dataesr/scientific-paragraphs-categorization huggingface dataset:
Results: Sauropsid Kiss and KissR genes were predicted from multiple genome and transcriptome databases by TBLASTN. Phylogenetic and syntenic analyses were performed to classify predicted sauropsid Kiss and KissR genes and to reconstruct the evolutionary scenarios of both gene families across the sauropsid radiation. Genome search, phylogenetic and synteny analyses, demonstrated the presence of two Kiss genes (Kiss1 and Kiss2 types) and of two KissR genes (KissR1 and KissR4 types) in the sauropsid lineage. These four genes, also present in the mammalian lineage, would have been inherited from their common amniote ancestor. In contrast, synteny analyses supported that the other Kiss and KissR paralogs are missing in sauropsids as in mammals, indicating their absence in the amniote lineage. Among sauropsids, in the avian lineage, we demonstrated the existence of a Kiss2-like gene in three bird genomes.
Following these events, the evolutionary history of Kiss and KissR was marked by multiple gene loss events in the various vertebrate lineages  [17, 21]. Notably, due to massive gene loss, there is no impact of the teleost specific third round of genome duplication (3R) on the current number of Kiss or KissR in teleosts  [17, 21]. Strikingly among sauropsids, a complete loss of the kisspeptin system may have occurred in birds, as suggested by the lack of Kiss and KissR homologs in the current bird genomic databases  [4, [17] [18] [19] [20] [21] 24]. Nevertheless, some immunocytochemical and experimental studies suggest the existence of a kisspeptin system in birds. Previous studies, using polyclonal antibodies against human Kp  (10) , have reported the observation of Kiss immunoreactivity in mallard duck hypothalamus  [25]  and hen cultured granulosa cells  [26].
In contrast, in adult male chicken, peripheral injections of human Kp  (10)  led to decreased circulating testosterone level  [30]. The effects of exogenous Kp  (10)  treatments on the bird gonadotropic axis could be obtained using other RF-amide peptides such as human 26RFa suggesting that they could be non-specific  [30]. Furthermore, it has recently been suggested that this inhibitory effect of exogenous Kp  (10)  on testosterone levels could be mediated by GnIH receptor in bird, since kisspeptin was shown to bind to this receptor  [31]. As functional approaches suggest that exogenous kisspeptins could exert physiological effects on birds while no Kiss or KissR has been evidenced in the different bird genomic databases so far, the question of the existence of a kisspeptin system in birds remains open.
In order to address this question in an evolutionary context, we choose to investigate the Kiss and KissR gene diversity in the genomic databases from representatives of different sauropsid groups, including ophidian, chelonian, crocodilian, and bird genomes. This integrated approach enabled us to improve the evolutionary history of both Kiss and KissR families in the sauropsid lineage and to demonstrate, for the first time, the presence of a Kiss2-like gene in the genome of three different bird species.
One Kiss-like gene in birds At first, the TBLASTN performed in bird genomic databases, using previously characterized kisspeptin sequences and newly described sauropsid mature kisspeptins as queries, resulted in no hit except for the mallard duck genome, pointing out a 99 bp long ORF. We then amplified and cloned the genomic region encompassing the corresponding mallard duck ORF (Figure  2 ). It encodes a peptidic sequence presenting the following 10 a. a. -sequence: FIFNPFGLGF (Figures  1  and 2 ). The C-terminal end of this sequence is followed by a typical GKR motif (Figures  1  and 2 ). The mallard duck Kp(10)-like sequence shares 40% identity with crocodilian, chelonian and ophidian Kp1  (10)  while it shares 80% identity with the anole Kp2  (10) , the painted turtle Kp2  (10)  and the Chinese turtle Kp2 (10) (Figure  1 ).
for crocodilian Kiss2-like pseudogene  [33]. Nevertheless, the presence of two putative splice acceptor sites, between the 5′ stop codon and the sequence encoding the mallard duck Kp(10)-like (Figure  2 ), opens the possibility that the mallard duck Kiss-like gene possesses a different exon/intron structure compared to the other vertebrate Kiss genes. However no potential exon coding for a signal peptide has been predicted so far that could support this hypothesis. Similarly, it is notable that the coelacanth Kp3  (10)  is encoded by a 81 bp-long ORF, that also suggests a different structure or a loss of function for this gene  [17]. As a second step, using the mallard duck Kiss-like sequence as query, we performed TBLASTN in the other bird genomic databases. The results of this second TBLASTN series pointed out a 279 bp-long ORF in the zebra finch genome (Additional file 1: Figure  S1 ) and a 117 bp-long ORF in rock pigeon (Additional file 1: Figure  S1 ).
In contrast, no putative splice acceptor site has been predicted between the 5′ stop codon and the sequence encoding the zebra finch Kp(10)-like (Additional file 1: Figure  S1 ). In addition, no potential exon coding for a signal peptide could be predicted in this study, neither for rock pigeon Kiss-like nor zebrafinch Kiss-like. As suggested above for the mallard duck sequence, this could reflect either a different structure or a loss of function for these genes. Finally, using the zebra finch and rock pigeon Kiss-like sequences as queries, we performed a novel TBLASTN search in the other genomic databases of birds, including chicken. This third TBLASTN series returned no hit, suggesting that the Kiss-like genes of the other birds are even more degenerated than those found in the mallard duck, zebra finch and rock pigeon genomes.
Phylogenetical analysis of Kiss genes Based on an alignment of 68 predicted long mature kisspeptin (Additional files 2 and 3: Figure  S2  and Table  S1 ), and assuming sea lamprey Kiss1 sequence as the out-group, a phylogenetic tree was generated (the list of the sequences and accession numbers is provided in Additional file 2: Figure  S2 ). Our in silico prediction of N-terminal proteolytic cleavage sites did not enable us to predict mature kisspeptin from the bird Kiss-like (this study), the crocodilian Kiss2-like  [33]  and the coelacanth Kiss3  [17] , which were excluded from this analysis. As shown in Figure  3 , gnathostome mature kisspeptins cluster into three clades, Kiss1, Kiss2 and Kiss3, which are supported by significant bootstrap values: 70%, 94% and 87%, respectively. The Kiss1 clade encompasses the chondrichtyan and actinopterygian Kiss1 sequences including the spotted gar and teleost Kiss1 sequences.
The human Kiss1, crocodile Kiss1, Chinese turtle Kiss1, Xenopus Kiss1a, coelacanth Kiss1 and spotted gar Kiss1 are positioned in genomic regions containing common loci, including TEAD3, NAV1, PPP1R12B, PPFIA4, MYBPH, KCNC4, REN, GOLT1A, PLEKHA6, PPP1R15B, PIK3C2B and SYT6 (Figure  4A ). The painted turtle Kiss1, alligator Kiss1 and garial Kiss1 are also located in the vicinity of those genes (data not shown). This supports the orthology relationship between all these Kiss genes, all considered as Kiss1 genes. Syntenic analysis supports the absence of Kiss1 gene in the chicken, mallard drake, zebra finch and green anole, although the above-mentioned neighbouring genes are present in their respective genomic databases (Figure  4A ). The Kiss-like genes of mallard duck and zebra finch, the Chinese turtle, green anole, coelacanth and spotted gar Kiss2 genes are positioned in genomic regions containing common loci including TEAD4, STRAP, PLEKHA5, GOLT1B, C12orf39, GYS2, LDHB, KCNJ8, ABCC9, CMAS and SYT10 (Figure  4B ).
The painted turtle Kiss2 and the Kiss-like sequence of the rock pigeon are also located in the vicinity of these genes (data not shown). The crocodile Kiss2-like is located in an isolated scaffold, in the vicinity of the LDHB gene. This supports the orthology relationship between all these Kiss, considered as Kiss2 genes, or as Kiss2-like genes in the case of crocodile, mallard drake, zebra   S1 ) using the Neighbour Joining method with 1,000 bootstrap replicates. The number shown at each branch node indicates the bootstrap value (%); only values and branching above 70% are indicated. The tree was rooted using the sea lamprey Kiss1 sequence. The sauropsid kisspeptins are in red. finch and rock pigeon. The syntenic analysis suggests that chicken would not possess any Kiss2 gene, although the above-mentioned neighbouring genes are present in their respective genomic databases (Figure  4B ).
As already demonstrated, the coelacanth Kiss3 and the Xenopus Kiss1b genes are positioned in genomic regions containing common loci, including NTF4, KCNA7, PPFIA3, TEAD2, PIH1D1, ALDH16A1, KCNC3, MYBPC2 and SYT3 (Figure  4C  and  [17] ). This supports the orthology relationship between these two Kiss genes, both considered here as Kiss3 genes. Syntenic analysis supports the absence of Kiss3 gene in human, crocodile, and green anole, although the above-mentioned neighbouring genes are present in their respective genomic databases (Figure  4C ). Syntenic analysis also indicates that the considered region is largely missing from the bird and turtle genomic databases likely due to incomplete genome sequencing (see below). Indeed, among the fourteen considered genes in the Kiss3 syntenic region, only KCNC3 gene is present in mallard drake genomic databases, only SYT3 gene is present in zebra finch genomic databases and only KCNA7, PIH1D1 and SYT3 genes are present in Chinese turtle genomic databases (Figure  4C ).
This is in agreement with the higher sequence identity between bird Kp(10)like and the other sauropsid Kp2  (10). Synteny analyses also highlighted the absence of Kiss1 and Kiss4 genes in birds, while the corresponding genomic environment is well conserved. In contrast, our syntenic analysis revealed an urgent need for genomic information in the putative region of bird Kiss3 (Figure  4C ). In the human genome, this region is located in the chromosome 19q (HSA19q) (Figure  4C ). It has been well documented that most parts of the homologous region of HSA19q are not represented in the whole genome shotgun reads and BAC libraries used to build the bird genome databases  [37, 38]. However, it has been demonstrated that this region does exist and is split into micro-chromosomes in the bird genomes  [38, 39].
Investigation of the Kiss existence in bird transcriptome databases As kisspeptins are considered as neuropeptides, the Kiss gene expressions were mainly investigated in the brain of vertebrates (for reviews:  [40, 41] ). In human, in addition to its cerebral expression, Kiss1 transcript is found in placenta, intestine, testis, pancreas, spleen, kidney and liver  [3, 42]. In Xenopus, Kiss1a (Kiss1-type), Kiss1b (Kiss3-type) and Kiss2 genes are expressed in brain, testis, heart and liver  [18]. In order to investigate the potential existence of Kiss transcripts in birds, we performed TBLASTN searches in twelve released transcriptomic databases, including brain transcriptomes from ten different bird species. We also investigated chicken transcriptomes including embryo, muscle, brain and liver, and zebra finch transcriptomes including blood and spleen.
Using as query the same sequences as already used for the search against the sauropsid genomic databases as well as the bird Kiss2-like sequences, we performed TBLASTN in the twelve bird transcriptomic databases, but they resulted in no hit. In contrast, TBLASTN search using other RFamide sequences as query, allowed us to retrieve transcripts of the corresponding peptides including GnIH, NPFF, 26RFa and PrRP (data not shown). The absence of result concerning kisspeptin, in all released databases from a large number of species, supports the potential loss of functional Kiss gene in birds. To further understand whether Kiss genes have been lost in birds and to improve the Kiss evolutionary scenario, we focused on the diversity of newly described Kiss genes among the sauropsids.
Evolutionary scenario of Kiss genes in the sauropsid lineage Our analyses demonstrate that Kiss1 and/or Kiss2 genes are present in the sauropsid lineage. No trace of Kiss4 was found in agreement with the hypothesis that this loss-event would have taken place at the early stage of vertebrate evolution  [17]. Among sarcopterygians, the Kiss3 existence has been demonstrated in the coelacanth and the Xenopus genomes,  [17, 18]  indicating that Kiss3 gene has been inherited by the sarcopterygian ancestor and more recently by the tetrapod ancestor. No trace of Kiss3 was found in the mammalian and sauropsid genomic databases, suggesting the loss of this gene in the amniote ancestor. However, the fact that the whole Kiss3 genomic region is missing in the current bird genome databases, does not allow us to confirm the absence of Kiss3 in birds by synteny analysis.
Considering the alternative hypothesis of the presence of Kiss3 in birds, the phylogenetic position of birds among amniotes implies that Kiss3 gene would have been conserved in birds and lost independently in mammals, squamates (lizards and snakes), chelonians (turtles) and crocodilians lineages. This hypothesis represents four evolutionary events, as compared to only one event if considering the loss of Kiss3 gene in the amniote ancestor (Figure  5 ). This scenario is therefore the most parsimonious and is also supported by the absence of Kiss3 mRNA in the bird transcriptomic databases. Following the most parsimonious scenario (Figure  5 ), only Kiss1 and Kiss2 genes are present in the sauropsid lineage. Both Kiss1 and Kiss2 are present in chelonians. Kiss1 and Kiss2 genes were also inherited by squamate ancestor since python still possesses both Kiss1 and Kiss2 genes, while Kiss1 would have been lost in lizards.
No KissR gene in birds The TBLASTN analysis performed in bird genomic databases, using previously characterized KissR and newly described sauropsid KissR as queries, only returned genes corresponding to already characterized G-protein coupled receptors different from KissR with identity percentages below 40%. This percentage corresponds to the sequence identity shared with the galanin receptor, one of the KissR closest relatives  [5]. Thus, we did not find any KissR gene candidate in the bird genomic databases.
The painted turtle, alligator and garial KissR1 are also located in the vicinity of those genes (data not shown). This supports the orthology relationship of all these KissR genes, all considered as KissR1 genes. This syntenic analysis also supports the absence of chicken, mallard drake, zebra finch and green anole Kiss1 gene, although the above-mentioned neighbouring genes are present in the respective genomic databases (Figure  7A ). The Xenopus (GPR54-2), coelacanth and spotted gar KissR2 are positioned in genomic regions containing common loci, including PTPRF, KDM4A, ST3GAL3 and DPH2 (Figure  7B ). As already demonstrated  [21] , this supports the orthology relationship of these KissR genes, considered as KissR2. Syntenic analysis also supports the hypothesis that the human, chicken, mallard duck, zebra finch, crocodile, Chinese turtle and green anole genomes do not contain KissR2 gene, although the above-mentioned neighbouring genes are present in the respective genomic databases (Figure  7B ).
The Xenopus (GPR54-1b) and spotted gar KissR3 are positioned in genomic regions containing common loci, including ISCA and ZCCHC6 (Figure  7C ). As already demonstrated  [21]  this supports the orthology relationship of these two KissR genes, both considered as KissR3 genes. The coelacanth KissR3 is split into scaffolds JH131603. 1 and JH131921. 1, which are too small to contain any other gene (  [21]  and Figure  7C ). The syntenic analysis also supports the hypothesis that the human, chicken, mallard duck, zebra finch, crocodile, Chinese turtle and green anole genomes do not contain any KissR3 gene, although the above-mentioned neighbouring genes are present in the respective genomic databases (Figure  7C ). The green anole, coelacanth and spotted gar KissR4 are positioned in genomic regions containing common loci, including CAMSAP3, XAB2, STXBP2, RETN, RAB3D, PALM3, NOTCH3, EPHX3, WIZ and LPAR2 (Figure  7D ), as already shown  [21].
The sauropsid KissR are in red. KissR4 gene, although the above-mentioned neighbouring genes are present in the respective genomic databases (Figure  7D ). Syntenic analysis reveals that almost the whole considered region is absent from the bird genomic databases, likely due to incomplete genome sequencing. Indeed, among the eleven considered genes in the KissR4 syntenic region, only WIZ and LPAR2 genes are present in the chicken and mallard duck genomic databases (Figure  7D ). The results of this syntenic analysis are in agreement with our conclusions based on our phylogenetic analysis, that the sauropsid lineage possesses KissR1 and KissR4 genes. These results based on the study of multiple sauropsid genomes, represent the first large-scale investigation and classification of KissR genes in sauropsids.
We were not able to find any evidence demonstrating the existence of KissR gene in the bird genomic databases. However, similarly to what was observed in the syntenic region of Kiss3 gene (section Syntenic analysis of Kiss genes), our syntenic analysis also emphasizes a drastic lack of genomic information in the bird putative KissR4 region. In human, this region is also located in the HSA19q. All these analyses lead to the hypothesis that, if birds still possessed a KissR gene, it could only be a KissR4-type gene. To further investigate the KissR existence in birds, we looked for evidence of KissR mRNA in the released bird transcriptomic databases.
Investigation of the KissR existence in the released bird transcriptomes In tetrapods, the KissR genes are expressed in a wide range of tissues depending on the species. Kiss1r (KissR1) transcript is found in placenta, brain, spinal cord, pituitary and pancreas of human  [2, 3, 42] , and in rat brain, liver and intestine  [18]. In Xenopus, the three KissR genes are all expressed in the brain. In addition, GPR54-1a transcript (KissR1) is found in pituitary, testis and intestine, GPR54-1b (KissR3) in testis and intestine, and GPR54-2 (KissR2) in pituitary and heart  [18]. To further investigate the existence of KissR in birds, we looked for evidence of KissR mRNA in the released bird transcriptomic databases. We performed TBLASTN searches in the same twelve bird transcriptomic databases we used to investigate the potential existence of Kiss mRNA).
Using the predicted sauropsid KissR   S4. peptidic sequences as query, the TBLASTN returned only hits corresponding to other G-protein coupled receptors different from KissR, as already observed when performing a BLAST search on the genomes (see No KissR gene in birds). Thus, we did not find any KissR transcript candidate in the bird transcriptomic databases. Although a transcriptomic database cannot totally reflect the gene diversity of a species, the absence of result, in all current databases, is in favour of the loss of KissR4 in birds.
Both KissR1 and KissR4 genes were also inherited in the chelonian lineage, as observed in the turtles. The KissR4 gene would have been lost in the crocodilian lineage since crocodile, alligator and garial only possess KissR1 (Figure  8 ). The KissR1 gene would have been lost in the avian lineage, as shown by synteny analyses (Figure  8 ). Concerning KissR4, the fact that the whole putative KissR4 genomic region is missing in the current bird genome databases (Figure  7D ) does not allow us to assess the absence of this gene in birds by synteny analysis. Thus, even if the absence of KissR4 in the various genomic and transcriptomic databases is in favour of the loss of this gene, its existence in birds cannot yet be fully excluded (Figure  8 ).
Genomic databases The following dedicated genomic databases were investigated from e!ENSEMBL genome browser (http://www. ensembl. org/index. html): human, Homo sapiens; chicken, Gallus gallus; turkey, Meleagris gallopavo; zebra finch, Taeniopygia guttata; Chinese turtle, Pelodiscus sinensis; green anole, Anolis carolinensis; Xenopus, Xenopus tropicalis; and coelacanth, Latimeria chalumnae. The following dedicated genomic databases were investigated from Pre!ENSEMBL genome browser (http://pre. ensembl. org/index. html): collared flycatcher, Ficedula albicollis; mallard duck, Anas platyrhynchos; budgerigar, Melopsittacus undulates; painted turtle, Chrysemys picta; and spotted gar, Lepisosteus oculatus. The following dedicated genomic databases were investigated from NCBI genome browser (http://www.
ncbi. nlm. nih. gov/genome/browse/): medium ground finch, Geospiza fortis; rock pigeon, Columba livia; saker falcon, Falco cherrug; peregrine falcon, Falco peregrinus; Tibetan ground-tit, Pseudopodoces humilis; Puerto-Rican parrot, Amazona vittata; and Indian python, Python molurus. The following dedicated genomic databases were investigated from croc genomes web site (http://crocgenomes. org/): Alligator, Alligator mississippiensis; garial, Gavialis gangeticus; and crocodile, Crocodylus porosus.
Transcriptomic databases The following bird transcriptomic databases were investigated from NCBI Trace browser (http://www. ncbi. nlm. nih. gov/Traces/sra/?view=run_browser): pied flycatcher, Ficedula hypoleuca (SRX012275); zebra finch (SRP003283); ring-necked dove, Streptopelia roseogrisea (SRX012364); blue tit, Parus caeruleus (SRX012276); European crow, Corvus corone (SRX006755 and SRX006756); American crow, Corvus brachyrhynchos (SRX012416); ruby-throated hummingbird, Archilochus colubris (SRX012361); Anna's hummingbird, Calypte anna (SRX012362); golden collared manakin, Manacus vitellinus (SRX012420); emu, Dromaius novaehollandiae (SRX012419); and budgerigar, Melopsittacus undulatus (SRX012363). The chicken transcriptomic databases were investigated from the archive at ftp://www. chick.
Gene predictions TBLASTN search The TBLASTN algorithm (search sensitivity: near exact match short) of the e!ENSEMBL website (http://www. ensembl. org/index. html) was used on the genomic databases available at the e!ENSEMBL or pre!ENSEMBL website. The TBLASTN algorithm of the CLC DNA Workbench software (CLC bio, Aarhus, Denmark) was used on the other investigated genomic and transcriptomic databases.
Considering that the Kiss gene sequences are highly variable among species except for the sequence encoding Kp  (10)  and the flanking proteolytic cleavage and alpha-amidation site, we focused our predictions on the genomic ORF encoding these sequences. The peptidic sequences of Xenopus Kiss1a, Kiss1b and Kiss2 (Kiss1, Kiss3 and Kiss2 in this study, respectively)  [18] , coelacanth Kiss1, Kiss2 and Kiss3, and elephant shark Kiss1, Kiss2 and Kiss3  [17]  were used as query in TBLASTN search to identify the ORF encoding Kp  (10)  in the various investigated genomic and transcriptomic databases. We applied ORF-finder tool of the CLC DNA Workbench software to retrieve ORF encompassing Kp  (10). Novel sauropsid Kiss sequences, predicted in the present study, were used as query to blast again bird genomic and transcriptomic data bases.
KissR genes The exon-intron structure of the KissR genes is well conserved among vertebrates and it is made of five exons and four introns (for review:  [19] ). To retrieve the CDS of new KissR genes from the various investigated databases, the TBLASTN searches were performed using the human Kiss1r (KissR1 in this study), the three xenopus KissR, the four coelacanth KissR and the green anole KissR4 peptidic sequences as query. The splice junctions were predicted using the HSF (Human splicing Finder) v2. 4. 1  [59]  and manually checked using the empirical nucleotidic splicing signatures, i. e. intron begins with "GT" and ends with "AG"  [21]. Novel sauropsid KissR sequences, predicted in the present study, were used as query to blast again bird genomic and transcriptomic data bases.