Evolutionary Genetic Studies Research Articles

Comparative linkage mapping to investigate synteny and recombination in social Vespidae

Abstract Genetic linkage maps are valuable resources for investigating chromosomal structure, quantifying karyotype, estimating recombination rates, and improving preexisting genome assemblies. Comparative linkage mapping, in turn, broadens our understanding of the phylogenetic history of these genomic features. Through an assessment of synteny (the conservation of gene order on homologous chromosomes in different species) and variation in recombination rate, we can begin to understand how genomic features change during the evolution of distinct species. Here, we construct high-density genetic linkage maps for 3 Vespidae wasp species from the Vespula genus: Vespula consobrina, Vespula pensylvanica, and Vespula vidua to investigate shared genomic architecture between these 3 yellowjacket wasp species. We show that these species exhibit high levels of collinearity, often in chromosome-length blocks of synteny, with some evidence for small interchromosomal rearrangements. We also identify 2 “inversions” in all 3 species that are likely artifacts from the genome assembly process. In addition, we map genome-wide recombination rates and reveal the recombination landscape to be highly variable on intrachromosomal, interchromosomal, and interspecific scales. Genome-wide recombination rates are high for all three Vespula species, (V. pensylvanica: 22.7 cM/Mb, V. consobrina: 24.3 cM/Mb, and V. vidua: 24.7 cM/Mb), which is consistent with findings of high recombination rates for other eusocial species. Our high-quality linkage maps will be resources for ongoing evolutionary genetics studies interested in the genome evolution of social wasps.

Liberties of the genome: insertions of mitochondrial DNA fragments into nuclear genome.

The transition of detached fragments of mitochondrial DNA into the nucleus and their integration into chromosomal DNA is a special kind of genetic variability that highlights the relation between the two genomes and their interaction in a eukaryotic cell. The human genome contains several hundreds of insertions of mtDNA fragments (NUMTS). This paper presents an overview of the current state of research in this area. To date, evidence has been obtained that the occurrence of new mtDNA insertions in the nuclear genome is a seldom but not exceptionally rare event. The integration of new mtDNA fragments into the nuclear genome occurs during double-strand DNA break repair through the non-homologous end joining mechanism. Along with evolutionarily stable "genetic fossils" that were integrated into the nuclear genome millions of years ago and are shared by many species, there are NUMTS that could be species-specific, polymorphic in a species, or "private". Partial copies of mitochondrial DNA in the human nuclear genome can interfere with mtDNA during experimental studies of the mitochondrial genome, such as genotyping, heteroplasmy assessment, mtDNA methylation analysis, and mtDNA copy number estimation. In some cases, the insertion of multiple copies of the complete mitochondrial genome sequence may mimic paternal inheritance of mtDNA. The functional significance of NUMTS is poorly understood. For instance, they may be a source of variability for expression and splicing modulation. The role of NUMTS as a cause of hereditary diseases is negligible, since only a few cases of diseases caused by NUMTS have been described so far. In addition, NUMTS can serve as markers for evolutionary genetic studies. Of particular interest is the meaning of NUMTS in eukaryotic genome evolution. The constant flow of functionally inactive DNA sequences from mitochondria into the nucleus and its significance could be studied in view of the modern concepts of evolutionary theory suggesting non-adaptive complexity and the key role of stochastic processes in the formation of genomic structure.

Relationships within Bolbitis sinensis Species Complex Using RAD Sequencing.

Species identification and phylogenetic relationship clarification are fundamental goals in species delimitation. However, these tasks pose challenges when based on morphologies, geographic distribution, and genomic data. Previously, two species of the fern genus Bolbitis, B. × multipinna and B. longiaurita were described based on morphological traits; they are phylogenetically intertwined with B. sinensis and fail to form monophyletic groups. To address the unclear phylogenetic relationships within the B. sinensis species complex, RAD sequencing was performed on 65 individuals from five populations. Our integrated analysis of phylogenetic trees, neighbor nets, and genetic structures indicate that the B. sinensis species complex should not be considered as separate species. Moreover, our findings reveal differences in the degree of genetic differentiation among the five populations, ranging from low to moderate, which might be influenced by geographical distance and gene flow. The Fst values also confirmed that genetic differentiation intensifies with increasing geographic distance. Collectively, this study clarifies the complex phylogenetic relationships within the B. sinensis species complex, elucidates the genetic diversity and differentiation across the studied populations, and offers valuable genetic insights that contribute to the broader study of evolutionary relationships and population genetics within the Bolbitis species.

ProSeq4: A user-friendly multiplatform program for preparation and analysis of large-scale DNA polymorphism datasets.

Preparation of DNA polymorphism datasets for analysis is an important step in evolutionary genetic and molecular ecology studies. Ever-growing dataset sizes make this step time consuming, but few convenient software tools are available to facilitate processing of large-scale datasets including thousands of sequence alignments. Here I report "processor of sequences v4" (proSeq4)-a user-friendly multiplatform software for preparation and evolutionary genetic analyses of genome- or transcriptome-scale sequence polymorphism datasets. The program has an easy-to-use graphic user interface and is designed to process and analyse many thousands of datasets. It supports over two dozen file formats, includes a flexible sequence editor and various tools for data visualization, quality control and most commonly used evolutionary genetic analyses, such as NJ-phylogeny reconstruction, DNA polymorphism analyses and coalescent simulations. Command line tools (e.g. vcf2fasta) are also provided for easier integration into bioinformatic pipelines. Apart of molecular ecology and evolution research, proSeq4 may be useful for teaching, e.g. for visual illustration of different shapes of phylogenies generated with coalescent simulations in different scenarios. ProSeq4 source code and binaries for Windows, MacOS and Ubuntu are available from https://sourceforge.net/projects/proseq/.

PyPop: a mature open-source software pipeline for population genomics.

Python for Population Genomics (PyPop) is a software package that processes genotype and allele data and performs large-scale population genetic analyses on highly polymorphic multi-locus genotype data. In particular, PyPop tests data conformity to Hardy-Weinberg equilibrium expectations, performs Ewens-Watterson tests for selection, estimates haplotype frequencies, measures linkage disequilibrium, and tests significance. Standardized means of performing these tests is key for contemporary studies of evolutionary biology and population genetics, and these tests are central to genetic studies of disease association as well. Here, we present PyPop 1.0.0, a new major release of the package, which implements new features using the more robust infrastructure of GitHub, and is distributed via the industry-standard Python Package Index. New features include implementation of the asymmetric linkage disequilibrium measures and, of particular interest to the immunogenetics research communities, support for modern nomenclature, including colon-delimited allele names, and improvements to meta-analysis features for aggregating outputs for multiple populations. Code available at: https://zenodo.org/records/10080668 and https://github.com/alexlancaster/pypop.

Ectopic expression in commonly used transgenic Drosophila GAL4 driver lines.

Transgenic tools such as the GAL4/UAS system in Drosophila have been used extensively to induce spatiotemporally controlled changes in gene expression and tissue-specific expression of a range of transgenes. We previously discovered unexpected expression of the commonly used dilp2-GAL4 line in tracheal tissue which significantly impacted growth phenotypes. We realized that few GAL4 lines have been thoroughly characterized, particularly when considering transient activity that may have significant impact on phenotypic readouts. Here, we characterized a further subset of 12 reportedly tissue-specific GAL4 lines commonly used in genetic studies of development, growth, endocrine regulation, and metabolism. Ten out of 12 GAL4 lines exhibited ectopic activity in other larval tissues, with seven being active in the larval trachea. Since this ectopic activity may result in phenotypes that do not depend on the manipulation in the intended target tissue, it is recommended to carefully analyze the outcome while taking this aspect into consideration.

Microsatellite Content in 397 Nuclear Exons and Their Flanking Regions in the Fern Family Ophioglossaceae.

Microsatellites or SSRs are small tandem repeats that are 1-6 bp long. They are usually highly polymorphic and form important portions of genomes. They have been extensively analyzed in humans, animals and model plants; however, information from non-flowering plants is generally lacking. Here, we examined 29 samples of Ophioglossaceae ferns, mainly from the genera Botrychium and Sceptridium. We analyzed the SSR distribution, density and composition in almost 400 nuclear exons and their flanking regions. We detected 45 SSRs in exons and 1475 SSRs in the flanking regions. In the exons, only di-, tri- and tetranucleotides were found, and all of them were 12 bp long. The annotation of the exons containing SSRs showed that they were related to various processes, such as metabolism, catalysis, transportation or plant growth. The flanking regions contained SSRs from all categories, with the most numerous being dinucleotides, followed by tetranucleotides. More than one-third of all the SSRs in the flanking regions were 12 bp long. The SSR densities in the exons were very low, ranging from 0 to 0.07 SSRs/kb, while those in the flanking regions ranged from 0.24 to 0.81 SSRs/kb; and those in the combined dataset ranged from 0.2 to 0.81 SSRs/kb. The majority of the detected SSRs in the flanking regions were polymorphic and present at the same loci across two or more samples but differing in the number of repeats. The SSRs detected here may serve as a basis for further population genetic, phylogenetic or evolutionary genetic studies, as well as for further studies focusing on SSRs in the genomes and their roles in adaptation, evolution and diseases.

The value of hybrid genomes: Building two highly contiguous reference genome assemblies to advance Canis genomic studies.

Previous studies of canid population and evolutionary genetics have relied on high-quality domestic dog reference genomes that have been produced primarily for biomedical and trait mapping studies in dog breeds. However, the absence of highly contiguous genomes from other Canis species like the gray wolf and coyote, that represent additional distinct demographic histories, may bias inferences regarding interspecific genetic diversity and phylogenetic relationships. Here, we present single haplotype de novo genome assemblies for the gray wolf and coyote, generated by applying the trio-binning approach to long sequence reads generated from the genome of a female first-generation hybrid produced from a gray wolf and coyote mating. The assemblies were highly contiguous, with contig N50 sizes of 44.6 and 42.0 Mb for the wolf and coyote, respectively. Genome scaffolding and alignments between the two Canis assemblies and published dog reference genomes showed near complete collinearity, with one exception: a coyote-specific chromosome fission of chromosome 13 and fusion of the proximal portion of that chromosome with chromosome 8, retaining the Canis-typical haploid chromosome number of 2n = 78. We evaluated mapping quality for previous RADseq data from 334 canids and found nearly identical mapping quality and patterns among canid species and regional populations regardless of the genome used for alignment (dog, coyote, or gray wolf). These novel wolf and coyote genome reference assemblies will be important resources for proper and accurate inference of Canis demography, taxonomic evaluation, and conservation genetics.

The interest of informative ancestry markers (AIM) and their fields of application

This review focuses on the study of biogeographic ancestry using the Accurate Ancestry Identification Panel. Autosomal markers may provide little information about the nature of an individual's admixture due to ongoing human recombination and migration. Biogeographic ancestry assessment (BGA) is a term used to describe ancestry through DNA testing. This is usually accomplished by testing specific regions of DNA called ancestry information markers (AIMs). AIMs are chosen because they expose significantly different frequencies between different populations in different parts of the world. The panels of these AIMs can be assessed using next-generation sequencing (NGS) to predict the geographical origins of a person of interest's ancestors, usually in terms of continent of origin, and sometimes by smaller geographic regions. The use of ancestry informative markers (AIM) to identify genomic ancestry can be useful for a variety of studies in evolutionary genetics, biomedical research, and forensic analyses. However, there remains a major challenge in determining AIMs for populations with complex and highly mixed ancestry.

The ribosomal transcription units of five echinostomes and their taxonomic implications for the suborder Echinostomata (Trematoda: Platyhelminthes).

Five newly obtained nuclear ribosomal transcription unit (rTU) sequences from Echinostomatidae and Echinochasmidae are presented. The inter- and intrafamilial relationships of these and other families in the suborder Echinostomata are also analyzed. The sequences obtained are the complete rTU of Artyfechinostomum malayanum (9,499bp), the near-complete rTU of Hypoderaeum conoideum (8,076bp), and the coding regions (from 5'-terminus of 18S to 3'-terminus of 28S rRNA gene) in Echinostoma revolutum (6,856bp), Echinostoma miyagawai (6,854bp), and Echinochasmus japonicus (7,150bp). Except for the longer first internal transcribed spacer (ITS1) in Echinochasmus japonicus, all genes and spacers were almost identical in length. Comprehensive maximum-likelihood phylogenies were constructed using the PhyML software package. The datasets were either the concatenated 28S + 18S rDNA sequences (5.7-5.8kb) from 60 complete rTUs of 19 families or complete 28S sequences only (about 3.8-3.9kb) from 70 strains or species of 22 families. The phylogenetic trees confirmed Echinostomatoidea as monophyletic. Furthermore, a detailed phylogeny constructed from alignments of 169 28S D1-D3 rDNA sequences (1.1-1.3kb) from 98 species of 50 genera of 10 families, including 154 echinostomatoid sequences (85 species/42 genera), clearly indicated known generic relationships within Echinostomatidae and Echinochasmidae and relationships of families within Echinostomata and several other suborders. Within Echinostomatidae, Echinostoma, Artyfechinostomum, and Hypoderaeum appeared as monophyletic, while Echinochasmus (Echinochasmidae) was polyphyletic. The Echinochasmidae are a sister group to the Psilostomidae. The datasets provided here will be useful for taxonomic reappraisal as well as studies of evolutionary and population genetics in the superfamily Echinostomatoidea, the sole superfamily in the suborder Echinostomata.

Developmental genetic studies on rice morphogenesis

Developmental genetic studies on rice morphogenesis

Concurrent invasions of European starlings in Australia and North America reveal population‐specific differentiation in shared genomic regions

AbstractA species' success during the invasion of new areas hinges on an interplay between the demographic processes common to invasions and the specific ecological context of the novel environment. Evolutionary genetic studies of invasive species can investigate how genetic bottlenecks and ecological conditions shape genetic variation in invasions, and our study pairs two invasive populations that are hypothesized to be from the same source population to compare how each population evolved during and after introduction. Invasive European starlings (Sturnus vulgaris) established populations in both Australia and North America in the 19th century. Here, we compare whole‐genome sequences among native and independently introduced European starling populations to determine how demographic processes interact with rapid evolution to generate similar genetic patterns in these recent and replicated invasions. Demographic models indicate that both invasive populations experienced genetic bottlenecks as expected based on invasion history, and we find that specific genomic regions have differentiated even on this short evolutionary timescale. Despite genetic bottlenecks, we suggest that genetic drift alone cannot explain differentiation in at least two of these regions. The demographic boom intrinsic to many invasions as well as potential inversions may have led to high population‐specific differentiation, although the patterns of genetic variation are also consistent with the hypothesis that this infamous and highly mobile invader adapted to novel selection (e.g., extrinsic factors). We use targeted sampling of replicated invasions to identify and evaluate support for multiple, interacting evolutionary mechanisms that lead to differentiation during the invasion process.

Complete mitochondrial genome of Trematomus newnesi (Perciformes, Nototheniidae)

The complete mitochondrial genome of Trematomus newnesi was sequenced using an Illumina platform. The 18,602 bp mitogenome contains 13 protein-coding genes, two rRNAs, and 23 tRNAs (tRNAMet is duplicated). The eight stop codons are TAA, TAG, CTT, GTA, AAT, ACT, AGG, and TTA. Two start codons ATG and GTG are present. The GC content is 44.4% and AT content is 55.6%. A phylogenetic tree was generated using 13 species from three families. The results showed that T. newnesi is closely related to Pagothenia borchgrevinki in Nototheniidae. This study provides fundamental data for further genetic evolutionary studies on T. newnesi.

Spatio-temporal expression of candidate genes for nectar spur development in Tropaeolum (Tropaeolaceae: Brassicales).

Tropaeolaceae (Brassicales) comprise ~100 species native to South and Central America. Tropaeolaceae flowers have a nectar spur, formed by a late expansion and evagination of the fused proximal region of the perianth (i.e. the floral tube). This spur is formed in the domain of the tube oriented towards the inflorescence axis, which corresponds to the adaxial floral region. However, little is known about the molecular mechanisms responsible for the evolution of spurs in Tropaeolaceae. In this study, we examined the spatio-temporal expression of genes putatively responsible for differential patterns of cell division between the adaxial and abaxial floral regions in Tropaeolaceae. These genes include previously identified TCP and KNOX transcription factors and the cell division marker HISTONE H4 (HIS4). We found a TCP4 homologue concomitantly expressed with spur initiation and elaboration. Tropaeolaceae possess two TCP4-like (TCP4L) copies, as a result of a Tropaeolaceae-specific duplication. The two copies (TCP4L1 and TCP4L2) in Tropaeolum longifolium show overlapping expression in the epidermis of reproductive apices (inflorescence meristems) and young floral buds, but only TlTCP4L2 shows differential expression in the floral tube at early stages of spur formation, restricted to the adaxial region. This adaxial expression of TlTCP4L2 overlaps with the expression of TlHIS4. Later in development, only TlTCP4L2 is expressed in the nectariferous tissue of the spur. Based on these results, we hypothesize that Tropaeolaceae TCP4L genes had a plesiomorphic role in epidermal development and that, after gene duplication, TCP4L2 acquired a new function in spur initiation and elaboration. To better understand spur evolution in Tropaeolaceae, it is critical to expand developmental genetic studies to their sister group, the Akaniaceae, which possess simultaneously an independent duplication of TCP4L genes and a spurless floral tube.

Heterogeneity and functions of the 13 IFN-α subtypes - lucky for some?

Type I IFNs are critical for host responses to viral infection and are also implicated in the pathogenesis of multiple autoimmune diseases. Multiple subtypes exist within the type I IFN family, in particular 13 distinct IFN-αgenes, which signal through the same heterodimer receptor that is ubiquitously expressed by mammalian cells. Both evolutionary genetic studies and functional antiviral assays strongly suggest differential functions and activity between the 13 IFN-αsubtypes, yet we still lack a clear understanding of these different roles. This review summarizes the evidence from studies describing differential functions of IFN-αsubtypes and highlights potential reasons for discrepancies between the reports. We examine both acute and chronic viral infection, as well as autoimmunity, and integrate a more recent awareness of the importance of anti-IFN-αautoantibodies in shaping the type I IFN responses in these different conditions.

Overview of SNPs Associated with Trans Fat Content in Cow’s Milk

Lipids consumed with milk derivatives are one of the main parts of the human diet. Trans fatty acids in milk are causing a debate about their impact on the incidence of cardiovascular disease, pathological abnormalities, and cancer. The fatty acid profile of milk is influenced by a large number of different factors, one of which is genetic. The development of genetic studies, including Genome-Wide Association Studies (GWAS), may help define genomic regions associated with fatty acid content in milk, including trans fatty acids. This article provides an overview of international studies on the identification of genomic regions and SNPs associated with the trans fatty acids in cow’s milk. The results are based on research of cattle such as Norwegian Red cattle, Holstein, Jersey, and Brown Swiss. The presented review shows that 68 SNPs were localized on chromosomes 1, 2, 4–6, 8–10, 12, 14–20, 22–25, and 27–29. Further research in this direction will provide new information that will serve as an impetus for the creation of modern breeding technologies and increase the performance of the manufacture of high-quality dairy products. The search for genetic markers associated with the content of TFA in milk is a promising direction in agricultural science and will allow more complete breeding work with cattle.

Mitogenomic and nuclear ribosomal transcription unit datasets support the synonymy of Paragonimus iloktsuenensis and P. ohirai (Paragonimidae: Platyhelminthes).

The complete mitogenome (mtDNA) of nominal Paragonimus iloktsuenensis (Paragonimidae: Trematoda) and the nuclear ribosomal transcription unit (rTU) coding region (rTU*: from 5'-terminus of 18S to 3'-terminus of 28S rRNA gene, excluding the external spacer region) of this species and of P. ohirai were obtained and used to further support the previously suggested synonymy of these taxa in the P. ohirai complex. The complete mitogenome of P. iloktsuenensis was 14,827bp long (GenBank: ON961029) and nearly identical to that of P. ohirai (14,818bp; KX765277), with a 99.12% nucleotide identity. The rTU* was 7543bp and 6932bp in these two taxa, respectively. All genes and spacers in the rTU were identical in length, with exception of the first internal transcribed spacer, which contained multiple tandem repeat units (6.7 for P. iloktsuenensis and 5.7 for P. ohirai). There was near 100% identity for the rTU genes. The phylogenetic topology inferred from the mtDNA and from individual gene regions (partial cox1 of 387bp and the ITS-2 of 282bp - 285bp) indicated a very close relationship consistent with synonymy of P. iloktsuenensis and P. ohirai. The datasets provided here will be useful for taxonomic reappraisal as well as studies of evolutionary and population genetics of the genus Paragonimus and family Paragonimidae.

Variation in mutation, recombination, and transposition rates in Drosophila melanogaster and Drosophila simulans.

The rates of mutation, recombination, and transposition are core parameters in models of evolution. They impact genetic diversity, responses to ongoing selection, and levels of genetic load. However, even for key evolutionary model species such as Drosophila melanogaster and Drosophila simulans, few estimates of these parameters are available, and we have little idea of how rates vary between individuals, sexes, or populations. Knowledge of this variation is fundamental for parameterizing models of genome evolution. Here, we provide direct estimates of mutation, recombination, and transposition rates and their variation in a West African and a European population of D. melanogaster and a European population of D. simulans Across 89 flies, we observe 58 single-nucleotide mutations, 286 crossovers, and 89 transposable element (TE) insertions. Compared to the European D. melanogaster, we find the West African population has a lower mutation rate (1.67 × 10-9 site-1 gen-1 vs. 4.86 × 10-9 site-1 gen-1) and a lower transposition rate (8.99 × 10-5 copy-1 gen-1 vs. 23.36 × 10-5 copy-1 gen-1), but a higher recombination rate (3.44 cM/Mb vs. 2.06 cM/Mb). The European D. simulans population has a similar mutation rate to European D. melanogaster, but a significantly higher recombination rate and a lower, but not significantly different, transposition rate. Overall, we find paternal-derived mutations are more frequent than maternal ones in both species. Our study quantifies the variation in rates of mutation, recombination, and transposition among different populations and sexes, and our direct estimates of these parameters in D. melanogaster and D. simulans will benefit future studies in population and evolutionary genetics.

Whole Genome Sequence of an Edible Mushroom Oudemansiella raphanipes (Changgengu).

Oudemansiella raphanipes, considered as a well-known culinary edible mushroom with a high content of natural bioactive substances, is widely cultivated in China with the commercial name Changgengu. However, due to the lack of genomic data, molecular and genetic study on O. raphanipes is rare. To obtain a comprehensive overview of genetic characteristics and enhance the value of O. raphanipes, two mating-compatible monokaryons isolated from the dikaryon were applied for de novo genome sequencing and assembly using Nanopore and /or Illumina sequencing platforms. One of the monokaryons, O. raphanipes CGG-A-s1, was annotated with 21,308 protein-coding genes, of which 56 were predicted to be involved in the biosynthesis of secondary metabolites such as terpene, type I PKS, NRPS, and siderophore. Phylogenetic and comparative analysis of multiple fungi genomes revealed a close evolutionary relationship between O. raphanipes and Mucidula mucid based on single-copy orthologous protein genes. Significant collinearity was detected between O. raphanipes and Flammulina velutipes on the synteny of inter-species genomes. 664 CAZyme genes in CGG-A-s1 were identified with GHs and AAs families significantly elevated when compared with the other 25 sequenced fungi, indicating a strong wood degradation ability. Furthermore, the mating type locus analysis revealed that CGG-A-s1 and CGG-A-s2 were conserved in the gene organization of the mating A locus but various in that of the mating B locus. The genome resource of O. raphanipes will provide new insights into its development of genetic studies and commercial production of high-quality varieties.

Sex differences in the coexpression of prokineticin receptor 2 and gonadal steroids receptors in mice.

Loss-of-function mutations in prokineticin 2 (PROK2) and the cognate receptor prokineticin receptor 2 (PROKR2) genes have been implicated in reproductive deficits characteristic of Kallmann Syndrome (KS). Knock out of Prokr2 gene produces the KS-like phenotype in mice resulting in impaired migration of gonadotropin releasing hormone (GnRH) neurons, olfactory bulb dysgenesis, and infertility. Beyond a developmental role, pharmacological and genetic studies have implicated PROKR2 in the control of the estrous cycle in mice. However, PROKR2 is expressed in several reproductive control sites but the brain nuclei associated with reproductive control in adult mice have not been defined. We set out to determine if ProkR2 neurons in both male and female mouse brains directly sense changes in the gonadal steroids milieu. We focused on estrogen receptor α (ERα) and androgen receptor (AR) due to their well-described function in reproductive control via actions in the brain. We found that the ProkR2-Cre neurons in the posterior nucleus of the amygdala have the highest colocalization with ERα and AR in a sex-specific manner. Few colocalization was found in the lateral septum and in the bed nucleus of the stria terminalis, and virtually no colocalization was observed in the medial amygdala. Our findings indicate that the posterior nucleus of the amygdala is the main site where PROKR2 neurons may regulate aspects of the reproductive function and social behavior in adult mice.