The fluctuating climates, rising human population, and deteriorating arable lands necessitate sustainable crops to fulfil global food requirements. In the countryside, legumes with intriguing but enigmatic nitrogen-fixing abilities and thriving in harsh climatic conditions promise future food security. However, breaking the yield plateau and achieving higher genetic gain are the unsolved problems of legume improvement. Present study gives emphasis on 15 important legume crops, i.e., chickpea, pigeonpea, soybean, groundnut, lentil, common bean, faba bean, cowpea, lupin, pea, green gram, back gram, horse gram, moth bean, rice bean, and some forage legumes. We have given an overview of the world and India’s area, production, and productivity trends for all legume crops from 1961 to 2020. Our review article investigates the importance of gene pools and wild relatives in broadening the genetic base of legumes through pre-breeding and alien gene introgression. We have also discussed the importance of integrating genomics, phenomics, speed breeding, genetic engineering and genome editing tools in legume improvement programmes. Overall, legume breeding may undergo a paradigm shift once genomics and conventional breeding are integrated in the near future. Rising human population, fluctuating climates, and depleting arable land, coupled with lower productivity and post-harvest losses, pose a serious threat to global food security. Furthermore, surging pests and diseases challenge global researchers to develop stress resilient, high-yielding, nutritious crops to alleviate hunger and deprivation . LegumesLegumes have the capability to fix atmospheric nitrogen and are also believed to be involved in carbon sequestration and soil amelioration. Hence, they are considered an essential feature of sustainable agricultural production, particularly in arid regions . Their aPisum sativum L.), was the first experimental crop used by Gregor Johann Mendel (Father of Genetics) in his pioneering work to understand the basics of heredity and variation, which paved the way for establishing new branch of biological science, i.e., Genetics  over last two decades has been very slow compared to that achieved in cereals  Figure 1In addition, indeterminacy, plant morphology, environmental sensitivity, slow growth rate, and lack of management practises and government policies will further hinder legume improvement. Indeterminacy leads to non-synchronous flowering and maturity, which will affect the crossing and mechanical harvesting, respectively. Legume crops have poor photosynthetic efficiency, slow dry matter accumulation, reduced seedling vigour and canopy development, poor source-sink relationship, and rapid leaf senescence. Furthermore, they have reduced leaf area index (0.7-2.0) compared to cereal crops (3-6), which will reduce the gas exchange and photosynthetic efficiency. Legumes produce many flowers, but due to a lack of significant nutrient assimilation, most of them will fall before setting into fruit. For instance, about 70-90% and 80-95% of the produced flowers will drop in green gram and pigeon pea, respectively . Legume Given the importance of legume crops in a sustainable agricultural system and food security, we prepared a comprehensive review on challenges and strategies to overcome these challenges in legume breeding programmes. Furthermore, we discussed the advantages of integrating advanced genomics and phenomics approaches into distant hybridization to accelerate alien gene introgression and achieve higher genetic gain in these crops.Gene pool (GP) is a relationship among crop plants and related taxa that could be useful to breeders for crop improvement . Based oCicer arietinum L.) is the only domesticated species of the genus Cicer and was evolved from its immediate wild progenitor, C. reticulatum Ladiz. through natural selection. The genus Cicer consists of 46 species, of which 10 are annual and 36 are perennial in nature , GP-2 , and GP-3  M. Pop. and C. cuneatum Hochst. Ex Rich), respectively (Chickpea (n nature . Of the Anatolia . Among tectively .Cajanus cajan (L.) Millspaugh) is an important grain legume crop which includes nearly 13,200 cultivated and 555 wild accessions in the gene bank. Based on their crossability relationship with cultivated pigeonpea, all accessions were classified into three GPs. The C. cajana and C. cajanifolius (Haines) van der Maesen belongs to GP-1, whereas 10 species each belongs to GP-2 and GP-3, respectively (Pigeonpea (ectively .Glycine max (L.) Merr.) belongs to the genus Glycine, which has two subgenera, i.e., Glycine and Soja. The subgenus Glycine has 26 perennial species native to Australia  and its wild progenitor (G. soja Sieb.)  to GP-1 (G. max)  . Furtherertility . Majorit(G. max) .Arachis hypogaea L.) belongs to the genus Arachis, with 80 species. All groundnut species are divided into total nine taxonomic sections on the basis of their sexual compatibilities, morphological and cytogenetic features, and geographic distributions  as GP-3 . The genus Lens has seven closely related taxa and were categorized into four GPs  Hand.-Mazz and L. odemensis (Ladiz.) are crossable with cultivated lentils and may share the common gene pool (Wild species/sub-species of the genus four GPs . It has domensis . Furtherene pool .Phaseolus vulgaris L.) is the important legume crop with limited germplasm characterization. Over the course of its evolution and domestication, the common bean eventually formed two separate GPs, namely the Mesoamerican and the Andean  is an important legume crop. On the basis of seed weight, seed shape, and pod characteristics, faba bean has been divided into four groups, i.e., major, equine, minor, and paucijuga  belongs to the genus, Pisum, present in the tribe Fabeae, and is the oldest domesticated crop, with an estimated domestication of about 10,000 years ago  Asch. & Graebn., and P. abyssinicum A. Braun from their cultivated genepools (P. elatius) in the GP-1 , a member of GP-3  of these five sub-genera in Asia, Africa, and America. Domesticated cowpea (Vigna unguiculata (L.) Walp.) belongs to the section Catiang of sub-genus Vigna  and unguiculata (grain and vegetable cowpea) differ for their pod length and are cultivated in Asia and central Africa, respectively  R. Wilczek) and black gram (V. mungo (L.) Hepper) are the two most important legumes, which were believed to be originated from the single ancestor (V. radiata var. sublobata (Roxb.) Verdc and V. mungo var. silvestris Lukoki, Marechal & Otoul, respectively  Marechal)  Ohwi & H.Ohashi and V. stipulacea (Lam.) Kuntze with rice bean (V. umbellata (Thunb.) Ohwi & H.Ohashi)  Ohwi & H.Ohashi  , V. tenu.Ohashi) , and V. H.Ohashi . List ofAgricultural practises have domesticated hundreds of crop plants from their wild relatives with altered phenotype and genotype . During Favourable alleles stored in the wild repositories can be explored to break the yield plateau Figure 2http://www.nbpgr.ernet.in/Research_Projects/Base_Collection_in_NGB.aspx). A huge amount of chickpea germplasm  is conserved at various gene banks (https://www.croptrust.org/). Globally, ICRISAT, Hyderabad has the largest collections of cultivated chickpeas  and wild Cicer species (308 accessions from 18 species) from 60 countries. These reservoirs can be utilised in breeding programmes to boost the genetic gain in legumes. However, lack of appropriate germplasm characterization and pre-bred lines may limit their effective utilization in breeding programmes. Therefore, it has become indispensable to characterize the crop germplasm and develop the pre-breeding population.Huge germplasm collections of Chickpea , Groundnut , Pigeonpea , Soybean , Pea , Green gram , Common bean , Cowpea , Horse gram , and Rice bean  are stored in the National Bureau of Plant Genetic Resources (NBPGR), New Delhi (New combinations of genes resulting from spontaneous hybridization (between cultivars and their wild relatives) and trait introgression are considered as the major avenues for gene flow to expand the genetic diversity of cultivated crops . HoweverPre-breeding involves all the activities associated with identification of desirable traits/genes from unadapted germplasm  and transfer of these traits/genes into well-adapted, cultivar backgrounds . Pre-breeding offers a great opportunity to broaden the GP-1 by utilising genetic variability available in the wild species. This will ensure a steady flow of new and useful genetic variability into the breeding pipelines for the development of new cultivars with high levels of resistance and wider adaptability . Pre-breAs mentioned earlier, huge genetic variation is available in the wild relatives of legumes with several important traits. Despite their potential applications in crop improvement, legume breeders are reluctant to use wild species in their breeding programmes. This is owing to a lack of proper germplasm characterization, cross-incompatibility, and associated linkage drags Figure 3Chickpea is a cool-season, low-input demanding crop that ranks third in world production among the legumes . ChickpeCicer taxa. Furthermore, multi-parent populations such as MAGIC (Multiparent advanced generation inter-cross) and NAM (Nested Association Mapping) populations may enhance the allelic diversity and may also help in the inclusion of novel recombinants in GP-1  Desi and Kabuli ILs with enhanced resistance to wilt, foot rot, and root rot (C. arietinum and C. reticulatum (C. arietinum and C. echinospermum (C. reticulatum and C. echinospermum) were used to increase variability, transfer genes for several abiotic  and biotic  stresses, and also to exploit higher heterosis  Aba blight , cold  into JG-11 (foc2 and foc4) for Fusarium wilt resistance and pyramiding three genes, i.e., one for Ascochyta blight and two for Fusarium wilt resistance (foc1 and foc3), respectively   by transto JG-11 . This apectively . QTL-hot markers . Furtherchickpea . The Traultivars . A largedhal, seed husks used as cattle feed, dry stems used as fuel-wood, raw material to make huts and baskets, and soil mulch, and green leaves used for green manuring and medicinal purpose   . Seeds aitional) . Pigeonpitional) .Cajanus species are reservoirs of untouched diversity that can be explored to enhance the disease and insect pest resistance (C. scarabaeoides (L.) Thouars, a wild relative of pigeonpea has agronomically superior characteristics, including dwarfness, the number of fruiting branches and pods per plant, and resistance to pod borers and phytophthora stem blight (C. scarabaeoides have been successfully introgressed into the Pigeonpea (C. sericeus (Benth. ex Baker) Maesen for A1 CMS, C. scarabaeoides for A2 CMS, C. volubilis (Blanco) Blanco for A3 CMS, C. cajanifolius (Haines) Maesen for A4 CMS, C. cajan for A5 CMS, C. lineatus (Wight & Arn.) Maesen for A6 CMS, C. platycarpus (Benth.) Maesen for A7 CMS, C. reticulatus (Aiton) F.Muell for A8 CMS, and C. lanceolatus (W.Fitzg.) Maesen for A9 CMS  (G. canescens F.J.Herm. for powdery mildew (G. tomentella Hayata for nematode and leaf rust (G. latifolia (Benth.) Newell & T.Hymowitz for stem rot (G. formosana Hosok.) after screening 5,000 lines (2:3 and BC1F2 mapping populations (G. soja was identified on chromosome-14 and was found to increase the soybean yield by 9.4 per cent (G. tomentella (Kunitz trypsin inhibitor (kti) genes to develop trypsin inhibitor free lines (The wild accessions of us (YMV) , nematodus (YMV) , aphids us (YMV) , and brous (YMV)  It additus (YMV) . Other wy mildew , G. tomeeaf rust , and G. stem rot . These s00 lines . In addiulations . A yieldper cent . Genes fmentella . MABC haee lines .A. duranensis Krapov. & W.C.Greg. (AA) and A. ipaensis Krapov. & W.C.Greg. (BB)  . The kerrovement . FurtherA. cardenasii Krapov. & W.C.Greg. to cultivated peanuts  with A. hypogaea help to develop an IL  resistance to multiple diseases  and repeated use of the same genotypes in breeding programmes  into the cultivars  Grande) (Stemphylium blight (L. lamottei Czefr. and L. ervoides) (L. nigricans (M.Bieb.) Webb & Berthel. and L. ervoides) (L. ervoides) , high-micronutrient content  (L. odemensis)   (L. culinaris ssp. culinaris)  ( Grande) , resistarvoides) , resistarvoides) , architervoides) , extra eILWL401) , floweriemensis) , yield aentalis) , and tolgricans) , heat (Llinaris)  and salientalis) . Distantentalis) . Furtherentalis) .L. orientalis  , micronutrient content, and reduced generation time (80-100 days) (L. culinaris cv. Alpo and L. odemensis was constructed by using RNAseq methodology (Ascochyta blight resistance (on chromosome-6) were mapped in the interspecific crosses.Soil salinity affects the nodulation and nitrogen fixation in lentils. Collaborative research of ICAR, New Delhi  and ICARDA, Lebanon  identified the salt-tolerant accessions of -136670) . Success00 days) . Recentlhodology . SimilarPhaseolus is native to America, with 80 identified species (P. vulgaris L.), lima bean (P. lunatus L.), runner bean (P. coccineus L.), tepary bean (P. acutifolius A. Gray), and year bean (P. dumosus Macfad.), were domesticated for cultivation. Two of these species, P. vulgaris and P. lunatus, were independently domesticated at least twice in Mesoamerica and the Andes. This suggests that some domestication traits, such as determinacy, might be selected multiple times in common beans (Common bean is a popular legume crop that can be eaten as green pod vegetable or dried seeds (dry bean/Rajmash). Because of its medicinal properties, it can also be used to treat diabetes, heart problems, and bladder burn . The gen species . Among ton beans . Furtheron beans .Phaseolus species. For instance, P. acutifolius is known to possess relatively high levels of tolerance to most of the abiotic and biotic stresses  . In spitstresses . Therefoe traits .P. vulgaris and P. acutifolius was not successful without in vitro embryo culture  without the aid of embryo rescue to produce interspecific crosses between these two species  (P. coccineus cv. PI255956 to P. vulgaris cv. OR91G (PvPdh1 (Phaseolus vulgaris Pod dehiscence-1) located on chromosome-3, identified from the middle American domesticated beans was used to develop shattering tolerant common bean (Interspecific hybrids of  VRW-32) . In addiv. OR91G . Resistav. OR91G . Recentlmon bean .Faba bean is less resource consuming, cool season crop grown on marginal soils. It is also known as broad bean, bakala, horse bean, and tick bean . Middle-V. faba and V. narbonensis L. , L. albus L. (white lupin), L. luteus L. (yellow lupin), and L. mutabilis Sweet (Andean lupin) have been domesticated and are under cultivation (Lupin seeds have been used for thousands of years as food and feed. It has originated from the Old World (Mediterranean region), as well as from the New World (North America and Andean highlands) . Lupin itivation . There ativation .L. albus), thin seed coats and pod walls (L. angustifolius) (L. luteus), and high pod set . Genes for several agriculturally important traits, such as, vernalization independence (Ku and Julius), low-alkaloid content (iucundus), reduced pod shattering (tardus and lentus), soft seededness (mollis), white flower colour (leucospermus), and anthracnose resistance (Lanr1), have been identified and transferred into L. angustifolius (Crossing wild species with cultivated lupins is rarely successful . With huifolius) , reducedtifolius .iucundus and tardus, prevented the development of shattering resistant, alkaloid-free lupin lines  and pea cultivar, Alaska-81 to transfer the resistance genes for pea weevil into the cultivar background  from wild germplasm accessions. Introgressing these mutants into cultivar background may reduce the antinutritional factors. The P. elatius mutant has very low seed protease inhibitory activity, and this mutation has been successfully introgressed into cultivated pea , dry pea, or green manure crop and will also help in improving soil fertility by fixing atmospheric nitrogen. Peas are neglected by many farmers and will be grown on marginal lands, despite their importance in health benefits and sustainable crop production. Furthermore, due to limited genetic base coupled with changing climate conditions, wide range of biotic and abiotic stresses affects peas production. Novel genetic variations can be either exploited from the available wild relatives or can be created through mutagenesis . Distanta blight , P. satiium wilt , and Pis pinodes . Phenotya weevil . Geneticeritance . The resckground . In addi. fulvum . Howeverated pea . Furtherated pea .Vigna is an important genus in the Leguminaeceae family and it helps in restoring soil fertility by fixing atmospheric nitrogen. Cowpea (Vigna unguiculata (L.) Walp.) is a warm-season legume crop and is a rich source of nutrients and minerals. It can be used as a pot herb or as livestock feed. It has the ability to thrive well in drought-prone areas and in poor soils. Cowpea is a self-pollinated crop with a monophyletic origin. Several insect-pests, including pod borers, pod sucking bugs, flower bud thrips, and cowpea aphids, will greatly affect the cowpea yield  and V. sesquipedalis (L.) Fruwirth  A.Rich. as the resistance source to aphids, flower bud thrips, pod borers, and bruchids (symbiosis receptor-like kinase (SYMRK), through null mutants produced by CRISPR/Cas-9 technology  and abiotic factors  will cause severe yield reduction in these crops. Distant hybridization has allowed us to broaden the genetic base and offers the opportunity to deal with these problems.Green gram and black gram are the important short-duration legumes, and they can be intercropped with cereal crops to improve soil fertility . It is tears ago . Green gVigna, has useful genes for vigorous and erect growth, sturdy stems, broad leaves, and long and profuse pods with biotic and abiotic stress tolerance (V. radiata var. sublobata) (V. unguiculata subsp. dekindtiana (Harms) Verdc.), nematode (V. angularis (Willd.) Ohwi & H.Ohashi) (V. sublobata)  Benth., V. marina (Burm.) Merr., and V. vexillate) were identified (V. sublobata accessions were successfully introgressed using high-throughput genotyping and phenotyping tools (Wild relatives of olerance . Wild acblobata) , pod bug.Ohashi) , MYMD (mblobata) , and heaentified . Subsequng tools . Furtherng tools . Furtherng tools .V. aconitifolia) is largely cultivated as multi-purpose crop in arid regions of India. The prostrate, vining, and semi-trail growing patterns of moth bean help in reducing soil erosion. It can be used as a green manure crop and also helps to improve soil fertility by fixing atmospheric nitrogen. Moth bean has been considered as a vital source of amino acids, vitamins, and minerals (qVacBrc2.1) and minor (qVacBrc5.1) QTLs for seed resistance to Callosobruchus chinensis. Resistance genes for C. chinensis were further successfully introgressed into cultivars  is an underutilized legume that has gained attention due to its numerous applications, including profitability, diversity, and agricultural sustainability. It is also known as “climbing mountain bean,” “red bean,” or “Oriental bean.” It is used as a green manure crop as well as a cover crop. Rice bean is mostly grown for beans, although they serve as vegetable (green pod), fodder, and folk medicine. Despite these benefits, rice bean is grown in fewer areas owing to lack of ideal plant types, non-synchronous maturity, seed shattering, and insufficient marketing facilities (Rice bean (cilities . FurtherV. tenuicaulis)  , bruchidicaulis) . Interspmbellata . HoweverV. mungo . By emplmbellata . Furthermbellata .Macrotyloma uniflorum (Lam.) Verdc.) is an underutilised legume crop that originated in south-west India and it serves as a major source of vegetable protein for millions of rural residents in the Indian subcontinent  Verdc. and M. ciliatum (Willd.)Verdc. However, M. uniflorum, is the only cultivated species distributed in the Indian subcontinent   (M. africanum (Brenan ex R.Wilczek) Verdc. and M. axillare)  and lipid (10.85%) content in the horse gram (The genus ontinent . Wild spxillare) , increasDangwal) , and higxillare) . Introgrrse gram . Furtherrse gram .Fusarium spp., Pythium spp., root knot nematode, Anthracnose, collar rot, powdery mildew, Stemphylium leaf spot, Alfalfa leaf curl virus, and Alfalfa weevil)  ; (ii) fo weevil) ; and (ii weevil) . Forage Trifolium repens (L.). For instance, reproductive traits (such as flowering and seed yield) from T. nigrescens Viv.   .Medicago sativa L.) is possibly the world’s most important cultivated temperate perennial forage legume. M. arborea L. is another perennial forage legume, that serves as major source of genes for high level of heterosis (M. rugosa Desr. and M. scutellata (L.) Mill. into M. sativa (M. rugosa-M. truncatula Gaertn. and M. scutellate-M. truncatula were successfully produced by protoplast fusion to transfer the genomic fragments from the these species (through intergenomic recombination) into M. truncatula , selection intensity (I), and trait heritability (h2). Genetic variation is the fundamental requirement for crop improvement. However, domestication followed by intensive selection and utilization of same parental lines has significantly reduced the genetic diversity in the legume gene pool. Narrow genetic diversity of legumes can be widened either by unlocking the hidden variation present in the germplasm resources or by creating novel alleles or haplotypes not present in the crop germplasms  for specific traits of interest . Several2, RILs, BCs, DH, NILs, BILs, and immortalised F2), multi-parental (NAM and MAGIC), and unstructured populations (association panels), help to identify significant alleles (QTLs) or haplotypes. Multi-parent populations will combine allele richness and increase the mapping resolution by enhancing recombination frequency  (AhNFR1 and AhNFR5) and cowpea (SYMRK)  and genome editing can be effectively used to transfer favourable genes from unrelated species or produce novel genetic variations not present in the nature. Transgenesis has been successfully employed in broad range of legume species and it can potentially stack multiple genes for targeted traits . Mutatio century . CRISPR-C genes) . This ca (SYMRK) .Identified or created genetic variations  can be transferred into breeding populations by employing genomics-assisted breeding tools  . The MASHigh-throughput phenotyping platforms such as spectral reflectance provide an opportunity to utilize the vast amount of genomic data accumulating in databases. Furthermore, by combining phenomics and envirotyping with refined field trails and experimental designs, one can significantly improve heritability estimation and reduce spatial heterogeneity in the field, and, as a result, achieve higher genetic gain. However, decoding and using the information from such tremendous dataset generated from phenomics demands data science, particularly artificial intelligence and machine learning, to develop model-based breeding methods .via rapid generation advancement tools such as, single seed decent, shuttle breeding, doubled haploid  offers an avenue to broadening the genetic base of these crops. Taking into account the number of wild accessions available in gene banks, a rational sampling of target donors would have to be achieved. Reduced representation sequencing and high-throughput phenotyping offer the advantage of characterizing all germplasm lines stored in gene banks . FurtherGlycine (GP-3) have not yet been fully exploited. A. cardenasii has probably been one of the most widely used sources of useful genes in groundnut to date, even if crosses involving other species have also been used.One of the major lessons learned from wild species is that the valuable alleles present in CWRs can only be identified when they are incorporated into cultivar background. Alien gene introgression have offered us dividend results in chickpeas. Still, the chickpea hybridization programme needs meticulous planning and implementation for full utilization of wild resources to break the yield plateau. Wild species of pigeonpea offer the potential use in hybridization programmes for introgression of multiple traits, including resistance to pod borers and diseases. Exploitation of GP-3 and GP-4 has been attempted in soyabean, but it ended at the amphidiploid stage . The novThe wild lentil taxa are potential reservoirs of valuable genes controlling several traits. To increase and sustain lentil production and productivity, new gene sources need to be identified and transferred from different GPs. To maximize and sustain common bean production, high-yielding, superior quality cultivars that are less dependent on water, fertilizer, and pesticides should be developed. A tiered breeding approach involving alien gene introgression, gene deployment, and multi-trait improvement programmes would be the most appropriate strategy to accomplish these goals. Cross incompatibilities are the major barriers to improve the productivity of faba bean. However, modern breeding tools succeeded in transferring genes across incompatible species and opened opportunities to develop high-yielding faba bean with reduced anti-nutritional factors. Marker-based assessment of genetic diversity in geographically specified germplasm sets of cultivated lupin species might speed up the process of assigning different genotypes to “Old World” and “New World” farmed species. Transgressive segregants identified in distant hybridisation programmes enable the development of high yielding pea cultivars.Vigna species helps to discover novel alleles for yield improvement in different Vigna crops. The application of genomic tools to harness genetic diversity from wild relatives of cowpea has yet to be accessed. Introducing molecular genetics tools into cowpea distant hybridization helps in bypassing the incompatibility barriers and introgressing desirable alleles or haplotypes. Horse gram and rice bean are the climate-resilient future crops for dryland areas. Present and future challenges in the breeding of forage legumes demand for the exploration of genetic variation in breeding programmes. The most dependable and desirable way to use their wild relatives is to introgress desirable traits into modern cultivars. Forage yield, quality, and persistence are the most important economic traits and have been shown to be good candidates for the future forage breeding.Exploitation of wild In conclusion, concerted integrated strategies should be used to identify and introgress the valuable genes housed in the CWRs of legume crops. This will provide us with a generation of a multitude of novel pre-bred lines that can be utilised in mainstream breeding to fight today’s climatic vagaries.IS, PS, GS, HA and SG conceived the idea. Authors have contributed in writing individual crops: GS (Pigeonpea), AD (Groundnut), PU (Chickpea), PKS (Lentil), GN (Moth bean and Rice bean), LG (Forage legumes), DK (Soybean), PK (Lupin), AK (Common bean), AT (Black gram), SS (Green gram), GK (Faba bean), MDP (Pea and Cowpea), and PH (Horse gram). SG and GS prepared the figures. HA, SG, RK, SK and AD collected the data and finalized the tables. HA, PS, GS, and SG compiled and edited the manuscript. IS supervised the study and finalized the manuscript. All authors contributed to the article and approved the submitted version.The authors would like to thank everyone who contributed to improving the quality of the review article at various stages of writing manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Habermas’s argument from human nature, which speaks in favour of holding back the use of human germline editing for purposes of enhancement, has lately received criticism anew. Prominent are objections to its supposedly genetic essentialist and determinist framework, which underestimates social impacts on human development. I argue that this criticism originates from an instrumentalist reading of Habermas’s argument, which wrongly focuses on empirical conditions and means-ends-relations. Drawing on Habermas’s distinction of a threefold use of practical reason, I show how an alternative—the ethical—reading avoids essentialist and determinist objections by addressing an existential level of sense making. I present three reasons that speak in favour of the ethical reading and I demonstrate how it incorporates social aspects of character formation. Habermas’s account therefore offers exactly what the critics claim is missing. The paper concludes with a conceptual challenge that the ethical reading has to face within Habermas’s overall approach to genetic engineering. The Future of Human Nature (FHN) has gained renewed attention. Habermas argues that the natural contingency of the biological genetic setup of humans plays a fundamental role for their self-understanding as moral agents. This is why one should proceed with caution when intentionally introducing changes to the human genome, especially for the purpose of enhancement.Recent developments in human germline editing n Nature  (FHN) haLately, this argument has received criticism anew. One major objection questions the supposed relationship between genetic contingency of natural procreation and our self-understanding as autonomous and equal members of a moral community and it imputes genetic essentialism and determinism to Habermas’s account. It furthermore accuses it of neglecting social aspects of character formation , second, it relates to the aspects of “chance” and “contingency” characteristic of biological procreation , and third, it relates to the circumstance that the genetic setup of humans is out of their reach (22). Habermas contrasts the concept of human nature as that which is given, contingent, and beyond one’s influence to the concept of “the made”. The latter refers to the fact that humans entertain in actively choosing, planning and conducting various activities (44). The meaning of human nature therefore remains at a rather abstract level and does not characterize certain genetic features or character traits of humans as natural. What is worthy of preservation is thus not human nature in the sense of specific human characteristics but the way these characteristics come about. This will be important when discussing the objection that Habermas’s argument relies on genetic essentialist and determinist assumptions.3In his FHN, 42). Habermas sees the sole authorship of an individual’s life to be threatened by genetic interventions parents chose for their offspring. By deciding on certain genetically bound characteristics, parents introduce an aspect of intentional design into the parent-child relationship that has formerly not been there. For parents, this equals to a shift from chance to choice; for treated children, it might mean loss of contingency as well as a burdensome awareness of parental expectations . Since germline editing introduces permanent changes to the genome of an individual, gene edited children are thought to have no possibility to distance themselves from the expectations of their parents. Habermas sees a difference here between influences parents have on their children through upbringing and education and the biotechnological shaping of the human body. While the former, though also enormously powerful, might be critically reflected in communicative acts and to some extent rejected through a change of cultural practices, determinations of the germline are irreversible for that specific individual. What is missing here is a way to distance oneself from genetic determination in the space of communicative reason, which is why grown-up children and parents might not be experiencing themselves as equals .Habermas’s argument from nature confers a positive value to human nature in the following sense: he understands the uncontrollability of the genetic makeup in natural reproductive processes to be a condition of the self-understanding of individuals as being free and morally equal persons in society. According to him, it is the “nature-like growth which alone allow[s] us to conceive of ourselves as the authors of our own lives and as equal members of the moral community” .Please note that Habermas’s threefold distinction of a pragmatic, an ethical, and a moral use of practical reason has a partial analogy in Immanuel Kant’s distinction of hypothetical and categorical imperatives as introduced in his While Habermas refers to Kant’s distinction of the three kinds of imperatives of reason, his terminology differs.Habermas’s reference to an ethical employment of reason does not find an analogy in Kant’s distinction of the three imperatives. The author of reference here is Aristotle and an ethics of the good life , 4. The FHN, 5, 25). His specific form of “moralizing nature” is rather meant as an “assertion of an ethical self-understanding of the species which is crucial for our capacity to see ourselves as the authors of our own life histories, and to recognize one another as autonomous persons” . The normative aspect of the notion of human nature is thus derivative insofar as genetic contingency and uncontrollability serve as a requirement for an autonomous and egalitarian human self-understanding. Because the latter is considered a condition for moral practices at all, that kind of normativity transfers back to its condition, namely human nature .There have been many critical remarks concerning Habermas’s appeal to nature. For the most part, they concern the relationship between genetic contingency of human procreation and perceived autonomy and equality. Here, I will focus on the attempt of critics to show that Habermas’s argument from nature wrongly asserts essentialist and genetic determinist premises. According to Elizabeth Fenton, Habermas wrongly assumes that human nature is something fixed and stable and that the normative claim to preserve it must fail. As technological interventions to what is considered a fixed human nature challenge exactly that idea, “it seems that we cannot be certain that human life as it is currently lived simply is the way it ought to be” , 39. FenRecently, several authors have resumed the objection of genetic determinism and essentialism with reference to empirical sciences. Oliver Feeney calls arguments from human nature that speak against enhancement as grounded in a myth “without sufficient empirical basis,” which leads to “groundless speculation and overreactionary normative responses” , 239. Acnecessary presupposition for being-able-to-be-oneself and for the fundamentally egalitarian nature of our interpersonal relationships” . A genetic determinist interpretation therefore does have some textual evidence; nevertheless, in the same book, Habermas also makes claims that are more moderate when he evaluates the impact genetic interventions could have on personality formation. He states that “the egocentric intervention takes on the meaning of a communicative action which might have existential consequences for the adolescent” (Habermas’s emphasis) and that “knowledge of the temporal prius of being made does not necessarily result in self-alienation” . Therefore, there is a more charitable interpretation, which does not support strict genetic determinism. Notwithstanding, even within this charitable reading, Habermas still brings forward reservations against genetic engineering for purposes of enhancement: “As long as we cannot be sure that this harmony between one’s own intentions and those of a third party will inevitably be produced, we cannot rule out the possibility of dissonant cases” .While Habermas clearly rejects a concept of human nature as intrinsically valuable, his remarks concerning the relationship between genetic contingency and an autonomous and egalitarian human self-understanding unfortunately remain ambiguous. On the one hand, he holds that natural “contingency proves to be—in the very moment we can master it—a What becomes obvious at this point is that the above disagreement about the normative force of Habermas’s appeal to nature is about the burden and scope of proof. On the one hand, critics claim that Habermas’s reasoning relies on problematic premises. Genetic determinism has been widely rejected, and psychological studies do not seem to confirm a close connection of genetic contingency and perceived autonomy as of yet. According to the more charitable reading, on the other hand, ruling out strict genetic determinism is not sufficient to turn down Habermas’s concerns about genetic engineering for purposes of enhancement. The pure possibility that there might be cases in which genetically engineered individuals will not confirm their parents’ genetic intervention is reason enough for him to hold on to the claim that human nature is normative in the sense of it being a possible condition of the perception of autonomy and equality. As long as the pure possibility of dissonant cases is not ruled out, one should refrain from using the technology for purposes of enhancement.In order to resolve the dispute between the two parties, one would have to ask: do we need proof that there is a possible connection between genetic contingency and perceived autonomy and equality in order to protect human nature? Or do we rather need proof that such a relation does not exist to rebut this argument from nature? Either way you look at it, the normative analysis at this point aims at an evaluation of the empirical consequences of using the technology, without discussing the ends for its use. I therefore argue that both—the critical reading underlying essentialist and determinist objections as well as the charitable reading, which sees Habermas as making a more moderate claim—operate within an instrumentalist employment of reason. This kind of reasoning is concerned with finding adequate means to undebated goals by solving empirical questions as well as by deciding on adequate technology. The critical instrumentalist reading does exactly that: it refers to empirical sciences as well as to technological possibilities in order to rebut reservations against germline editing. The end itself, the protection of human nature from enhancement interventions, is not itself debated. Fenton  and FeenThe charitable reading of Habermas’s argument operates at this level as well: it is concerned with the empirical possibility of disagreement between future children and parents resulting from the usage of germline editing. According to this charitable reading, germline editing is a problematic means because consequences of its use are uncertain. The argument itself is not about the evaluation of the possible end itself. The end—change of human nature for enhancement purposes—is already supposed to be problematic within this interpretation.Within the instrumentalist use of reason, the question “What should I do?” is a matter of deciding empirical questions and choosing appropriate means. In case of human germline editing, answers to these questions remain rather speculative now, considering the undue risks of clinical germline editing . What would be an attitude that allows for a morally admissible use of germline editing? According to Habermas, it is an attitude that is dedicated to the logic of healing. The logic of healing, including preventing severe diseases, allows for morally admissible uses of technology because establishing health is considered to be a common good by Habermas. As a common good, it is mediated by communicative processes within a moral community. The challenging task of a moral community is therefore to develop convincing criteria to distinguish healthy from sick forms of bodily existence in order to identify legitimate uses of genetic engineering in humans .While Habermas’s argument from human nature speaks in favour of a conservative attitude towards human genetic engineering in general, he does approve of a restricted use of this technology for purposes of therapy as opposed to purposes of enhancement. It is therefore not genetic engineering per se, as a means, which is critically assessed by Habermas but the different purposes for which it can be used: “The problem, of course, is not genetic engineering, but the mode and scope of its use. It is, moreover, the FHN, 52).Because there is supposed to be a common logic of healing, Habermas argues that one can assume consensus of a future person in case of therapeutic interventions; however, for purposes of enhancement, “virtual consent” of the child cannot be assumed, because it is impossible to foresee the child’s preferences. What is more, in this case, an intervention “according to the sole preferences of a third person” would be made, taking “on the form of an instrumentalization of human nature” Irrespective of how far genetic programming could actually go in fixing properties, dispositions, and skills, as well as in determining the behavior of the future person, post factum knowledge of this circumstance may intervene in the self-relation of the person, the relation to her bodily or mental existence. The change would take place in the mind. Exercising the power to dispose over the genetic predispositions of a future person means that from that point on, each person, whether she has been genetically programmed or not, can regard her own genome as the consequence of a criticizable action According to Habermas, a shift of awareness could take place irrespective of the actual causal influence a specific genetic setup might have on character formation. The mere fact that parents made an active choice is what could cause a problematic self-image of a child. Leon-Philip Schäfer has convincingly argued that even children who themselves have not been genetically engineered can experience this shift of awareness once the technology is available , 1061. HIn comparison to genetically edited individuals, a non-edited child might feel disadvantaged and impaired in its sense of equality. By choosing to refrain from genetic engineering, parents might therefore be confronted with the accusation that they did not use all the available means to augment certain features in the child.The challenge of mere controllability has a follow-up-challenge. Robert Sparrow  has receSeveral open peer commentaries critically discuss Sparrow’s article. Amongst them are objections to Sparrow that accuse him of genetic reductionism and determinism from within an instrumentalist reading, for instance Savulescu  and ChapIntroducing the aspect of choice into the genetic lottery of procreation raises multiple possibilities to affect the self-understanding and identity of individuals. Habermas’s remarks on shifting the experience of genetic contingency to genetic choice therefore speaks in favour of an ethical reading of his argument from human nature.FHN, 11, 28, 37ff.). Along with his claim that an ethics of the species is needed, Habermas therefore constrains a credo he otherwise endorses, namely the “priority of the just over the good” . Because, for Habermas, germline editing touches the presuppositions of morality, coming to terms with an ethical evaluation of this technology is prior to questions of justice.There is a third reason to interpret Habermas’s approach to genetic engineering from the perspective of an ethical employment of reason, and it is the most obvious one: he explicitly states that genetic engineering touches the ethical self-understanding of humans in its entirety, which is why an ethics of the species is needed. According to him, it is the overall structure of human moral experience that is at stake insofar as presuppositions of moral judgment and action in general might be affected by genetic engineering. These presuppositions include a self-understanding of moral individuals as autonomous and equal as well as being guided by norms and reason . This common image of what it is and should be like to be human, though intuitively clear to many, might not be as self-evident as Habermas claims. Daniel C. Henrich, while he opposes genetic determinist objections against Habermas, critically remarks that he lacks an elaborate normative anthropology or account of a good life. This is why his attempt to preserve our ethical self-understanding and with it human nature becomes rather decisionistic 5Since man, biologically speaking, is born “unfinished” and subject to lifelong dependency on the help, care, and respect of his social environment, individuation by DNA sequences is revealed as incomplete as soon as the process as social individuation sets in. Individuation, as part of life history, is an outcome of socialization. […] It takes entrance in the public sphere of a linguistic community for a natural creature to develop into both an individual and a person endowed with reason.  for individual and societal identity formation are also evident in the above introduced three reasons that speak in favour of an ethical reading of his argument from nature. The distinction of therapy and enhancement, the shift from chance to choice, and an ethics of the species connect ethical evaluation of germline editing to social structures as follows.The importance Habermas places on “relations of mutual recognition of communicatively acting persons” . For another thing, the argument from mere controllability shows that children that are not genetically modified might experience the non-intervention as an omission in comparison to other children who have been modified. Habermas’s normative claims therefore do not in the first place address relations that might or might not hold between certain genetic conditions and specific character traits. His evaluation of the technology draws much more on developments that occur at the level of society.Recalling the other paring of words reinforces this conclusion. Habermas’s remarks about a possible shift from chance to choice and about possible changes in the self-understanding of individuals also refer to structures at the level of society. For one thing, he understands the impact parents have by choosing genetic interventions for their children primarily in psychological and sociological, not so much in biological terms: going back to a quotation from above: “The egocentric intervention takes on the meaning of a FHN, 11). It is therefore evident that Habermas’s argument from nature does not neglect processes and structures of socialization, as some critics claim. Much to the contrary, it refers to social structures, when a) considering germline editing as a biogenetic technique that can be used for different purposes, b) when evaluating impacts that using this technique has on individuals, and c) on the human species.Social structures are furthermore essential to establishing an ethical self-understanding of the species. According to Habermas, it is especially the communicative sphere of reason, established by language-using agents, that constitutes this kind of species ethics. So both, germline editing as a form of communicative action as well as a species ethics are within the “logos of language,” which “embodies the power of the intersubjective” . While the ethical reading reminds us that germline editing touches on fundamental human values that lie beyond a means-ends rationality, it remains an open question whether germline editing as a communicative act indeed subverts presuppositions of moral judgment and action. Asking and engaging in answering it at the level of society is well worth it.6I would like to close by pointing to a conceptual challenge that arises within the ethical reading of Habermas’s argument. It relates to his distinction of genetic engineering and education. Habermas separates the two due to the irreversibility of genetic engineering. Given that an ethical reading of Habermas’s argument does not build on genetic determinist assumptions about character formation but refers much more to the level of social interaction, how does this distinction of education and genetic engineering still make sense? I see two options. The first is that the ethical reading must include empirical assumptions about the impact genetic interventions have on identity formation and self-understanding, even if genetic determinism is rejected. In this case, Habermas’s argument is in need of a more detailed empirical account of how exactly certain biological conditions can irreversibly influence the self-understanding of an individual. The drawback of this option is that it must rely on empirical speculation about the impact genes have on character formation. The second option I see is to refrain from reference to speculative biological assumptions and deny that the distinction of education and genetic intervention supports his argument in the sense described above. In this case, both education and genetic engineering are understood as forms of parental influence that reveal specific, negotiable conceptions of the good life. Since, for Habermas, the distinction between therapy and enhancement draws the line between legitimate and illegitimate uses of the biotechnology and since ruling out dissonance is the main criterion to distinguish the two, Habermas cannot offer a coercive argument against enhancement. For, if a moral community decides to include genetic enhancement as part of a good life, which is expected to be agreed upon by future individuals, then the use of germline engineering for purposes of enhancement would be ethically acceptable. Habermas himself admits that he is not offering a “conclusive moral argument” but rather “an orientation relying on an ethics of the species, which urges us to proceed with caution and moderation” (In this article, I show how genetic essentialist and determinist objections to Habermas’s argument from human nature originate from an instrumentalist reading, which, though there is some textual evidence for it, is neither very plausible nor very convincing. I draw on Habermas’s distinction of a threefold use of practical reason and provide three reasons from within his approach that speak in favour of an ethical reading of his argument. This reading not only avoids essentialist and determinist objections but also fundamentally incorporates social aspects of character formation and of individual and collective self-understanding. I therefore argue that it offers exactly what the critics claim is missing in Habermas’s account.


This study aims to present Genome Access and discuss the importance of adopting digital technologies designed specifically for GC and what tools these solutions should include.Genetic counselors are healthcare professionals who are trained in both medical genetics and counseling to help guide individuals through what is known about genetic predispositions toward a variety of diseases, how they are inherited, and what impact this information can have on them and their partners and families. The range and scope of practice of GC has greatly expanded beyond where it started and now, it is employed in a variety of clinical and research settings. The traditional approach to GC involves meeting with a counselor in person. However, with the increasing availability of online resources, more people are seeking information about genetic disorders online. This shift has led to the development of online GC services. Indeed, genetic counselors are no strangers to improvements in terms of adopting digital solutions in their clinical routine, however, there are few studies assessing genetic counselors' attitudes regarding existing digital tools. Genome Access Clinical genomics is a fast-growing field and the simple access to the market of DNA testing (better known as “consumer genetics”) is becoming easier. In fact, we are facing a global-wide increased demand for genetic healthcare services that sometimes exceeds the clinical capacity (further exacerbated by the COVID-19 pandemic) .Genome Access uses a variety of AI technologies to manage the GC process, including an automated conversation model (chatbot) to enable contextual conversations on genetics. Together, these technologies cover the input, processing and analysis, and the output needed during GC. Genome Access is available as a SaaS (Software as a Service) stand-alone system.Kaleidos has recently released a digital solution fully compliant with privacy regulation (GDPR) and embedded with protocols entirely encrypted to protect users' personal identity. Genetic services facilities represent a perfect launching point for further development of digital solutions, as they characterize many routine telemedicine care processes that could be similarly automated. Moreover, Genome Access is expected to bring significant benefits for geneticists and healthcare staff by reducing their clinical burden. The Platform also has several benefits for patients. Genome Access will provide a reliable and consistent safety net before in-person visits (when needed). Patients will be able to ask questions about their risk recurrency just as they would with a in person clinician. The system is convenient because it does not require them to travel to the site of care and can be used at the time and/or for the duration that suits them.The platform is based on innovative AI algorithms, specifically developed to revolutionize the counseling process for patients affected by genetic diseases . Genome Information and Education. Genome Access is committed to finding innovative and more efficient ways to counsel and educate patients. Therefore, the goal behind the first module aims to streamline, by digitalization, the process of delivering privacy information, collecting the patient's consent and letting them have a better understanding of DNA tests results and to understand what they mean. Indeed, patients can consult “Genebot,” a custom chatbot made for genetics, developed to provide a platform for individuals to ask questions relevant to genetic testing. This includes understanding the meaning of a genetic test, or what to expect from genetic testing, as well as information about genetic disorders or conditions. Patients can also enjoy several interactive videos realized for genetics and DNA pathogenic variants risk recurrence.Data retrieval. The second module provides tools for guided self-anamnesis and for collecting the pedigree. Moreover, the second module provides digital support to the professional in the genetic “raw” data analysis and phenotyping through bioinformatic software and specific algorithms.Tele-genetics. This is an interoperable telemedicine solution aimed at virtualizing the relationship between patients and healthcare providers through a televisit system as well as offering software for elaborating a final clinical report. Genome Access can easily be implemented by a variety of customers, such as clinical facilities, patient advocacy organizations of rare diseases, laboratories for molecular analyses, and research centers: the platform can be personalized to meet the specific needs of our customers for developing custom-tailored solutions. Moreover, Genome Access can be easily integrated into third-party applications through API applications.With pioneering technological advancements, such as tools for drawing electronic pedigrees (genogram), geneticists have digitalized the process of collecting relevant personal, family, and clinical information, reducing the time spent on these duties during in-person appointments. The digitalization of the medical history taking procedure resulted in genetic counselors being able to more easily retrieve complex family histories, to review relevant information more quickly, and to reduce appointment times for patients .Many clinics send questionnaires to patients before their appointments; in most cases these forms are even accessible online so that patients do not have to wait for a mailed copy .There is an increasing number of private healthcare companies that are employing counselors specifically to provide telemedicine . TelevisA chatbot is a computer program that uses AI and natural language processing to simulate conversations with “human” users. There are currently several chatbots that have been developed and are in use by genetic counselors. Chatbots are already being used as part of patient-initiated testing journeys, to allow patients to give consent to participate in research studies, and to provide valid support to counselors on disclosing positive genetic test results to patients  and discBesides Genome Access, several genomic platforms have flourished around the world in the past few years with the same goal: to streamline genetic counseling through the digitization of time-consuming processes and, in most cases, with the support of AI tools. Digital solutions in genetic counseling fall into two main categories: platforms that offer both direct and remote counseling digitally delivered by “in-person” counselors and web-based software offering a variety of services, such as ancestry tracing, genetic health risk assessment, and carrier screening. There are already available some different digital platforms focused on genetic counseling: some clear differences between them are highlighted . Most ofThe pattern of informed consent acquisition on the use of genetic data is becoming more and more prevalent as technology advances. However, while the benefits of using such data are clear, the privacy measures that need to be applied are not always given the attention they deserve . GeneticTechnological advances have already served to help genetic counselors to see more patients and spend less time on repetitive steps of face-to-face appointments and facilitate the access to genetic counseling services to patients . As in oThe original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.AC and MP contributed both to draft the manuscript and performed data collection. MC conceived and designed the manuscript, supervised data collection, and drafted the manuscript. AC, MP, and MC confirm that they had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. All authors gave final approval of this version to be published and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved and approved the final manuscript.This study was supported by the grants Artemisia (CESVI-Bergamo) and SI4.0 2021 (Unioncamere Lombardia) awarded to MC.Authors AC, MP, and MC were employed by Kaleidos SCS onlus.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Apis cerana is widely distributed in China and, prior to the introduction of western honeybees, was the only bee species kept in China. During the long-term natural evolutionary process, many unique phenotypic variations have occurred among A. cerana populations in different geographical regions under varied climates. Understanding the molecular genetic basis and the effects of climate change on the adaptive evolution of A. cerana can promote A. cerana conservation in face of climate change and allow for the effective utilization of its genetic resources.A. cerana workers from 100 colonies located at similar geographical latitudes or longitudes were analyzed. Our results revealed an important relationship between climate types and the genetic variation of A. cerana in China, and a greater influence of latitude compared with longitude was observed. Upon selection and morphometry analyses combination for populations under different climate types, we identified a key gene RAPTOR, which was deeply involved in developmental processes and influenced the body size.To investigate the genetic basis of phenotypic variations and the impact of climate change on adaptive evolution, A. cerana to actively regulate its metabolism, thereby fine-tuning body sizes in response to harsh conditions caused by climate change, such as food shortages and extreme temperatures, which may partially elucidate the size differences of A. cerana populations. This study provides crucial support for the molecular genetic basis of the expansion and evolution of naturally distributed honeybee populations.The selection of RAPTOR at the genomic level during adaptive evolution could allow The online version contains supplementary material available at 10.1186/s12864-023-09167-x. Apis cerana is an important crop-pollinating insect in China. Its long collection cycle and ability to utilize sporadic nectar sources have enabled A. cerana to play a key role in the development of Chinese agriculture. During the long-term natural evolutionary process, many unique phenotypic variations have occurred among A. cerana populations in different regions under different climatic conditions [A. cerana gradually shows a trend of weakening migration, slightly larger size, darker body color, and stronger cold resistance [A. cerana an excellent research subject for the study of climatic adaptation on adaptive evolution [A. cerana can help effectively conserve A. cerana against climate change challenges. Simultaneously, understanding the genetic basis of these unique phenotypic variations is beneficial for their conservation and provides insight to better harness their genetic resources.Over the past few decades, the numbers of many insects have shown a rapid decline , includinditions , 15. In sistance . The chavolution . Thus, tA. cerana. Based on direct measurements of the phenotypic and morphological characteristics of A. cerana in different regions [A. cerana was published in 2015 (GCA_001442555.1) [A. cerana with special traits—A. cerana abansis (Aba strain) [A. cerana with cold resistance [A. cerana from different regions. A 2020 study identified the role of the leucokinin receptor (Lkr) in influencing the foraging division of labor [A. cerana.So far, many studies have used different methods to elucidate the basis of the unique phenotypic differences in  regions , nine va regions –21. Once42555.1) , genome 42555.1) , 24. Stu15 GCA_0042555.1 [42555.1) , 18, 25  strain)  and the sistance —progressA. cerana. Simultaneously, we chose collection sites at similar latitudes or longitudes to explore the influence of geographical coordinate factors on the adaptive radiation of A. cerana. Upon genetic structure, genetic differentiation, and population diversity analyses of these 10 populations, as well as through selective sweep analysis and morphometry analysis combination of the populations with high genetic differentiation, we were able to identify the key signaling pathways and genes involved in the process of adaptation to different climatic conditions in China. This study provides a useful reference for the improvement of A. cerana conservation and breeding.In this study, we collected 100 samples from 10 regions in China (covering the most important climate types) and utilized population genetics methods to identify the key traces in the process of adaptation to different environments at the genome level; we aimed to reveal the genetic basis of the strong environmental adaptability of We collected and resequenced samples from 10 regions of China and included previously published resequencing data from three regions (Table The admixture model-based software (Admixture) was used to estimate the population structure. With K = 2, MK, JL, and WC formed an ancestral cluster, while the other populations showed different degrees of mixed lineage. As K increased from 3 to 5, MK, JL, and WC showed distinct lineages from the other populations, with ZZ and MY forming an ancestral cluster Fig. . In partFST , and the minimum value was less than 0.01 . These results also showed that JL, MK, and WC were in a state of high genetic differentiation (FST > 0.15), which is consistent with the analysis of population structure and PCA. JL displayed the highest degree of genetic differentiation (average FST = 0.28), followed by WC (average FST = 0.19) and MK (average FST = 0.16). The remaining regions were in a state of low genetic differentiation. The parameter calculation results for genetic diversity were greatest for SZ (PIC = 0.205) and GZ (PIC = 0.206) among the 10 populations. The genetic diversity of JL (PIC = 0.099), MK (PIC = 0.140), and WC (PIC = 0.148) was lower than that of other regions, indicating that bees in these three areas have been subjected to a higher intensity of natural selection.To understand the genetic differentiation between the 10 groups, we calculated pairwise ST Table  and geneIC Table . The maxA. cerana, we estimated their historical effective population sizes , all other indicators were significantly different among the three groups (P < 0.01) , the right forewing width (FB), the sixth sternum length L6), the sixth sternum width (T6), the third sternum length (S3), the third tergum length and the fourth tergum length T3 + T4), the femur length (Fe), the tibia length (Ti), the basitarsus length (ML), and the basitarsus width (MT) Fig. S. The res, the fem, the sixA. cerana from different regions of China and the genome-level changes that occurred during the evolution of adaptation to the unique climate of each region under natural selection pressure, we estimated pairwise genetic differentiation (FST) and differences in nucleotide diversity (π ratios) from the four different types of populations to identify key selective sweeps. For the 40 samples from JL, MK, WC, and SZ, we performed the linkage disequilibrium (LD) decay analysis. The LD analysis results showed that when r2 was half of the maximum value, the decay rate was MK > JL > WC > SZ  with a threshold of the top 5% level. The results of the selection signal analysis of the highly differentiated populations under different climate types, with the SZ population  as the reference population, identified 839 candidate regions involving 527 genes  and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed on these 33 candidate genes. The 33 shared genes were involved in 846 GO and 44 KEGG pathways, with 138 GO and 6 KEGG pathways significantly enriched at a threshold of P < 0.05  related to energy metabolism in the body, the thermogenesis pathway related to adaptation to changes in ambient temperature, and the Hedgehog signaling pathway involved in regulating gene expression  could have independently formed a subpopulation from the analyses of the population structure, genetic diversity, and genetic differentiation based on whole-genome resequencing data. Furthermore, we confirmed the significant differences in the body size of A. cerana by comparing morphological data. This evidence shows that the JL, MK, and WC populations were highly differentiated compared with other populations, which may be a consequence of different climate types and environmental selection pressures. This is in line with the findings of a study on the adaptive evolution of A. cerana in which isolated groups showed a high degree of differentiation and lower genetic diversity, whereas other groups were less differentiated and had higher genetic diversity [PIC of the 10 populations was 0.17, and the PIC values of all populations were lower than 0.5, representing low polymorphism and low genetic diversity of A. cerana. Human activities are considered to be an important reason for the decrease in the genetic diversity of A. cerana in recent years. Human activities led to the reduction of the habitat area of A. cerana, which directly led to the reduction of wild resources of A. cerana. A large reduction in the number of wild bees leads to a decrease in the effective population, which may directly lead to a decrease in the genetic diversity of A. cerana. In addition, the introduction of A. cerana from other regions also affected the original genetic structure and genetic diversity of local populations. A rich genetic diversity can enhance the adaptability of species to their environment, more stably maintain population balance [A. cerana.Our study results reveal an important link between climate type and the adaptive evolution of iversity . In the  balance , 29, and balance . TherefoA. cerana at high and low latitudes also reflect the different impacts of sudden climate change on populations at different latitudes. Our study shows that this discrepancy first originated around 70 to 80 ka years ago. Since the last glacial period of the Quaternary Ice Age [It is also worth noting that the geographic latitude factor had a high degree of influence on the biodiversity of biological groups. This is supported by the estimations of the effective population size. The different change patterns of the effective population size of  Ice Age , 32, difA. cerana in Jilin is lower than that of other populations. This means that A. cerana in Jilin may have experienced serious bottleneck events, and the evolution is more affected by genetic drift. This is consistent with the previously reported situation that human capture has greatly changed the population dynamics of A. cerana in the area around JL in recent decades [FST between populations in the sliding window.The main driving forces in the process of species evolution include selection and genetic drift. The joint action of multiple forces may not only make the mutant increase its frequency faster, but also may be that genetic drift slows down the speed of selection, or even reduces the frequency of the mutant. The intensity of genetic drift depends on the effective population size. The larger the effective population size, the weaker the genetic drift; the smaller the effective population size, the higher the probability of genetic drift. According to our estimation of the effective population size, the effective population size of  decades , 25. TheA. cerana as the research object rather than Apis mellifera to study climatic adaptation. Because the domestication history of A. cerana is shorter than that of A. mellifera, and the degree of domestication is weaker than that of A. mellifera. In addition, during the sampling process, the samples we collected were A. cerana living in different geographical environments in the wild or semi-wild state. These samples are relatively less affected by artificial domestication. We believe that such samples are suitable for studying climatic adaptation during the adaptive evolution of A. cerana, and the analysis results based on these samples are representative.In this study, we mainly focus on the relationship between genetic variations and climatic conditions. Hence, we pay attention to the factors like latitude, longitude, and altitude related to climate conditions. It is worth noting that in the process of adaptive evolution, especially in the case of short generation interval of bees, the impact of artificial domestication cannot be ignored. Both the changes in genome structure characteristics caused by artificial selection and natural selection are called selection signals. For these selection signals, it is difficult to distinguish whether their source is artificial selection or natural selection. Therefore, when using the selective sweep analysis to analyze the evolution of species, it is necessary to select appropriate research objects according to the direction of focus. We selected A. cerana. Positive selection will lead to an increase in the frequency of the dominant allele at the locus, which in turn will lead to a decrease in the genetic polymorphism of the selected locus. Therefore, for the studied colonies of A. cerana under the four climatic types, we set the SZ population as the reference, based on the calculated population polymorphism (SZ > WC > MK > JL). An abundance of polymorphisms means that the selection pressure received is less; hence using the SZ population with less selection pressure as the background facilitates the detection of traces of selection in the remaining populations. In addition, we use different selective signal methods (FST and π ratios) for selective signal detection. The adoption of an overlapping detection strategy can further avoid the occurrence of false positives through mutual verification between methods to a certain extent. It is undeniable that the results obtained after these pairwise comparisons are relatively rich, but not all signals are related to adaptation to the climate environment and body size; therefore, we pay more attention to the signals detected in all three selective sweep analyses. These signals will help gain insights into the climatic adaptation mechanisms of A. cerana. According to the results of GO and KEGG pathway analyses, we speculate that the adaptive evolution of A. cerana may be preferentially reflected in its response to the abundance of food resources and changes in temperature. A. cerana actively regulates its metabolism to cope with food shortages in harsh environments and maintain its body temperature. This also indicates that A. cerana energy utilization patterns may differ in different climate types.Long-term natural selection often causes changes in the allele frequencies of selected loci and their linked loci. Upon analyzing these traces of natural selection in the genome, we can better understand some of the important events in evolution. We used selective sweep analysis to determine the relationship between climate type and the adaptive evolution of A. cerana adapt to the complex climatic environment in China. A total of 33 candidate genes related to climatic adaptation were screened using selective elimination analysis. Upon annotation results combination with existing research reports, we identified RAPTOR as a candidate gene related to body size in climate adaptation. RAPTOR is the intersection of the AMPK and mTOR signaling pathways. The evidence to date suggests that the mTOR signaling pathway is involved in the development of A. mellifera [A. cerana in different regions is inconsistent. The selection of RAPTOR at the genome level could be in response to changes in climate and food sources. Early studies also reported the functions of the RAPTOR and TOR pathways in Drosophila [Drosophila [Drosophila showed that downregulation of TOR activity produces smaller flies with smaller wing epidermal cells, and flies with small cells can maintain superior performance in metabolically demanding activities, such as flying under hypoxic conditions [A. cerana has adopted the same strategy as other insects to combat different climate types requires further investigation.Based on our findings, we speculate that the selection of RAPTOR could help ellifera . Studiesellifera –41, and ellifera . Under nellifera . The mTOellifera , 44. Theellifera , 46. Beeellifera . Native ellifera , 47. As osophila , 49. Decosophila . In addiosophila . A recennditions . WhetherA. cerana. It is highly probable that food and nutrition deficits caused by diverse climates are the predominant driving forces for unique geographic phenotypes. Further research is necessary to verify the molecular mechanism between RAPTOR and the body size.In summary, our findings suggest that climate type, geographic location, and food resources are all related to the adaptive radiation distribution and unique geographic phenotypes of A. cerana are related to the distribution of climate and environment in China and are strongly affected by latitude. A key gene, RAPTOR, plays an important role in this process. The selection of RAPTOR at the genomic level helps A. cerana respond to nutritional problems caused by harsh environments by modulating TOR activity to alter body size, which partly explains the difference in body size among A. cerana distributed in China. These results help us understand the genetic basis of the climatic adaptation of A. cerana and provide a reference for the protection and utilization of germplasm resources and future genetic improvement.Our study shows that the genetic structure and genetic differentiation of A total of 100 honeybee samples were studied in this experiment, 70 of which were collected from Tibet, Hubei, Anhui, Jiangsu, Guangdong, and Hainan provinces in China. The collection sites for each sample were centered in Suzhou, Jiangsu, and were located at similar latitudes and different longitudes or similar longitudes and different latitudes. In addition, 30 samples from sites with similar longitude and different latitude as Suzhou were included for comprehensive data analysis , 18. Tenhttp://broadinstitute.github.io/picard/).Total genomic DNA was extracted from samples, and at least 3 µg genomic DNA was used to construct paired-end libraries with an insert size of 500 bp using a Paired-End DNA Sample Prep kit . These libraries were sequenced using the HiSeq X10 NGS platform . Raw reads were processed to obtain high-quality clean reads according to two stringent filtering standards: 1) removing reads containing > 50% of low-quality bases (Q < 20) or > 10% unidentified nucleotides (Ns); and 2) removing reads aligned to the barcode adapter. The Burrows-Wheeler Aligner (BWA) was used to align the clean reads against the reference genome (GCA_011100585.1) with the settings ‘mem 4 -k 32 -M’ . DuplicaVariant identification was performed using the Genome Analysis Toolkit (GATK) . SNPs weThe admixture model-based software, Admixture V1.3.0 , was useHo and He for each group or population were calculated using Plink [Ne and PIC were calculated using the Perl script. The pairwise FST matrix was calculated using Genepop software [ng Plink . Ne and software .Ne over the past one million years (Ma). The mutation rate was set to 5.27 × 10−9 per base pair per generation, following a divergence estimate of 7 Ma between A. mellifera and A. cerana [SMC +  + 1.13.1  was used. cerana . The genA total of 130 worker bees  were used for morphometric measurements. The bees were dissected to make samples of each tissue, observed and photographed under a stereo microscope, and measured by a measurement system . The measure of each sample in parallel was repeated 3 times and the mean value was taken.2) using Haploview 4.2 [To evaluate the LD pattern, we estimated the squared allele frequency correlation (rview 4.2 . LD decaFST) [FST and π ratios), with a threshold of the top 5% level. All related graphs were drawn using R scripts. Candidate genes within sweep regions were extracted for GO and KEGG enrichment analysis.For the 40 samples from JL, MK, WC, and SZ, we performed selective sweep analysis, with SZ as the reference population. We estimated pairwise genetic differentiation (FST)  and diffFST)  from theFST)  with theAdditional file 1:Figure S1. The cross-validation error rate of genetic structure analysis of 100 samples. Figure S2. The diagram of 10 morphological indicators of A.cerana. (A) The right forewing length (FL) and width (FB). (B) The sixth sternum length (L6) and width (T6). (C) The third sternumlength (S3). (D) The third tergum length (T3). (E) The fourth tergum length (T4). (F) The femur length (Fe), the tibia length (Ti), the basitarsus length (ML), and the basitarsus width (MT). Figure S3. Analysis of linkage disequilibrium. Figure S4. GO classification of candidate genes. Figure S5. The top 20 enriched KEGG pathways.Additional file 2:Table S1. Sequencing data production and quality statistics of 100 samples. Table S2. Summary of sequencing data depth, coverage, and mapping rate of 10 sampling sites. Table S3. 527 selected genes of A. cerana in Jilin. Table S4. 565 selected genes of A. cerana in Mangkang. Table S5. 311 selected genes of A. cerana in Wenchang. Table S6. 33 common selected genes of the three geographical populations. Table S7. GO enrichment of the 33 common selected genes of A. cerana (P < 0.05). Table S8. KEGG enrichment of the 33 common selected genes of A. cerana (P <0.05).


HD) and population pairwise genetic differentiation (FST) indices. Genetic diversity and divergence landscape surfaces were then generated based on the HD and FST, respectively, to clarify the patterns of genetic structure in the region. Subsequently, we analyzed the relationships between plastid genetic diversity and 16 explanatory variables . We found that the highest genetic diversity occurred in the Yulong Mountain region, with a significant proportion (~74.81%) of the high diversity land area being located outside of protected areas. The highest genetic divergence was observed approximately along the 25°N latitudinal line, with notable peaks in the western and eastern edges of the LRGR. Genetic diversity (HD) was weakly but significantly positively correlated with both Latitude (lat) and Annual Mean Wet Day Frequency (wet), yet significantly negatively correlated with all of Longitude (long), Annual Mean Cloud Cover Percent (cld), Annual Mean Anthropogenic Flux (ahf), and Human Footprint Index (hfp). A combination of climatic, topographic, and anthropogenic factors explained a significant proportion (78%) of genetic variation, with topographic factors (lat and long) being the best predictors. Our analysis identified areas of high genetic diversity (genetic diversity “hotspots”) and divergence in the region, and these should be prioritized for conservation. This study contributes to a better understanding of the features that shape the distribution of plastid genetic diversity in the LRGR and thus would inform conservation management efforts in this species-rich, but vulnerable region.Intra-specific genetic diversity is a fundamental component of biodiversity, and is key to species adaptation and persistence. However, significant knowledge gaps still exist in our understanding of the patterns of genetic diversity and their key determinants. Most previous investigations mainly utilized single-species and/or a limited number of explanatory variables; so here we mapped the patterns of plastid genetic diversity within 15 plant species, and explored the key determinants shaping these patterns using a wide range of variables. Population-level cpDNA sequence data for 15 plant species from the Longitudinal Range Gorge Region (LRGR), southwest China, were retrieved from literature and used to estimate haplotype diversity ( Intra-specific genetic variation is a key prerequisite for species persistence, since it provides the raw material for evolution to act upon . Since tThe interplay of topography and climate is known to influence the patterns of genetic variation e.g., . Geologi2 and is located in southwest China, mostly in Yunnan Province. The region supports a rich biodiversity, with high levels of species endemicity (www.cbd.int/convention), its patterns and determinants remain unexplored in the LRGR.The Longitudinal Range Gorge Region (LRGR) covers a total area of 410,251.64 kmdemicity , and is demicity , but onldemicity . Additiodemicity . The Thrdemicity . Based odemicity , areas tdemicity . TherefoAlthough the LRGR is associated with rich biodiversity and endemism, the ecosystem is known to be highly fragile , especiaThe unique landforms of the LRGR, coupled with the intensified human activities in the region, provide an ideal opportunity to simultaneously test the relative effects of mountain-valley vicariance, the associated climatic variations, and anthropogenic influences on the genetic structure of plant communities. Although the influence of topographic and climatic factors on genetic diversity patterns in some parts of the LRGR (as well as its surroundings) has been explored to a considerable degree , a holisca. 10.33 degrees of longitude (106.12–95.79°E) and ca. 9.45 degrees of latitude  SRTM data . According to the boundary defined by RTM data , we estiWe retrieved published articles on population genetics and phylogeography from Google Scholar.N ≥ 5 were retained for downstream analyses.Based on the haplotype frequencies for each population and the mutation information in the haplotype sequences, we reconstructed the population and species sequence matrices in Geneious v2020.0.3HD  between HD and π, we only used HD in the subsequent analyses, since its calculation is insensitive to sample size, evolutionary forces, mode of reproduction, and ploidy level  and pairwise divergence (FST) values were used to generate genetic diversity and divergence landscapes using a modification of the Genetic Landscapes GIS Toolbox (FST) using Mantel tests in the vegan package in R . After this, a total of 16 explanatory variables  were retained for subsequent analysis  were extracted from various databases at various spatial resolutions . To avoianalysis .HD) were extracted from the extrapolated plastid genetic diversity landscape at the same spatial resolution as the corresponding explanatory variables  with integrated smoothness estimation using the “gam” function in the mgcv R package  of the populations lacking genetic variation , and ~5% showing maximum haplotype diversity (FST) ranged from 0.39 to 0.59 and showed high divergence approximately along the 25°N latitudinal line, with notable peaks at the western and eastern ends of this line , Ligularia vellerea , Quercus kerrii , Rosa soulieana , and Sophora davidii , we identified seven genetic diversity “hotspots” (labeled A-G in descending order of size) having a total area of 15,686.81 km2  and weakly positive (lat and wet) correlation with genetic diversity, though the relationships were significant in all cases , there was a marked decrease in genetic diversity at ~22.5–25° and at 28.5°. A similar trend was observed for cld and hfp, in which genetic diversity increased slightly before decreasing.The model selection analysis showed that In the subsequent RDA, the three categories of variables explained 78% of the total genetic variation, with topography alone accounting for the highest proportion (40%) of the variation. Climatic and anthropogenic factors explained 3 and 1% of the observed genetic variation, respectively . NotablyWe mapped patterns of plastid genetic diversity and divergence in the LRGR and determined the relative contributions of climatic, topographical, and anthropogenic factors in shaping the observed diversity patterns. Our results indicated that, within the LRGR, the Yulong Mountain region harbored the highest genetic diversity , 4. The The location of genetic diversity “hotspots” in the northern LRGR may also be explained by the fact that the Hengduan Mountains served as glacial refugia for many plant species in the region . Since tFST estimates. However, we found that the majority of the species did not show isolation-by-distance, suggesting the involvement of environment-driven differentiation in shaping the genetic structure of the majority of the species. The genetic divergence landscape in our study indicates the presence of gene flow barriers along a latitudinal range located approximately centrally in the LRGR, which is generally consistent with the findings of Moreover, the uneven distribution of populations in our study area might also have biased Q. kerrii and Q. schottkyana).Understanding the forces that shape the genetic structure of species across a given landscape is a fundamental goal in landscape genetics. Our analyses indicated that genetic diversity was weakly but significantly correlated with both latitude and longitude, tending to increase northwards and westwards . The norA similar trend in genetic diversity along a latitudinal gradient was recently found in subtropical evergreen oaks in southwest China . Howeverwet) (cld), thus reinforcing the link between genetic diversity and water. Although it is somewhat counterintuitive for wet and cld not to be correlated (they show discordant relationships with genetic diversity), the finding is not entirely unexpected, as cloud-precipitation anti-correlations have been noted particularly for low, optically thin cloud types  . Such a wet) , though ud types . Nonetheud types . Althougud types . Our resud types , though long) and latitude (lat) were the best-fitting models in the topography category, other variables such as terrain roughness index (tri) and elevation (elev) might have a more practical and direct effect on genetic diversity patterns (long and lat are simply proxies of these two variables (tri and elev).The LRGR is divided latitudinally into two climatic zones , which might account for the relatively high proportion of genetic variation (14%) that was explained by the combination of climatic and topographic variables. Moreover, a far higher proportion (78%) of the observed genetic variation could be explained by the joint effect of climatic, topographic, and anthropogenic factors . This fipatterns . We arguBesides climate and topography, our analysis also found a weakly negative (but significant) relationship between the two anthropogenic variables and genetic diversity, suggesting the role of human activities in shaping the genetic structure of plant communities in the LRGR. However, this relationship appears to be driven partly by the huge sample size as well as other unknown outliers; thus, more anthropogenic variables may be required to clarify and validate the relationship. Though our data indicate a negative effect of human activities on the distribution of genetic diversity in the LRGR, some recent studies have alluded to the positive impact of ancient anthropogenic forces on biodiversity e.g., , a trendHD value of >0.38 averaged across the study LRGR  is likely to have influenced the overall genetic structure to a considerable degree. Secondly, variations in life form, pollination, and dispersal modes may have biased the distribution of genetic variation, as the 15 species are not evenly distributed in the study area. We recommend that future explorations of this topic in the region should test the effect of climatic, topographic, and anthropogenic factors on genetic diversity in light of these differences in species traits. Moreover, further insights could be gained from studies that use biparentally inherited markers, which usually show higher population-level variation, and therefore allow for more meaningful inferences about the future evolutionary potential of populations within the region. Future research in the region may also benefit from genomic data, which provide more accurate estimations of genetic parameters, thus allow for more targeted measures during conservation planning.The original contributions presented in the study are included in the article/Z-YW, HW, L-MG, D-ZL, and JL designed the research and acquired funding. MW, JL, Y-HL, and G-FZ carried out literature search and data analysis. MW, RM, FW, and JL wrote the first draft of the manuscript. All authors contributed to interpretation of the results and editing of the manuscript.This study was supported by the Key Research Program of Frontier Sciences, CAS (ZDBS-LY-7001), the Strategic Priority Research Program of Chinese Academy of Sciences (XDB31010000), the National Natural Science Foundation of China , and the Top-notch Young Talents Project of Yunnan Provincial “Ten Thousand Talents Program” (YNWR-QNBJ-2018-146), the CAS ‘Light of West China’ Program, and the International Partnership Program of the Chinese Academy of Sciences (151853KYSB20190027). Z-YW was supported by CAS’ Youth Innovation Promotion Association (2019385). RM thanks the CAS President’s International Fellowship Initiative (2022VBA0004). MW was supported by the Postdoctoral International Exchange Program of the Office of China Postdoctoral Council, and the Postdoctoral Targeted Funding and Postdoctoral Research Fund of Yunnan Province.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.