrogen for 2 min. Samples were homogenized using a Bead Mill 24  for 20 s twice with 5 min in liquid nitrogen between the homogenization steps. DNA extraction was completed using a modified protocol of the DNeasy Plant Mini kit  by including 2% v/v liquid polyvinylpyrrolidone and 4 μl of RNase A in the lysis buffer. After addition of P3 buffer, samples were frozen (−20°C) for 1 h. Genomic DNA was quantified using a NanoDrop ND-1000 spectrophotometer  and stored at −20°C.H. annuus  was completed in 10 μl reactions with 1 μl of (10 ng/μl) genomic DNA, 2.5 μl (10 μM) of both forward and reverse primers, 0.5 dimethyl sulfoxide , 4 μl (0.8×) GoTaq Colorless Master mix , and brought to a final volume of 10 μl with sterile Nanopure water (Thermo Fisher Scientific). Reactions were performed using a Mastercycler Pro Automatic Thermal Cycler  using the following touchdown-PCR (TD-PCR) conditions: 95°C for 3 min, followed by 10 cycles of 94°C for 30 s, 65°C lowering 1°C per cycle to a final 55°C for 30 s, then 72°C for 45 s, another 30 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 45 s and a final elongation step at 72°C for 20 min (Forty eight microsatellite loci developed from . annuus  were scrr 20 min . Amplicor 20 min . A sampln = 206) were then grouped into 14 collection zones: 5 representing the 1 × 1 m quadrants sampled at the first site and 9 for each row of the grid at the second site (FLEXIBIN v2  was usedond site . All binond site  to remov5) of a collection zone (exp)   were alson (exp)  was usedon (exp) .H. verticillatus were assessed with STRUCTURE v2.3.4  with Arlequin v3.5.2.2 . To asseon zones , was calH. verticillatus collection zones. Sign and Wilcoxon tests were employed to establish whether or not the loci used in this study were in the mutation-drift equilibrium  removed three samples of the same MLG from analyses. Samples removed through clone correction were from three collection zones . The clone corrected data (n = 203) was used for all subsequent analyses with 14 polymorphic loci. H. verticillatus individuals were divided into 14 different collection zones based on location and quadrant in which samples were gathered .Clone correction of the original dataset (5) was 1 for all collection zones and 21 private alleles were identified in 8 of the collection zones (n = 7) (exp) ranged from 0.36 (collection zone C5) to 0.59 (collection zone R8) with an average of 0.50. However, site one had lower gene diversity (0.44) compared to site two (0.53) on average. The standardized index of association (P = 0.07)  ranged from 1.61 in collection zone C3 to 3.26 in collection zone C1, with an overall average of 2.58 . Evenneson zones . Only on (n = 7) . Nei’s g = 0.07) . Values Helianthus verticillatus individuals grouped into two distinct genetic clusters (ΔK = 2), based on the STRUCTURE results. STRUCTURE results were subsequently visualized using the POPHELPER v , individuals from the R1 collection zone indicated the presence of admixture, which was very limited within other H. verticillatus collection zones  and 3 clusters (K = 3) (HELPER v  platformHELPER v . When thon zones . Though d ΔK = 4 . Clusterd ΔK = 4 . BAPS pr (K = 3) . However (K = 3) .P < 0.001; Fst = 0.24) (P < 0.001), rather than among individuals within collection zones , and among collection zones  , compared to among individuals within two genetic clusters , and within individuals  (P < 0.001)  . When da< 0.001) . When da< 0.001) . When sa< 0.001) .Fst) corresponded with Nei’s genetic distance, with the highest amount of differentiation between collection zones C5 and R9, and the lowest between collection zones C4 and C5 . The Sign, Wilcoxon, and the standardized difference tests across three mutation models showed a significant heterozygote excess when the collection zones were grouped by sampling site, with both shifting from the mutation equilibrium model . When thH. verticillatus individuals, thus supporting our hypothesis of clonality and population structure. Both sampling sites harbored moderate levels of genetic diversity and in contrast to our proposed hypothesis, a high number of distinct genets . In all but three collection zones, each sample was a unique MLG. The moderate diversity observed within these collection sites appears to contradict much of the current research concerning rare or endangered plant species , which is native to Southern California and Mexico, was investigated by exp = 0.31) in H. niveus spp. tephrodes when compared to other rare  and endemic species   than studies on H. niveus spp. tephrodes (n = 119) or H. verticillatus (n = 71) studies, which could result in biasing the outcomes of genetic diversity analyses . The lower number of H. verticillatus samples could bias the outcome of the study, therefore resulting in misrepresentation of species diversity and overall fitness  is an example of high linkage disequilibrium in a plant species which, like H. verticillatus, has been subject to a genetic bottleneck and is propagated mainly through asexual means  versus sexually, which can influence demography and genetics of natural populations , 2008. Loduction . Evidencoduction . Sugarcaal means .Helianthus species  and site one (C1–5). With the close proximity (less than 1 km) of these two sites, it is possible that pollinators in the forested site could be transferring pollen between the two locations. It would be presumed that native and honey bees would be the primary pollinators for this as well as other sunflowers, but very little research has been done on that subject in wild  species . Some be species . However species .exp = 0.50) in these collection zones suggested that the bottleneck occurred recently and a reduction in variation has not been able to take place (Dryopteris remota (wood fern) or tree species like Populus tremuloides (trembling aspen)  . Becauseg aspen) .H. verticillatus and its relation to the environment. Further research into seed viability and in vitro propagation could prove to be crucial for preservation of this species in seed banks and botanic gardens  continued research of the biology and ecology of H. verticillatus; (2) placement of this species in botanical gardens and seed banks; (3) exploration for unknown populations; (4) continued monitoring of known populations; and (5) in vitro propagation and germplasm preservation.Combining previous research on https://doi.org/10.5061/dryad.f4qrfj6s5.The datasets generated for this study can be found in the Dryad – RT, PW, and DH conceived and planned the experiments. TE carried out the experiments, took the lead in writing the manuscript and other authors provided critical feedback, edits, and helped shape the research, analysis and manuscript. TE, CW, and DH planned and carried out the simulations. TE, RT, CW, PW, and DH contributed to sample collection and preparation. All authors contributed to the interpretation of the results.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Manihot esculenta Crantz) accessions. We conducted a microsatellite marker analysis of 89 cassava accessions collected from Ghanaian and exotic sources. These accessions were assayed using 35 simple sequence repeat (SSR) markers. A total of 167 alleles were detected from 35 polymorphic markers with an average of 4.77 alleles per locus. High allelic frequency was detected across the accessions, ranging from 0.32 to 0.99 with an average of 0.62 per marker. Observed heterozygosity ranged from 0.03 - 0.97 across the accessions. Polymorphism information content (PIC) ranged from 0.03 to 0.78 with a mean of 0.45, indicating high level of polymorphism across the accessions. Comparatively, higher number of alleles, gene diversity and observed heterozygosity were detected among the local accessions compared with the exotic accessions indicating rich genetic diversity among them. Population structure analysis based on STRUCTURE identified two subpopulations and a large number of admixtures. Cluster analysis based on the neighbour joining algorithim further separated the collection into seven sub-groupings irrespective of geographical origin. This indicates the possible sharing of common genomic regions occurring across the accessions. High allelic frequency differences and levels of heterozygosity were observed among the germplasm. These findings indicated significant genetic variability in the germplasm to warrant selection.Genetic diversity is fundamentally important in crop improvement and provides plants with the capacity to meet the demands of changing environments. This work was carried out to assess the diversity and the extent of genetic relatedness among a number of assembled cassava ( Manihot esculenta Crantz; Heterozygosity; Genetic variability.Agriculture, Environmental Science; Molecular markers; genetic variability;  It is mostly grown by smallholder farmers often in marginal ecologies due to its ability to give better and appreciable yields than most staple crops in ecologies of drought and poor soils [−1) [Cassava  , compareils [−1) . Thus, tProgress made in a breeding programme depends on a better understanding of the genetic variability present in the population assembled . In somein situ germplasm conservation and farmer to farmer planting materials transfer [Several studies have reported the existence of diverse germplasm of crops on farmers fields as a result of transfer . Farmerstransfer , 16. Thrtransfer , accessitransfer . The samtransfer , 21, 22.transfer .Ability to remove duplicates during characterization of germplasm collections for breeding activities is very important. Characterization of crops can be done using agronomic, phenotypic descriptors or molecular markers , 24. PheMolecular markers are segments of DNA that can represent different functional classes and have been widely used to estimate genetic variation among different populations , 32. An 22.1The study involved 89 cassava accessions obtained mainly from farmers’ field in Ghana, CSIR-Crops Research Institute , International Centre for Tropical Agriculture , CSIR-Savanna Agricultural Research Institute  and International Institute of Tropical Agriculture . Names of the various accessions and the place of collection are presented in 2.22.2.1DNA extraction was done at the Biotechnology Laboratory of CSIR-Savanna Agricultural Research Institute (SARI), Nyankpala. Genomic DNA was extracted from the 89 cassava accessions using CTAB method  with sli2.2.22 (1.5 mM at 1X), stabilizers and loading dye. An amount of 1 μl of genomic DNA and 0.5 μl each of forward and reverse primers were added to the PCR kits for DNA amplification. PCR amplifications were done with the following conditions: initial denaturation at 95 °C for 3 min, denaturing at 95 °C for 10 s, annealing at X0C  primers, widely distributed across the cassava genome , were us2.2.3Gel electrophoresis were carried out in Polyacrylamide gel (6%) using Galileo Bioscience (81–2325) horizontal tank, in 100ml 1X TE running buffer stained with ethidium bromide (3 μl). Electrophoresis was carried out at 120V for 150 min using 10 μl of the amplified PCR products. Then 50 bp and 100 bp molecular ladder (Ladder Plus) obtained from NBS Biologicals Ltd  were used to estimate the molecular weight of the amplified products. The PCR products were visualized and photographed on Benchtop UV trans-illuminator.2.3The DNA bands were scored based on the fragment length of each allele. The assignment of the fragment was based on its position relative to the 50 and the 100 bp standard molecular marker (DNA Ladder) used. Alleles were scored as present (1) or absent (0). Band sizes for each marker per genotype were scored as a/b where ‘a’ is the upper band and ‘b’ is the lower band. PowerMarker version 3.25  was subs33.1A total of 167 alleles were generated by the 35 SSR markers . Allele 3.2The analysis of molecular variance (AMOVA) based on the molecular data indicated higher within group variation which accounted for 97% of the total variation compared with variation between groups which accounted for only 3% of the total variation . Compara3.3The population structure analysis of the 89 cassava accessions estimated that the optimum number of subpopulation K which best explained the structure of the accessions was 2 (K = 2) using the Evanno method . MajoritCluster analysis based on simple matching coefficient and neighbour-joining algorithim revealed seven ‘distinct’ clusters or sub-groupings with no apparent connections to the place of origin or collection of accessions . Specifi4Manihot at large [The success of any crop breeding programme depends on the amount of genetic variability within the targeted traits for improvement and the extent to which these traits are heritable . For thiat large . The popThe average number of primers required to adequately assess genetic diversity in crops differ and depend on the level of out-crossing within the species . Crop spThe observed heterozygosity levels  were higher than those found for 64 accessions of cassava with 26 SSR markers, where the heterozygosity levels ranged from 0.47 to 0.66 with a mean of 0.57 . These lCassava is naturally highly heterozygous and diploid (2n = 36) with some few rare cases of polyploidy (2n = 3x = 54 and 2n = 108) being recorded . But varPrecise identification of phylogenic relationship and divergence of germplasm population(s) gives very useful information which helps decision making  in breeding programmes . The resThe presence of a large proportion of admixtures among the accessions confirms the heterogenous nature of cassava, being predominantly an outcrossing crop. However, larger proportion of admixtures was found among the IITA accessions compared with the Local collections and those from CIAT, which indicates that most of the accessions collected from IITA might share ancestors in earlier collections from several parts of Africa including Ghana. The population structure observed in the current study indicates the existence of similarities among accessions from different origins suggesting that it will be more useful to select parents based on genetic relatedness rather than based on their origins alone.5The simple sequence repeat markers used were informative enough and could detect genetic diversity among the cassava accessions to warrant selection. Greater genetic variation was detected within population than among populations. The Local accessions were more genetically diverse compared to those obtained from IITA and CIAT. The population structure of the cassava accessions did not show any apparent link to geographical origin (place of collection). Farmer-held germplasm accessions of cassava remain important sources of rich genetic resources for cassava improvement.Joseph Adjebeng-Danquah: Conceived and designed the experiments; performed the experiment; contributed reagents, materials, analysis tools or data, analysed and interpreted the data; wrote the paper.Joseph Manu-Aduening: Conceived and designed the experiments; analysed and interpreted the data; wrote the paper.Isaac Kwadwo Asante: Conceived and designed the experiments, wrote the paper.Richard Yaw Agyare: Contributed reagents, materials, analysis tools or data; wrote the paper.Vernon Gracen: Conceived and designed the experiments; wrote the paper.Samuel Kwame Offei: Conceived and designed the experiments; wrote the paper.WACCI) PhD program, which received funding from The Generation Challenge Programme (GCP) and the Alliance for a Green Revolution in Africa (AGRA).J. Adjebeng-Danquah was supported by the West Africa Centre for Crop Improvement (The authors declare no conflict of interest.No additional information is available for this paper.


The wild boar is native to the temperate region of Eurasia, which is now one of the most widely distributed mammals worldwide. The recent expansion in the wild boar population has attracted a lot of attention, which may cause great damage to ecosystems. Elucidating the patterns of the population structure, genetic diversity, population origin, and colonization route of wild boar is very helpful in the conservation and management of wild populations. Phylogeographic analysis has proven to be a powerful tool. Here, 82 samples of wild boars in 16 sampling locations were collected in Qinling Mountains (QM). Genetic analysis was conducted based on the mitochondrial control region and nuclear genes. The level of genetic diversity of wild boars in QM was lower than the total population in East Asia, but higher than European population. No obvious phylogeographic pattern were found. The effective population size was under demographic equilibrium in the past.Species dispersal patterns and population genetic structure can be influenced by geographical features. Qinling Mountains (QM) provide an excellent area for phylogeographic study. The phylogeography of Asian-wide wild boars revealed the colonization route. However, the impact of the QM on genetic diversity, genetic structure and population origin is still poorly understood. In this study, genetic analysis of wild boar in the QM was conducted based on the mitochondrial control region (943 bp) and twelve microsatellite loci of 82 individuals in 16 sampling locations. Overall genetic haplotype diversity was 0.86, and the nucleotide diversity was 0.0079. A total of 17 new haplotypes were detected. The level of genetic diversity of wild boars in QM was lower than in East Asia, but higher than in Europe. Phylogenetic analysis showed the weak genetic divergence in QM. Mismatch analysis, neutrality tests, and Bayesian Skyline Plot (BSP) results revealed that the estimates of effective population size were under demographic equilibrium in the past. Spatial analysis of molecular variance indicated no obvious phylogeographic structure. The Qinling Mountains (QM) are an important geographic and ecological barriers between northern and southern China, which were formed during the Mesozoic and Cenozoic periods extending over 1600 km from east to west in the southern Shaanxi province of China . BecauseSus scrofa) is a temperate species with diverse groups, and a widespread mammal currently distributed in Europe, North Africa and Asia . Th. Th19]. X/K) = −4085.17] , indicating moderate admixture  and twelve microsatellite loci of 82 individuals in 16 sampling locations. Overall genetic haplotype diversity is 0.86, and the nucleotide diversity is 0.0079. A total of 17 haplotypes were detected, and the genetic diversity was moderate in East Asia. For mitochondrial data, phylogenetic analysis showed a low genetic divergence of wild boar in QM, and there is no obvious phylogeographic pattern in those areas. Mismatch analysis, neutrality tests, and BSP results revealed that the estimates of effective population size were under demographic equilibrium in the past. For microsatellite data, STRUCTURE identified that most individuals showed admixed genetic structure and revealed no obvious genetic divergence in the microsatellite data.Batrachuperus tibetanus revealed three lineages corresponding to the northwestern, eastern and western lineages, which was likely caused by orogenesis of the QM during the late Cenozoic [Gekko swinhonis in the QM [QM plays on important geographical barriers for the species with low dispersal ability. For amphibians, the study of stream salamander Cenozoic . For repn the QM . For mamn the QM . Howevern the QM .In present study, the complete control region of mitochondrial DNA data was used to analysis the genetic diversity and structure of the wild boar in QM. The mtDNA genetic diversity was obvious lower than total of East Asian wild boar, but higher than European wild boar ,19. For Here, we may infer that the wild boar in QM derives from the complicated population history and population expansions in East Asia . In EastWe used 12 microsatellite loci to study the genetic diversity and population genetic structure of wild boar in QM. We revealed a moderate level of genetic diversity and a relatively large effective population size, suggesting a relatively high gene flow between populations in QM. In this study, the observed heterozygosity was obviously lower than the expected heterozygosity. Such a lack of heterozygosity may be attributable to the admixture of gene pools from different sources . For micOur results indicated that the genetic diversity of wild boars in both mitochondrial and microsatellite data of Qinling Mountains was moderate in East Asia, but higher than European wild boar. Phylogenetic analysis showed that there is no obvious phylogeographic structure. The effective population size was under demographic equilibrium in the past. We infer that population admixture and two-times colonization history of founder effect played important roles in shaping the spatial genetic structure of wild boars in QM. This study could be used as a case model for the research into the genetic diversity, genetic origin, and admixture genetic structure of wild animals.


Janzen's seasonality hypothesis predicts that organisms inhabiting environments with limited climatic variability will evolve a reduced thermal tolerance breadth compared with organisms experiencing greater climatic variability. In turn, narrow tolerance breadth may select against dispersal across strong temperature gradients, such as those found across elevation. This can result in narrow elevational ranges and generate a pattern of isolation by environment or neutral genetic differentiation correlated with environmental variables that are independent of geographic distance. We tested for signatures of isolation by environment across elevation using genome‐wide SNP data from five species of Andean dung beetles (subfamily Scarabaeinae) with well‐characterized, narrow thermal physiologies, and narrow elevational distributions. Contrary to our expectations, we found no evidence of population genetic structure associated with elevation and little signal of isolation by environment. Further, elevational ranges for four of five species appear to be at equilibrium and show no decay of genetic diversity at range limits. Taken together, these results suggest physiological constraints on dispersal may primarily operate outside of a stable realized niche and point to a lower bound on the spatial scale of local adaptation. We use genomic data to ask whether dispersal is reduced across elevation relative to within elevation in five species of Andean dung beetle. Contrary to a downstream prediction of Daniel Janzen's “mountain passes” hypothesis, we find high gene flow across the elevational ranges of all species. Dispersal and its absence shape range limits  at higher latitudes, though this effect is reduced in the more aseasonal southern hemisphere. On the other hand, evidence for reduced thermal plasticity in tropical organisms—which Janzen expected in aseasonal environments for the same reason as reduced thermal tolerance, that is, as its costs would outweigh its benefits—is mixed at best  with well‐characterized thermal physiologies and elevational distributions that conform to predictions of Janzen's seasonality hypothesis. We tested for reduced dispersal across elevational gradients relative to within elevational bands by estimating fine‐scale population genetic structure, Wright's neighborhood size, and by using a Bayesian approach for describing the relative contribution of geographic distance and environmental distance to neutral genetic differentiation. To understand whether observed patterns were instead produced by recent population expansion , two species in the tribe Dichotomini , and a single species (Eurysternus affin. flocossus) from a third tribe, Oniticellini. Species in the genus Deltochilum are ball‐rolling dung beetles that feed and breed using dung or carrion, though at least one tropical species of Deltochilum is predatory and kills millipedes  are increasingly popular organisms in studies of ecology and evolution . To prepare libraries for reduced representation high‐throughput sequencing, we used a double restriction enzyme digest approach adapted from Gompert et al.  and descWe placed 6 μl DNA from each sample (with a minimum concentration of 20 ng/μl) in separate wells of a chilled 384‐well plates, filling the remainder with samples from a separate but related study. We then added 2.6 μl of a restriction digest master mix consisting of 10× T4 buffer, 1M NaCL, 1 mg/ml BSA, water, and the MseI and EcoRI enzymes in a 1:0.52:0.52:0.2125:0.10:0.25 ratio. After sealing, vortexing, and centrifuging the plate, we incubated it for 2 hr at 37°C on a thermocycler with a heated lid, followed by 20 min at 65°C to denature the active enzymes. We next added 1.44 μl of an adaptor ligation master mix to each well. This master mix consisted of a MseI‐specific adaptor, water, 10× T4 buffer, 1 M NaCl, 1 mg/ml BSA, and T4 DNA ligase in a 1:0.072:0.1:0.05:0.05:0.1675 ratio. We independently added 1 μl of a set of uniquely barcoded EcoRI adaptors to each well, permitting pooled samples to later be identified and demultiplexed. Again sealing, vortexing, and centrifuging the plate, we incubated it on a thermocycler at 16°C for 2 hr and then diluted the reaction by adding 3 μl to 19.5 μl water on a new plate.2, 5 μM Illpcr1 and Illpcr2 oligos, iProof polymerase, and DMSO in a 10.4:4.0:0.4:0.4:1.3:0.2:0.3 ratio. We then performed an additional PCR cycle to eliminate any remaining single‐stranded DNA and reduce sequencing errors, adding 2.1 μl of a new master mix consisting of 5× iProof buffer, 5 μM Illpcr1 and Illpcr2 oligos, and 10 μM dNTPs in a 0.425:1.3:0.4 ratio to each well. We ran this additional cycle at 98°C for 3 min, 60°C for 2 min, and 72°C for 10 min.To enrich our libraries, we performed two separate 20 μl PCR amplifications, each of which was conducted according to the following protocol. We added 3 μl of our libraries to 17 μl of a PCR mix in a new plate and performed the reaction using a thermocycler profile of 98°C for 30 s, 30 cycles of 98°C for 20 s, 60°C for 30 s, 72°C for 40 s, and final extension at 72°C for 10 min. Our PCR mix consisted of water, 5× iProof Buffer, dNTPs, 50 mM MgClAfter merging our replicate PCR reactions to reduce stochastic differences in sequencing effort, we selected fragments between 250 bp and 550 bp using a Blue Pippin machine (Sage Science). We confirmed observed fragments matched the expected size distribution using a bioanalyzer and sent the plate for 100 bp single end read Illumina sequencing at the Genome Sequencing and Analysis Facility (GSAF) and DNA Sequencing Facility at The University of Texas .2.3http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/), itself a wrapper around the CutAdapt  tools. The pipeline then assembles reads into loci within individuals using the RADseq assembly program Rainbow , and the dDocent script dDocent_filters to filter this file to high‐quality SNPs alone. We first dropped any individuals with missing data at more than 30% of loci, any loci missing data at more than 25% of individuals, all SNPs with a minimum minor allele frequency of 0.05 and a minimum minor allele count of 3, all SNPs with a quality (Phred) score of <30, and any genotypes with fewer than 5 reads across all individuals. We subsequently dropped sites with an allelic balance of >0.75 or <0.25, where the proportion indicates the ratio of reference allele reads to all reads; sites with reads from both strands; sites with a ratio of mapping qualities between reference and alternate alleles that were >0.9 or <1.05, and sites with a quality score >¼ below its depth. Finally, we identified sites with a depth exceeding 3× the square of the mean and removed any from this subset that lacked quality scores exceeding 2× their depth or were outliers, which we identified qualitatively based on the approximate point at which a histogram of mean depth across loci began to asymptote. For the remainder of the paper, we refer to genotypes filtered in this way as our “primary” SNP datasets; we also generated a “secondary” SNP dataset without minor allele frequency filtering.We then used VCFtools, vcffilter  of genotypes using our primary SNP datasets using adegenet v. 2.1.1. We plotted samples on principal component axes 1 and 2 and visually identified outliers representing misidentified samples, cryptic species, or highly divergent subpopulations. Second, we tested for fine‐scale population subdivision within each species using fineRADstructure v. 0.3.2, which uses haplotype data to identify a closest relative for each allele and then sums these data into coancestry similarity matrix among all individuals. We again used our primary SNP datasets, but dropped individuals identified as potentially belonging to different species through our PCA.2.5r2 value of .1 and converted these files into allele counts per population  using adegenet v. 2.1.1 and custom R code (https://github.com/elinck/scarab_migration/blob/master/scripts/bedassle.R). We calculated geographic distances among all sampling localities using the earth.dist function in the R package fossil  model or both theta and an exponential population growth parameter α (our “growth”) model using the coalescent simulator framework coala v. 0.5.3 in R , where t is the time in generations since the growth has started. To approximate our empirical SNP data, we used scrm to simulate 50 three‐nucleotide diploid loci for a sample size equivalent to each of our five species after filtering using the sequential coalescent with recombination model  based on the mean homozygosity of gene frequencies in the R package pegas v. 0.1.1  is an estimator of 4πρσ2 .To describe genetic diversity across the elevational distribution of our focal species, we calculated the population‐scaled nucleotide diversity of each sampling locality using an estimator of theta , Dichotomius podalirius (n = 26), Dichotomius satanas (n = 100), Dichotomius podalirius (n = 26), Deltochilum speciosissimum (n = 49), Deltochilum tessellatum (n = 20), and Eurysternus affin. flocossus (n = 35). Coverage was largely even across species, with mean read count ranging from 824,833.5 for Eurysternus affin. flocossus to 917,563.1 for D. satanas. After assembly and quality filtering of data, we dropped 1 individual of D. satanas, 2 individuals of D. speciosissimum, 1 individual of D. tessellatum, and 11 individuals of E. affin. flocossus. Our primary SNP datasets showed marked heterogeneity in both the number of loci and the resulting number of SNPs across species, with a minimum of 73 assembled RAD loci and 147 SNPs for D. podalirius and a maximum of 27,379 loci and 54,890 SNPs for D. tessellatum. As exploratory tuning of assembly parameters did not qualitatively change our results, we believe this variation is an artifact of underlying template quality, relative genetic diversity, or structural variation. Our secondary SNP dataset, which was not filtered by minor allele frequency or count, also showed substantial variation , ranging from 347 loci and 1,314 SNPs for E. affin. flocossus to 19,068 loci and 68,373 SNPs for D. speciosissimum. Despite this variation, simulation studies indicate our data are sufficient to accurately characterize genetic differentiation among populations  and a maximum of 23.74% of variation . Sampling elevation was uncorrelated with PC1 across the 5 species .Principal component analysis and fineRADstructure found no evidence of population genetic structure associated with elevation in any species. After dropping outlier samples representing putative cryptic species, the first principal component of individual genotypes explained a minimum of 3.27% of variation (in 3.3SD) for the posterior probability of the parameter αE (reflecting the contribution of environment) to aD (reflecting the contribution of geographic distance) were 0.0006 (±0.0002) for D. satanas, 0.0303 (±0.4417) for D. speciosissimum, 0.0006 (±0.0001) for D. tessellatum, 0.0171 (±0.0406) for D. podalirius, and 0.0221 (±0.176) for E. affin. flocossus. Overall, autocorrelation between distance and environmental variables and low global FST values  and isolation by distance (IBD) to genetic differentiation in BEDASSLE found little evidence of IBE in any focal species Figure . Mean ra3.4D. satanas, 3.814 for D. speciosissimum, 7.605 for D. tessellatum, and 4.081 for D. podalirius, all consistent with moderate‐to‐strong support <2.5 and p > .1 for all species), indicating genetic diversity remains constant even in samples from near putative range limits  might represent a rare “windfall” event, attracting large numbers of individuals and providing opportunities for gene flow among individuals from relatively distant locations. Given the dramatic relief of the eastern Andes and concurrently short geographic distances between upper and lower elevational range limits of the beetles, this span could easily encompass populations otherwise subject to divergent selection on thermal physiologies. Additionally, our focal species’ demonstrated ability to detect and reach dung resources (such as our traps) may make them inherently less likely to show signatures of isolation by environment.What biological processes might explain rampant gene flow across elevation in Andean dung beetles? We suggest that, while initially unintuitive to us, this result could be considered a predictable consequence of their natural history. Dung beetles in Canthonini, Dichotomini, and Oniticellini are strong fliers with highly developed olfactory systems ; Data curation (lead); Formal analysis (lead); Visualization (lead); Writing‐original draft (lead); Writing‐review & editing (lead). Jorge Celi: Project administration ; Resources ; Writing‐review & editing (supporting). Kimberly Sheldon: Conceptualization (lead); Funding acquisition (lead); Resources ; Writing‐review & editing (supporting).


We observed high levels of genetic variability  and strong spatial structuring  across this transect. Based on the congruence of mitochondrial and nuclear differentiation, as well as differences in shell shape, we identified a primary dispersal barrier in the southern Atlantic subtropical gyre (15–18°S). This barrier is maintained despite the presence of expatriates, a gyral current system, and in the absence of any distinct oceanographic gradients in this region, suggesting that reproductive isolation between these populations must be strong. A secondary dispersal barrier supported only by 28S pairwise ΦST comparisons was identified in the equatorial upwelling region (between 15°N and 4°S), which is concordant with barriers observed in other zooplankton species. Both oceanic dispersal barriers were congruent with regions of low abundance reported for a similar basin‐scale transect that was sampled 2 years later. Our finding supports the hypothesis that low abundance indicates areas of suboptimal habitat that result in barriers to gene flow in widely distributed zooplankton species. Such species may in fact consist of several populations or (sub)species that are adapted to local environmental conditions, limiting their potential for adaptive responses to ocean changes. Future analyses of genome‐wide diversity in pteropods could provide further insight into the strength, formation and maintenance of oceanic dispersal barriers.Pteropods, a group of holoplanktonic gastropods, are regarded as bioindicators of the effects of ocean acidification on open ocean ecosystems, because their thin aragonitic shells are susceptible to dissolution. While there have been recent efforts to address their capacity for physiological acclimation, it is also important to gain predictive understanding of their ability to adapt to future ocean conditions. However, little is known about the levels of genetic variation and large‐scale population structuring of pteropods, key characteristics enabling local adaptation. We examined the spatial distribution of genetic diversity in the mitochondrial cytochrome  Limacina bulimoides across the Atlantic Ocean. Two dispersal barriers were identified that were congruent with regions of low abundance, suggesting that regions of suboptimal habitat may create barriers to gene flow in widely‐distributed zooplankton species.Shelled pteropods or 'sea butterflies' are proposed as bio‐indicators to monitor impacts of ocean acidification on open ocean ecosystems. Local adaptation is affected by the presence and geographic extent of distinct populations, however, little is known about the large‐scale population structure of pteropods. We thus examined the spatial distribution of genetic and morphometric variation in the species  The AMT cruises traverse both the northern and southern subtropical gyres, which are separated by the equatorial upwelling region. The subtropical gyres are oligotrophic systems characterized by clear ocean waters with very low primary productivity in the surface layer, high sea surface temperature, a deep thermocline, and the presence of a deep chlorophyll maximum. Conversely, the mesotrophic equatorial province is characterized by upwelling of nutrient‐rich deep waters that stimulate primary production. Several species of copepods show spatial genetic structuring congruent with these Atlantic oceanic provinces. For example, Pleuromamma abdominalis has clades that are endemic to the equatorial province  were identified in the Atlantic Ocean: P. souleyeti, which is abundant in the subtropical gyres, and P. sculpta, which is most abundant in the equatorial upwelling region  annual transects of the Atlantic Meridional Transect programme  across a latitudinal transect in the Atlantic Ocean. This species has a circumglobal warm‐water distribution from ~45°N to ~40°S, a preferred depth range of 80–120 m, and it performs diel vertical migration with higher abundance in surface waters at night  and the nuclear 28S genes and made geometric morphometric measurements of shell shape. Our objectives were to (a) identify the location of dispersal barriers based on two genetic markers, (b) test for congruence of genetic barriers with differences in shell shape, and (c) assess whether the spatial structure was better explained by shifts in abundance or oceanographic parameters. By identifying dispersal barriers and the possible drivers of population structure in holoplanktonic gastropods, we can better predict their capacity to respond to a rapidly acidifying ocean.In this study, we aim to identify barriers to dispersal and spatial population structure in the coiled‐shelled pteropod 22.1Limacina bulimoides is a sinistrally coiled holoplanktonic gastropod, well‐adapted to life in the open ocean. It has a small . The 14 samples included in this study were collected between 35°N and 36°S in the following Longhurst biogeochemical provinces  in October and November 2012 (Table ls Table .L. bulimoides was obtained from AMT24 (September–October 2014) as quantitative samples from AMT22 were not available. For this equivalent AMT transect, bulk plankton samples were collected using a bongo net (200 µm mesh size) with a General Oceanics flowmeter (2030RC) mounted in the mouth to quantify the volume of seawater filtered   fragment of the mitochondrial COI gene, and 344 individuals for a 938 bp fragment of the nuclear 28S gene (Table L. bulimoides specimens were sequenced for both COI and 28S (Table 2O (Ultrapure), 2.5 μl PCR buffer CL (10X) (Qiagen), 0.5 μl MgCL2 (25 mM) (Qiagen), 0.5 μl BSA (100 mM) (Promega), 1 μl of each primer (10 μM), 0.5 μl dNTPs (2.5 mM) and 0.25 μl Taq polymerase (5 U/μl) (Qiagen). The initial denaturation step of 3 min at 96°C was followed by 40 cycles of 15 s at 96°C, 30 s at 50°C and 40 s at 72°C, ending with a final extension step of 5 min at 72°C. Amplified products were sequenced in both directions at Baseclear B.V. , combined and checked for errors in Geneious v8.1.9  and nucleotide diversity (π) were calculated using DnaSP v6.12.01 . Specimens were positioned in a standardized apertural orientation and photographed using a Zeiss V20 stacking stereomicroscope with Axiovision software, prior to destructive DNA extraction. The resulting images were processed and digitized at eight (semi‐)landmarks in TpsUtil and TpsDig Rohlf,  Figure . The cooL. bulimoides. Additionally, a Canonical Variates Analysis (CVA) was conducted in R  using Euclidean distances and 9,999 permutations in R with vegan  of 0.9998 (range: 0.9820–1.000) and nucleotide diversity (π) of 0.05449 (range: 0.0283–0.0402) across all sites  and a π of 0.0027 (range: 0.0006–0.0035) across all sites . Pairwise ΦST values for COI ranged from −0.017 to 0.451 for comparisons along the Atlantic transect and from 0.724 to 0.783 for comparisons between Atlantic and Pacific samples  from 51–64 (South), as 35 of the 40 pairwise ΦST values were significant (ΦST ranging from 0.012 to 0.621). There is additional spatial structuring separating samples from stations 13–29 (North) and 43–49 , as 13 of 17 pairwise ΦST comparisons were significant  than among four 'ocean provinces' . Thus, we find a strong primary dispersal barrier separating stations 13–49  from stations 51–64 (South) located at 15–18°S that is supported by both genes , but only for the North + Equatorial group , the repeatable RWs accounted for 85.21% of the total shell shape variation (Table N = 136) and Atlantic specimens only (N = 111) was visualized on the first two axes, which explain the most variation: RW1  and RW2   = 1.66, p = 0.204). Shell shapes of the Pacific individuals (N = 25) fall within the variation observed for the Atlantic individuals  = 14.53, p = 0.0001). Shell shape was different between the North and South , whereas comparisons of shell shape between the North and Equatorial or South and Equatorial groups were not significant . This result could be due to the low sample size of the Equatorial group (N = 9). Individuals from the North population group generally had a higher, narrower spire compared to individuals from the South  = 29.79, p = 0.00000) based on the CVA. Significant shell shape differences were recorded for both North‐South (p = 0.00000) and Equatorial‐South (p = 0.00003) comparisons, but not between North and Equatorial groups (p = 0.889) based on Tukey HSD multiple pairwise comparisons  in the Atlantic Ocean. Specifically, we have evidence for a primary dispersal barrier located in the southern subtropical gyre at 15–18°S and a more subtle, secondary barrier located in the region 15°N–4°S. This finding is in stark contrast to the historical perception of the pelagic environment as a homogeneous habitat lacking strong isolating barriers, and of holoplanktonic organisms as characterized by high dispersal and broad biogeographic ranges  and congruent genetic and shell shape differences. Despite the significant IBD test for the COI gene across the Atlantic transect, the disappearance of this result when populations across the primary barrier were tested separately supports the inference that these populations are spatially discrete evolutionary lineages  appears more permeable than the other (the primary barrier). The locations of these barriers coincide with regions of low abundance, congruent with the hypothesis that areas of less suitable habitat contribute to the maintenance of population structure in oceanic plankton  was observed at 18°S species in different ocean basins. These subdivisions generally result from divergence in allopatry, that is separated by current systems or continental land masses, and are often congruent with global biogeochemical provinces  using a strict COI molecular clock (Hellberg & Vacquier, L. bulimoides, a presumably circumglobal species. This underlying genetic variability and structure will influence the response of populations to global change, and in‐depth study of their life‐history traits, ecology and historical demography, in combination with experimental exposure to future conditions, will be required to more accurately predict their ability to adapt to a rapidly changing ocean.Holoplanktonic organisms are useful to gain insight into global biogeographical patterns in pelagic dispersal (Álvarez‐Noriega et al., The authors have no conflict of interest to declare.https://publons.com/publon/10.1111/jeb.13735The peer review history for this article is available at Appendix S1Click here for additional data file.Video S1Click here for additional data file.


This study highlighted new insights into the ROH patterns and provides a basis for future breeding and conservation strategies of Chinese sheep breeds.Homozygosity of long sequence genotypes are a result of parents transmitting identical haplotypes, which can be used to estimate their auto-zygosity. Therefore, we used high-density SNP Chip data to characterize the auto-zygosity of each breed according to the occurrence and distribution of runs of homozygosity (ROH). Subsequently, we identified the genomic regions with high runs of homozygosity frequencies within individuals of each breed. We selected 96 sheep samples from five local Chinese sheep breeds belonging to different geographical locations. We identified 3046 ROHs within the study breed individuals, among which the longer segments (>1–5 Mb) were dominant. On average, ROH segments covered about 12% of the genomes; the coverage rate of OAR20 was the lowest and that of OAR2 was the highest. The distribution analysis of runs of homozygosity showed that the detected ROH mainly distributed between >26 and 28 Mb. The Hetian and Hu sheep showed the lowest ROH distribution. The estimation of homozygosity level reflects the history of modern and ancient inbreeding, which may affect the genomes of Chinese indigenous sheep breeds and indicate that some animals have experienced recent self-pollination events . In these sheep breeds, the genomic regions were assumed to be under selection signatures frequently in line with long ROH. These regions included candidate genes associated with disease resistance traits ( The inheritance of consecutive homozygous fragments, identical haplotypes, of genome by the offspring from a common ancestor is known as runs of homozygosity (ROH) . ROH is e) is a population genetics basic concept. It determines the rate of evolutionary change caused by genetic drift and the equilibrium level of genetic variation and effective selection. Ne is often much lower than census size [e is an important parameter for evaluation of population genetic diversity and can provide a powerful method to characterize and understand the genetic architecture underlying complex traits. It is very relevant to the conservation biology matrix because it is used to estimate of genetic drift rate and inbreeding rate as well as influence of systematic evolution such as selection, mutation, and migration and other demographic factors. Low Ne leads to inbreeding and reduces genetic variability [Effective population size (Nsus size . Ne is aiability . Identification of long regions of homozygous genotypes in genome using high density SNP arrays has been considered an effective method for identifying the identity by descent (IBD) haplotypes ,13. In tAll experiments carried out on animals in this study were fully approved by the Animal Protection and Use Committee (IAS-CAAS) of the Institute of Animal Science, Chinese Academy of Agricultural Sciences, Reference No: IASCAAS-AE-03, on 1 September 2014. In total, 96 Chinese sheep of five different local sheep breeds, four flocks for each breed, were selected and five individuals from each flock were sampled randomly. Ear tissue and/or whole blood samples were collected from Hetian, Karakul and Yabuyi sheep distributing in arid environment and Hu and Wadi sheep in humid environment . Ear marp < 10−5) were filtered out. In addition, more than 10% of the samples missing genotyping (--geno) were deleted from the dataset. Finally, according to the suggestion of Zhang et al. [r2 threshold: 0.2) were used to prune SNPs with high linkage disequilibrium (LD), which left 502,072 autosomal SNPs and 96 animals for further analysis. Pruning is necessary to produce a better comparison between populations, as some extended of SNPs have a lower minor allele frequency. LD decay patterns which can inform population demography were investigated for each sheep breed, with r2 values calculated with the parameter --r2--ld--window 1000--ld--window--r2 0 commands in PLINK software.DNA samples were genotyped using Ovine Infinium HD SNP Bead Chip , which contained 606,006 SNPs (600 K) of the Oar_v4.0 Ovine genome assembly. We used PLINK v1.9 software  for qualg et al.  in PLINKROH) were calculated using the method previously described by McQuillan et al. [ROH was as follows: FROH = (LROH/LAUT), where LROH refers to the total length of ROH of each individual in the genome and LAUT is the specific length of the autosomal genome covered by SNPs of sheep chips (2452.06 Mb). To verify the accuracy of (FROH), we also used PLINK program and command --het to calculate the expected E(HOM) and observed O(HOM) number of homozygous genotypes and calculated the inbreeding coefficient (FROH). ROHs were identified and characterized in each of five sheep breeds using --homozyg function in the program PLINKv1.90, and the following conditions were adopted: minimum SNPs density were set to one SNP every 1 kB, and the maximum gap length was 1 Mb. For each ROH, one heterozygote and two missing genotypes were accepted. The total number of ROHs, the length of ROHs (in megabases) and the sum of all ROH segments (in megabases) were measured for all animals classified by breed and ROH length category. To compare the distribution of ROH among sheep breeds, we categorized the length of ROH into four classes . The average length of ROH was estimated by summing all ROH segments for each ROH length class per breed and divided by the total number of individuals of that respective breed. The genome inbreeding coefficients , was used for the identification of genes in ROH regions. In addition, the genome regions most commonly associated with ROH were identified by selecting the first top 0.5% (occurring in over 45% of the samples) of the common SNP observed in ROH [p ≤ 0.05). Gene Ontology (GO) terms and KEGG pathways used the list of genes from ROH islands and Ovine annotation file as backgrounds. Finally, we investigated the functional enrichments analysis for annotated genes in ROH Island from accurate literature and NCBI (https://www.ncbi.nlm.nih.gov/gene/) database.The --homozyg group functions implemented in PLINK v1.90  was usedd in ROH ,38. Adjad in ROH . The perd in ROH ,40, and e) for each of five sheep breeds was evaluated based on the following formula: Ne = e is effective population size, c is the genetic distance in Morgan and E (r2) is the expected r2 for distance c, implemented in the software SNeP v1.1 [e time points representing the number of generations ago (T) was calculated as T = e was estimated for each chromosome and generation in the past.The historical and modern effective population size (NE(r2)−1) , where NNeP v1.1 . The Ne  T = 12c . Ne was −5), minor allele frequency (<0.05) and call rate (<0.95). About 250,487 SNPs were removed using LD-based pruning when an r2 threshold of 5 was exceeded. After filtering, the 251,404 remaining SNPs were used for LD analysis. Minor allele frequencies were almost similar across the five breeds, inbreeding coefficients ranged between −0.004 in Wad sheep and −0.04 in Karakul sheep , followed by Yabuyi (790) on average (means of 46.8 and 46.5 Mb respectively). The lowest number of ROH (259) was found in the Hetian sheep breed on average (mean of 15.23 Mb) .R2 = 0.683  having 13,838 SNPs. In general, the total number of ROHs per chromosome reduces with decrease in chromosome length. The highest percentages of ROH per chromosome was observed on chromosome OAR2 (11.39%) and OAR3 (11.31%), whereas the lowest length was on chromosome OAR20 (1.23%) . The cor = 0.683 . ANXA10, CRYL1, PAG4 and 5S_rRNA genes, respectively (Auto-zygosity islands are obvious across the whole genome and their distributions varied in length and location on chromosomes . The mosectively . The topectively .e over the past 1000 generations and then a slow increase up to 2000 generations ago (e displayed for Hetian (n = 3563) and Hu (n = 3432) animals, compared to the most current generations. The Ne for the last 2000 generations showed the same value (n = 3133) shared in Wadi, Yabuyi and Karakul populations, falling above the minimum value (n = 30) of individuals for any livestock species to ensure the viability and genetic improvement in breeding programs. Ancient and modern effective population sizes (Ne) were obtained in these populations. We observed a rapid increase in Nions ago . Its dece as useful tools for detecting genomic relatedness, providing population assessment historical information over time and predicting underlying genome architecture [r2 or D’ [IS = 0.04 for Brazilian Santa Inés sheep [In previous studies, we revealed the genomic diversity, population structure and selection signature characteristics of five sheep populations in China . Here, witecture . In thisitecture . Secondar2 or D’ . Therefor2 or D’  because r2 or D’ . Inbreedr2 or D’ . In the és sheep , as wellés sheep ,53,54. Iés sheep . The maiés sheep . When thés sheep .r2 values decreased rapidly with increasing genome distance and were constant after 200–300 kb. The most rapid decrease in r2 was observed for the first five bins. The phenomenon may be caused by the mixing effects. This result is consistent with that reported by Gibbs et al. [The linkage disequilibrium (LD) decay analysis showed that the breeding histories of five Chinese sheep breeds were distinct. Wadi, Karakul and Yabuyi displayed the highest LD decay, while Hetian and Hu sheep had the lowest LD across all genetic distance intervals . In genes et al. .e of the five research groups in the past 1000 generations, and then it slowly increased up to 2000 generations ago across the five studied populations [e estimates before 2000 generations. Wadi, Karakul and Yabuyi sheep breeds had lower Ne estimates in all generations due to population admixture within the breeds [e value across five sheep breeds generations ago suggest that these animals can ensure viability within breeds in breeding programs [e is very important for maintaining heterozygosity and heterosis of Chinese animal breeds [Effective population size is one of the most important parameters in population genetic and conservation biology. Its transforms census size of the actual population into the size of the ideal population, showing the same loss rate of genetic variation, increases of inbreeding, mutation accumulation and determination of the accuracy of genome selection . Our resulations . Interese breeds . Therefoe breeds . In geneprograms . In addil breeds . Estimation of ROH is useful tool for exploring genetic diversity, providing information about the history of population demographic assessment and predicting potential genome structure ,16,60. AROH value is Yabuyi sheep, followed by Wadi and Karakul sheep, which indicates that the inbreeding rate of these sheep breeds is very high. This may be due to a small number of rams being widely used in herds breeding. Therefore, extensive mating among relatives may be the reasons for the high proportion of fixed alleles, resulting in the low genetic diversity in Yabuyi, Karakul and Wadi. Attention should be paid to prevent loss of sheep genetic resources. However, Hetian sheep have limited inbreeding, followed by Hu sheep breeds, which reflects the proper management of breeds, sufficient effective population size (Ne) and large number [The total length of ROH in Yabuyi, Karakul and Wadi sheep were higher, which indicated that their genetic diversity was low. In general, the average ROH of all breeds ranged from 11.56 to 26.29 Mb, which was consistent with the results obtained in Swiss sheep . The dise number . The maximum frequency of Yabuyi, Karakul and Wadi sheep in the longest ROH category reflect that recent inbreeding probably exists in these breeds. Generally, the high proportion of long ROH segments in the population indicated strong inbreeding in recent generations. In animal breeding, if there is a genetic relationship between individuals, it will inevitably result in increased homozygosity of selective region and stronger genome selection signals . In addiThe distribution of ROH on each chromosome has a certain regularity. The first three chromosomes have the largest number of ROH, while the number of ROH gradually reduce with the decrease of chromosome length. The number of ROH on chromosome OAR20 is the lowest, with 13,838 segments. Our results are consistent with those reported in sheep  and goatTo identify genomic regions with the highest frequency of ROH and thus harboring candidate genes potentially under selection, we examined ROH genomic regions with inbreeding coefficients that span a variety of candidate genes directly or indirectly associated with adaptation to extremely dry and humid environments. Genes associated with innate and adaptive immune responses include humid and thermo-tolerance, disease resistance, energy and digestive metabolism and performance in extreme dry and humid environments. HERC2 and CYFIP1, were identified in this study, which are reported to regulate innate and acquired immune response, as well as cytokine signaling. In addition, HERC2 and CYFIP1, which are involved in hemostasis regulation and mucosal defense, are also detected, which are of great significance in protecting sheep from parasites [HERC2 is a marker gene reported for biological predictions, some of which are unique and need functional verification [Several overlapping genes related to innate and adaptive immunity in mammals, such as arasites . Furtherfication .5S_rRNA, which is a gene detected in several overlapped regions (OAR2 and OAR6) with high statistical significance under warmer and humid climates conditions. It causes changes in the distribution and abundance of the five most common ticks in livestock in northern Xinjiang region of China. These ticks can transmit Borrelia disease in domestic animals [Borrelia (Burgdorferi sensu lato complex is an important endemic zoonosis) in human, sheep and cattle [Multiples candidate genes have been found in the genomic regions involved in host defense mechanism, diseases resistance and inflammation. The most significant genes include  animals ; controld cattle ; and cond cattle . GJB2 and GJA3, which are related to body size and developments [CPAP, also named CENPJ), is a centrosomal protein that participates in the assembly of centrioles and is very important for centrosome functions such as mitosis, motility or intracellular communication [Growth and body weight are the most important economic traits of livestock specially used for meat production. Several breeds from dry area or tropics tend to have small body weight/size and growth rate compared to humid or temperate breeds . Bodyweilopments . Anothernication .In this paper, we highlight the homozygosity patterns, population inbreeding levels and effective population size of five diverse Chinese sheep breeds from dry and humid environments. Based on the study of the occurrence and distribution characteristics of ROH, different numbers and lengths of ROH were found. Through ROH analysis, we revealed different genomic regions and candidate genes, which are highly correlated with important traits. Yabuyi, Karakul and Wadi sheep had higher homozygosity, whereas the inbreeding of Hetian sheep was low, followed by Hu sheep, which shows that the breeding management is proper, and the effective population size is sufficient. Our work contributes to a better understanding of genetic variation among different domestic sheep breeds and provides new insights into the genetic inbreeding aspects of selection and population demographic events.