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. 2025 Apr 28;21(5):1300–1313. doi: 10.1111/1749-4877.12992

Genome‐Wide Mining of lncRNAs Reveals Their Potential Regulatory Role in the Evolution of Viviparity

Xiaogang Feng 1,2, Wei Gao 1,✉, Wenjie Dong 1,2, Yijing Chen 3, Robert W Murphy 1,4, Yaping Zhang 1,3,5, Jing Che 1
PMCID: PMC13574192  PMID: 40296238

ABSTRACT

Reproduction in vertebrates usually involves egg‐laying (oviparity) or live‐bearing (viviparity). Oviparity is the ancestral trait from which viviparity has independently evolved more than 100 times in squamate reptiles. This transition involves a series of physiological and structural changes, including the degeneration of eggshell and the evolution of a placenta and differences in the temporal and spatial expression patterns of some functional genes that drive the structural transformation. Long non‐coding RNAs (lncRNAs) play important roles in the regulation of gene expression, yet it remains unclear whether they participate in gene expression shifts during the transition from oviparity to viviparity, and if so how. Therefore, we employ deep mining to identify novel lncRNAs of a closely related oviparous‐viviparous pair of lizards (Phrynocephalus przewalskii and P. vlangalii). We construct cis‐ and trans‐regulatory networks between lncRNAs and target genes using the transcriptomic data of oviduct or uteri tissues across reproductive periods. Results show that lncRNAs that regulate eggshell gland developmental genes in the oviparous lizard are lost or less expressed in the viviparous lizard. A number of lncRNAs involved in the regulation of placental development and embryo attachment in viviparous species have no orthologs in oviparous species, and others show little or no expression. Accordingly, lncRNAs may play important regulatory roles in the physiological and structural changes in the transition from oviparity to viviparity. These results open doors to the further elucidation of genetic regulatory networks.

Keywords: lncRNA, oviparity, Phrynocephalus, viviparity

This study reveals that lncRNAs likely play crucial regulatory roles in the evolution of viviparity in squamate reptiles. In viviparous lizards, specific lncRNAs associated with placenta development emerge, while those involved in eggshell gland development in oviparous species are downregulated or absent. These findings suggest that lncRNAs may drive the expression change of genes related to structural and physiological shifts from oviparity to viviparity.

graphic file with name INZ2-21-1300-g004.webp

1. Introduction

The evolution of reproductive modes in vertebrates, whether giving birth directly or hatching eggs, remains a central theme in evolutionary studies (Van Dyke et al. 2014). Viviparity has evolved independently more than 150 times in vertebrates and most origins are in squamate species (Blackburn 2015; Wright et al. 2015; Whittington 2021). The repeated independent evolution of viviparity in vertebrates underscores the selective advantages conferred by this reproductive strategy. Viviparity significantly cuts embryo mortality by shielding them from external conditions outside the mother and ensures better offspring fitness by providing an ideal environment for embryonic development. Most origins of viviparity in squamates occur in lower taxonomic groups (subgenera), and this involves about 20% of extant squamate species (Blackburn 1999). Thus, squamate reptiles provide an ideal model system for studying the evolution of viviparous reproduction (Blackburn 2001, 2006).

The evolution from oviparity to viviparity mainly involves two major structural transitions. First, the outer calcified layer of the eggshell is absent or extremely reduced in viviparous species, unlike in oviparous reptiles which usually possess a thick inner envelope composed of protein fibers and a thin calcium carbonate outer shell (Heulin et al. 2005; Stewart and Blackburn 2014). In squamates, the eggshell gland that secretes the shell seasonally is degenerated in viviparous species; it develops during vitellogenesis and regresses gradually after ovulation. Estrogen secreted by the ovaries during vitellogenesis drives the development of eggshell glands in oviparity (Palmer and Guillette 1990; Motz and Callard 1991). Various estrogen‐induced growth factors can stimulate proliferation and differentiation in the female reproductive tract (Hom et al. 1998; Girling 2002).

Another structural change involves the emergence of a placenta, which serves to transport various nutrients, water, and oxygen from the mother to her embryos. In contrast, embryos of oviparous species obtain nutrition from the yolk (and some calcium from the eggshell) (Stewart 2015; Murphy and Thompson 2011). Primarily, squamates have two types of placenta that develop during mid‐gestation: an embryonic pole chorioallantoic placenta and an anti‐embryonic pole yolk sac placenta (Adams et al. 2007). These placentae mainly form in a close association between the uterus and extraembryonic membranes, with the embryos not invading the uterine tissue in most viviparous squamates (Van Dyke et al. 2014; Whittington et al. 2022). Although the structure of these placentae is quite simple, the uterine epithelium undergoes significant remodeling during development, which occurs during the mid‐stage of gestation when the embryo has reached approximately stages 30–31 (Adams et al. 2007; Gao et al. 2019).

Transcriptome studies have identified several important differentially expressed genes in both oviparous and viviparous species, suggesting that these genes underlie the structural transformation. Regarding eggshells, estrogens and their downstream growth factors are highly expressed during the eggshell gland developmental period. Differences in gene expression patterns from egg‐laying to live‐bearing during the reproductive cycles are closely associated with the degeneration of the eggshell gland and the reduction of the eggshell (Gao et al. 2019). Because placental development involves the remodeling of the uterine epithelium, previous research has mainly focused on the differential expression between pregnant and non‐pregnant uteri. Comparison of pregnant and non‐pregnant female Chalcides ocellatus suggests that the differential expression of ACTA2, CDH5, ESR1, IGFBP5, LGR5, and VEGFA may be involved in nutrient supply to the embryo and uterine remodeling of the placenta (Brandley et al. 2012). The expression of some of these genes was also identified in another pair of oviparous–viviparous species (Griffith et al. 2016). Investigations into differentially expressed genes during pregnancy and non‐pregnancy in uterine tissues of multiple taxa revealed a shared genetic basis of viviparity at the gene or functional level (Recknagel et al. 2021; Foster et al. 2022). Further, the estrogen receptor (ESR1) and two growth factor receptors (GHR and IGF1R) may play crucial roles in initiating uterine changes during placental formation in viviparous species of Phrynocephalus (Gao et al. 2019). The downstream genes KRT80, LRP6, and HOOK3 were found to be likely involved in subsequent uterine cell differentiation, and CELSR1, which encodes the cadherin receptor, likely plays a crucial role in embryo attachment to maternal tissue. However, the absence of further analysis limits our understanding of how expression changes control the transition from oviparity to viviparity throughout the reproductive and developmental process.

To further explore this transition, we focus on long non‐coding RNAs (lncRNAs), which are a class of transcripts longer than 200 nucleotides that cannot produce proteins (Palazzo and Koonin 2020; Rinn and Chang 2012). As important regulators of gene expression, lncRNAs play many roles in a variety of biological processes (Ma et al. 2013), including transcription control, chromatin modification, genomic imprinting, epigenetic regulation, cell differentiation, and cell cycle control (Patil et al. 2014). In general, lncRNAs function on target genes mainly through cis‐ or trans‐regulation. Cis‐regulating lncRNAs control the expression of neighboring protein‐coding genes via sequence complementary binding to the DNA‐transcribed locus by recruiting chromatin modification complexes; this regulates the chromatin state, which affects promoter activities (Ma et al. 2013; Maenner et al. 2010; Ye et al. 2022). Trans‐regulating lncRNAs affect distant target genes by influencing transcription via binding to transcription elongation factors or RNA polymerases (Ma et al. 2013). Much is known about how lncRNAs regulate some particular traits including healthy aging in centenarians (Jiang et al. 2021), differentiation of chicken intramuscular preadipocytes (Zhang et al. 2017), and pig domestication (Zhou et al. 2014). However, very little is known about lncRNAs in reptiles, let alone the roles they play in the major physiological and morphological aspects of the oviparity‐viviparity transition.

Herein, we analyze RNA sequence data from the oviducts of oviparous Phrynocephalus przewalskii and viviparous P. vlangalii across their reproductive periods. Analyses identify thousands of novel lncRNAs for each species and explore their regulation of target genes. By comparing lncRNAs from viviparous and oviparous species, results show that they play important roles in the eggshell degeneration and the emergence of the placenta during the evolution of viviparity. Our study provides new insights into understanding the evolution of viviparity in squamate reptiles.

2. Materials and Methods

2.1. Sample Type and RNA‐seq Data Sources

RNA sequence data were obtained from (Gao et al. 2019). A total of 50 oviduct tissues were evaluated using the high‐coverage RNA‐seq, including 30 samples for six stages of viviparous P. vlangalii and 20 samples for four stages of oviparous P. przewalskii (Figure 1A). In P. vlangalii, the stages are as follows: ovarian egg stage (pre‐ovulation, S1), middle gestation 1 (embryonic developmental stage 25–27, S2), middle gestation 2 (embryonic developmental stage 30–31, S3), middle gestation 3 (embryonic developmental stage 34–36, S4), parturition (embryonic developmental stage late 40, S5) and postpartum 24 h (S6). For P. przewalskii, the respective stages include ovarian egg stage (before ovulation, S1), after ovulation (embryonic developmental stage 25–27, S2), after ovulation, but before egglaying (embryonic developmental stage 30, S3), and postpartum 24 h (S4). The embryonic developmental stages for the two species were determined based on the embryonic staging scheme outlined by Dufaure and Hubert (1961). These stages provide a framework for understanding reproductive and developmental processes in both oviparous and viviparous Phrynocephalus species.

FIGURE 1.

FIGURE 1

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Features of oviparous and viviparous samples and the overall profile of lncRNAs. (A) Comparison and correspondence of reproductive stages between Phrynocephalus przewalskii (oviparity) and P. vlangalii (viviparity). Identification and classification of novel lncRNAs in P. przewalskii (B) and P. vlangalii (C).

2.2. RNA‐seq Reads Mapping and Transcript Assembly

High‐quality clean reads were mapped to the respective reference genomes of P. vlangalii and P. przewalskii using HISAT2 (v2.1.0) with the default parameters (Pertea et al. 2016). We then used Samtools (v1.10) to sort and convert the SAM (Sequence Alignment/Map) files to BAM (Binary Alignment/Map) files with parameters “sort ‐o” (Pertea et al. 2016). Next, Stringtie (v2.1.4) assembled the transcripts for each sample with parameters “‐G ‐o ‐l” and finally merged them into one GTF file using the merge function. After merging, we re‐estimated the abundances of the merged transcripts by Stringtie with the parameters “‐A ‐eB” (Pertea et al. 2016, 2015). In addition, read‐count data were extracted directly from the Stringtie output files for subsequent calculations by using prepDE.py in Stringtie.

2.3. LncRNA Identification

First, Gffcompare (v0.11.6) was employed to examine how the transcripts compared with the reference annotation using parameters “‐G ‐r” (Pertea and Pertea 2020). The assembled transcripts were evaluated, and only those satisfying the following three criteria were retained for subsequent analyses: (1) transcript length exceeds 200 nucleotides (nt), as a length threshold of 200 nt is a widely accepted standard for differentiating long non‐coding RNAs (lncRNAs) from small non‐coding RNAs, ensuring that retained transcripts conform to the defining characteristics of lncRNAs; (2) transcripts contain more than one exon since requiring multiple exons excludes potential assembly artifacts and increases the probability of identifying functional lncRNAs, thereby improving the accuracy and reliability of the selection process; (3) transcript class code is “i,” “o,” “u,” “j,” or “x,” because these class codes represent intergenic, intronic, and exon‐overlapping lncRNAs, while excluding protein‐coding transcripts and low‐confidence categories, which enhances the specificity of lncRNA identification by focusing on well‐defined non‐coding RNA types. These criteria were applied collectively to ensure that only high‐confidence lncRNA candidates were included in further analyses.

Next, we predicted the coding potential of retained transcripts. The GTF file of the candidate transcripts was converted to FASTA a sequences file, which was used for three tools: CNCI (Sun et al. 2013), CPC2 (Kang et al. 2017), and LGC (Wang et al. 2019). These analyses independently calculated the coding potential of candidate transcripts. Transcripts labeled “non‐coding” by any method were retained. We also made predictions based on the PFAM protein database. For this, the FASTA file was translated to a protein sequence using transeq with parameters “‐frame = 6”; The PFAM database was downloaded from (http://ftp.ebi.ac.uk/pub/databases/Pfam/current_release) and formatted by using HmmerPress (Potter et al. 2018). Afterward, Pfamscan was used to calculate the coding potential of the protein sequence (Mistry et al. 2021). We retained no‐hit sequences. To summarize the results from CNCI, CPC2, LGC, and Pfamscan, we intersected the four gene sets and obtained the overlap transcripts, which were further filtered based on the condition that FPKM> 0 in at least one sample. Transcripts meeting the requirement were retained as candidate lncRNAs for subsequent analysis.

Finally, to systematically name the newly identified lncRNAs from the oviduct tissue of the two species, we established a standardized nomenclature based on their class codes. The lncRNAs were first classified according to the class_code annotations generated by gffcompare. Each lncRNA was assigned a unique identifier following the format: Vlalnc_$code‐XXXX for P. vlangalii and Przlnc_$code‐XXXX for P. przewalskii, where $code corresponds to the class_code categories: “u” (intergenic lncRNA) as IG, “x” (overlap with opposite) as OP, “i” (intronic lncRNA) as IT, “j” (muti‐exon match lncRNA) as ME, and “o” (overlapping with exons lncRNA) as OE. Each lncRNA was sequentially numbered (XXXX) within its respective category, starting from 0001. For instance, the first intergenic lncRNA in P. vlangalii was named Vlalnc_IG‐0001. This naming convention ensures clarity, facilitates downstream functional analyses, and enables cross‐species comparisons.

2.4. Cis‐regulation Target Gene Prediction

WindowBed in Bedtools (v2.30.0) (Quinlan and Hall 2010) was used to identify protein‐coding genes that fell in the 10‐kb upstream and downstream regions of lncRNAs. These lncRNAs were considered as cis‐regulatory target genes (Kopp and Mendell 2018)

2.5. Trans‐regulatory Network Construction

Trans‐regulatory effects of lncRNAs on mRNA were identified by correlations between their expression levels. For this, we constructed lncRNA‐mRNA co‐expression networks using the Weight Gene Co‐expression Network Analysis (WGCNA) package (v1.5.1) (Langfelder and Horvath 2008). The minimum cluster size was set to 30 members and unsigned correlation was used as an option. We focused on stage‐specific modules based on trait relationships, in which further filtering was based on gene significance (GS) and module membership (MM) values, where |GS|> 0.6 and |MM|> 0.8 was set as the threshold to screen hub genes. All programs were run in the R environment. We then employed Cytoscape (v3.8.0) (Shannon et al. 2003) to visualize the network with the top 1000 weight of hub genes.

2.6. Orthologous lncRNAs and Protein Coding Genes Identification

The lncRNA FASTA files of two species were assessed using makeblastdb of BLAST (v2.5.0) with parameter “‐dbtype nucl.” Next, putative lncRNA orthologs were inferred via BLASTn from bidirectional best hits in direct comparison to P. vlangalii and P. przewalskii with parameters “‐max_target_seqs 1” and “‐max_hsps 1” and with a fairly stringent E‐value threshold of 10−10.

Orthofinder (v2.5.5) (Emms and Kelly 2019) was used to calculate orthologs of protein‐coding genes. The protein sequence files of both species were carried as input files and MSA method was chosen for gene tree inference.

2.7. Gene Function Enrichment

Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis on target genes were performed by Metascape (Zhou et al. 2019). GO terms and pathways with a p value less than 0.05 were considered significantly enriched.

3. Results

3.1. Identification and Classification of Novel lncRNAs

Novel lncRNAs in P. vlangalii and P. przewalskii were explored based on high‐coverage RNA‐seq data because no lncRNA database contained reptilian lncRNAs. We reassembled the transcript by mapping reads to the reference genome, and applied rigorous filtering based on the sequence length, exon number, and class code of the transcript. Using four prediction tools, 8996 and 7884 transcripts were considered as putative lncRNAs. After further filtering on the condition that FPKM > 0 in at least one sample, a total of 8989 novel lncRNAs were identified in oviparous P. przewalskii and 7863 in viviparous P. vlangalii (Figure 1B,C).

All novel lncRNAs were classified according to their positional relationship to genes on the reference genome. In P. przewalskii and P. vlangalii, 71.44% and 69.73% lncRNAs had unidentified intergenic relationships, respectively; 13% and 13.6% lncRNAs were exonic on the opposite strand, respectively; 10.28% and 9.04% lncRNAs were fully contained with a reference intron, respectively; 3.06% and 4.21% lncRNA were muti‐exon with a least one junction match, respectively; and 2.21% and 3.42% lncRNAs had exonic overlap with a reference sequence on the opposite strand, respectively (Figure 1B,C; Tables S1 and S2).

3.2. Characteristics of lncRNAs and mRNAs

We further assessed the genomic differences between lncRNAs and mRNAs using sequence lengths, exon numbers, and expression levels. Oviparous P. przewalskii had an average lncRNA sequence length of 1702 bp, while that of mRNAs was 1823 bp (Mann–Whitney U test, p value < 0.001) (Figure 2A). LncRNAs averaged 2.58 exons and transcripts of mRNAs averaged 7.19 (Mann–Whitney U test, p value < 0.001) (Figure 2C). The expression level of lncRNAs transcripts was significantly lower than those of mRNAs. The FPKM values of average expression quantity were 2.11 and 42.95, respectively (Mann–Whitney U test, p value < 0.001) (Figure 2E). Similar results were obtained for P. vlangalii, whose sequence lengths averaged 1595 and 1768 bp for lncRNAs and mRNAs, respectively (Mann–Whitney U test, p value < 0.001) (Figure 2B). LncRNA averaged 2.57 exons, but protein‐coding gene transcripts averaged 6.84 (Mann–Whitney U test, p value < 0.001) (Figure 2D). Meanwhile, lncRNAs had an FPKM value of 3.37 for the average expression quantity, and this was significantly lower than that of mRNAs, which was 40.25 (Mann–Whitney U test, p value < 0.001) (Figure 2F). Overall, lncRNA showed the following characteristics compared with mRNAs: shorter sequence length, smaller exon number, and lower gene expression.

FIGURE 2.

FIGURE 2

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Comparison of characteristics between lncRNA and protein‐coding genes. Statistics of sequence length, the exon number, and expression level of mRNA and lncRNA in Phrynocephalus przewalskii (A, C, E) and P. vlangalii (B, D, F), respectively. Log‐transformed FPKM was used as the value of expression level in (E) and (F).

3.3. Orthologs of lncRNAs and Genes Between Two Species

We obtained 16451 pairs of orthologs based on 21 937 and 22 994 protein‐coding genes of two species, accounting for about 80% of all genes. However, no such high proportion of orthologs occurred in lncRNA due to the low conservation. We only accessed 1232 pairs of lncRNA one‐to‐one orthologs between 8989 lncRNAs in P. przewalskii and 7863 lncRNAs in P. vlangalii after applying the extremely strict threshold for screening and calculation. Therefore, 1232 pairs of lncRNA orthologs were considered evolutionarily conserved lncRNAs and used for subsequent species comparison analysis.

3.4. Cis‐regulation by lncRNAs on Genes

Because lncRNA tends to regulate the expression of neighboring protein‐coding genes by cis‐acting, we explored the role cis‐regulation plays in viviparity. Analyses identified protein‐coding genes located within 10 kb of lncRNAs. In oviparous P. przewalskii, 5113 protein‐coding genes were located in the upstream and downstream 10 kb ranges of 6099 lncRNAs. Results also identified 4612 protein‐coding genes within 10 kb of 5241 lncRNAs in viviparous P. vlangalii. Further, lncRNAs significantly correlated with the expression of adjacent protein‐coding genes (average Spearman r 2 = 0.54 in P. przewalskii, r 2 = 0.43 in P. vlangalii, p < 0.01), and similarly between adjacent protein‐coding genes (average Spearman r 2 = 0.57 in P. przewalskii, r 2 = 0.47 in P. vlangalii, p < 0.01).

Efforts focused on phenotypic traits associated with viviparous evolution and determined if lncRNAs acted on the associated genes in a cis‐regulating manner. We calculated the highly expressed genes (HEGs) of S1 when the eggshell gland develops. In P. przewalskii, 50 HEGs of S1 were located in the upstream or downstream 10 kb of 107 lncRNAs (Figure 3A; Figure S1). Gene Ontology (GO) enrichment analysis showed that the functions of these lncRNAs may involve anatomical structure maturation (GO:0071695), response to growth factor (GO:0070848), carboxylic acid biosynthetic process (GO:0046394), and developmental maturation (GO:0021700) (Figure 3C; Table S3), which correlate with eggshell gland development. These lncRNAs may promote the high expression of these protein‐coding genes in a cis‐regulated manner. Orthologs in P. vlangalii mostly differed and only 10 protein‐coding genes were located in the same range of 12 orthologous lncRNAs. Meanwhile, viviparous species lost 95 lncRNAs. As a result, genes that are highly expressed at S1 in oviparous lizards do not exhibit the same high expression pattern at S1 in viviparous species (Figure 3B; Figure S1).

FIGURE 3.

FIGURE 3

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Cis‐regulatory role of lncRNA in eggshell degeneration. The expression pattern of protein‐coding genes within 10 kb upstream or downstream of lncRNAs and specific highly expressed (HEGs) at S1 in Phrynocephalus przewalskii (A) and orthologous genes in P. vlangalii (B). The y‐axis values represent the Z‐score–normalized expression for each gene across different samples. (C) Bubble diagram shows the enrichment GO terms of S1 HEGs cis‐regulated by lncRNAs in P. przewalskii. (D) Expression levels of the lncRNA Przlnc_IT‐0714 across all reproductive stages. The error bar represents the standard error (SE). (E) The gene structure diagram shows the positional relationship between lncRNA Przlnc_IT‐0714 and the cis‐regulated gene FGFRL1.

Among these S1‐specific highly expressed genes, analyses identified FGFRL1, a member of the FGFR gene family, which plays a crucial role in a multitude of processes during cell differentiation, adhesion, and proliferation pathways. Analyses identified cis‐regulation by lncRNA Przlnc_IT‐0714, an 1872‐bp intronic lncRNA located in the intron region of FGFRL1 (Figure 3E). Przlnc_IT‐0714 also showed high expression specific for S1 (Figure 3D), but in viviparous species, FGFRL1 did not exhibit significantly high expression. Analyses did not locate any lncRNA within a 10‐kb window upstream and downstream. Further, intergenic lncRNA Przlnc_IG‐3108 was located 805 bp upstream of the highly expressed gene SHISA2 during S1, overlapping with its promoter region. This lncRNA also had a high expression in S1, like its target (Figure S3). The targeted gene is a modulator of both Wnt and Fgf signaling (Hedge and Mason 2008), which are vital to embryonic development and tissue morphogenesis. It may influence the formation of the eggshell gland by regulating cell proliferation, differentiation, and migration. Similarly, the presence of lncRNA Przlnc_IG‐1131 occurred in the promoter region of GALNT14, situated at 422 bp upstream of the gene. It has been reported to play a significant role in gland tumors such as the breast gland and prostate gland by participating in processes like cell migration and invasion (Tian et al. 2015; Czyrnik et al. 2023). Consistent with its cis‐target GALNT14, Przlnc_IG‐1131 also had high expression during S1 (Figure S3).

The emergence of placenta is another important structural transition in the evolution of viviparity. During the gestation in P. vlangalii, the placenta may begin to develop at stages 30–31 (S3; Gao et al. 2019). Therefore, we obtained a set of genes that were specifically highly expressed only in S3 through differential expression analysis. Results identified 103 high‐expressed genes (HEGs) located in the upstream and downstream 10‐kb range of 191 lncRNAs (Figure 4A; Figure S2A), but orthologs of the 103 HEGs located within 10 kb of only 27 orthologous lncRNAs and the corresponding result is that the high expression pattern of S3 did not appear in P. przewalskii (Figure 4B; Figure S2B). GO enrichment analysis (Figure 4C; Table S4) of the 103 HEGs showed that many of them were involved in the morphogenesis of an epithelium (GO:0002009), sterol transport (GO:0015918), and regulation of epithelial cell differentiation (GO:0030856), each of which associated with the remodeling of the uterine epithelium during placental formation. Additionally, the enriched pathways, including the Wnt signaling pathway, estrogen signaling pathway, and the EGF/EGFR signaling pathway (Figure 4D; Table S5), indicated that these genes may be involved in functions related to placental development.

FIGURE 4.

FIGURE 4

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Cis‐regulatory role of lncRNA in placenta evolution. The expression pattern of protein‐coding genes within 10 kb upstream or downstream of lncRNAs and specific highly expressed (HEGs) at S3 in Phrynocephalus vlangalii (A) and orthologous genes in P. przewalskii (B). The y‐axis values represent the Z‐score normalized expression for each gene across different samples. Enrichment GO terms (C) and pathways (D) of S3 HEGs cis‐regulated by lncRNAs in P. vlangalii. LncRNAs marked with “*” are absent in oviparity. (E) The gene structure diagram shows the positional relationship between lncRNA Vlalnc_IG‐1300 and the cis‐regulated gene GLIS3.

ESR2, which is highly expressed during S3, has been found to be cis‐regulated by the lncRNA Vlalnc_IG‐2591, located in the upstream promoter region of ESR2. This lncRNA also showed relatively high expression during S3 (Figure S3). However, ESR2 exhibits significantly lower expression during S3 in oviparous species, and no lncRNAs were detected within a 10‐kb range upstream or downstream of ESR2.

GLIS3 (GLI‐similar 3) is another S3 highly expressed gene in P. vlangalii. Our results showed that this gene is regulated by lncRNA Vlalnc_IG‐1300, an intergenic lncRNA located approximately 3.7 kb upstream of GLIS3 (Figure 4E). Similarly, Vlalnc_IG‐1300 also showed significantly high expression at S3 (Figure S3). When we checked the expression of this gene in oviparous species, it was found to be down‐regulated at S3, and the orthologs of Vlalnc_IG‐1300 were also not found.

Additionally, we identified several genes involved in immune regulation that are subject to cis‐regulation by lncRNAs. In viviparous lizards, HLA‐DRB1, a key molecule essential for antigen presentation, plays a crucial role in immune responses (Arango et al. 2017). Its downregulation during pregnancy helps mitigate maternal immune stress toward the embryo. Notably, we observed that HLA‐DRB1 is regulated in cis by the lncRNA Vlalnc_OP‐0518, located 9 kb upstream of the gene (Figure S4C). Vlalnc_OP‐0518 exhibited specific high expression during the S1 stage, followed by a gradual decline throughout pregnancy. Consistently, HLA‐DRB1 also showed peak expression during S1, with a significant downregulation in later stages (Figure S4A). In contrast, no lncRNAs were detected in the upstream or downstream regions of HLA‐DRB1 in oviparous lizards, and the gene did not exhibit stage‐specific expression patterns across different developmental stages (Figure S4B).

3.5. Trans‐regulated co‐expression Networks

In addition to cis‐regulation on neighboring genes, lncRNAs can also regulate the expression of protein‐coding genes in distant locations through trans‐regulation. To understand the role trans‐regulated lncRNAs play in protein‐coding genes, we constructed co‐expression networks between lncRNAs and protein‐coding genes of the two species by WGCNA.

Expression data for the oviparous lizard involved all 21 937 protein‐coding gene transcripts and 8989 lncRNAs from 20 oviduct tissue samples from four time periods as input files to construct a co‐expression network. Analyses obtained 25 distinctly expressed modules (Figures S5 and S6). Among these, module epigengene in blue (MEblue) significantly associated with S1 (Figure 5A; Figure S6; cor = 0.67, p value = 0.001) when the eggshell gland developed. This led to investigating the roles lncRNAs played in the corresponding traits. The module had 3800 mRNAs and 546 lncRNAs. Gene significance (GS) was highly correlated with module membership (MM) (Figure S8), illustrating a strong degree of confidence between the module and transcripts of S1 (cor = 0.42, p value = 2.3 × 10−185). To screen genes most strongly associated with S1, we amplified the threshold for filtering hub genes (|GS|> 0.6 & |MM|> 0.8). Subsequently, we selected hub genes in the top 1000 relationships of weight value and visualized them using Cytoscape. The network included seven lncRNAs and 251 protein‐coding gene transcripts, which represented transcripts with the most relevant expressions in this module (Figure 5B; Table S6).

FIGURE 5.

FIGURE 5

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Trans‐regulatory role of lncRNA in eggshell degeneration. (A) Module–period relationships in Phrynocephalus przewalskii according to the WGCNA analysis. The color indicates the respective correlation coefficient, as shown in the scale bar. MEblue is the module with the highest correlation to S1. (B) Co‐expression network of hub genes and lncRNAs in P. przewalskii. The dots in the outer circle represent coding genes, and the genes in the top 30 of connectivity are highlighted. Triangles in the inner circle represent lncRNA. LncRNAs marked in red are absent in viviparity. (C) Enrichment GO term of genes in the co‐expression network.

Functional enrichment of target genes was used to investigate lncRNAs. GO analysis showed that most of the 255 genes involved morphogenesis and were mainly divided into several broad biological process categories: (1) epithelial‐related biological processes, including morphogenesis of an epithelium (GO:0002009); epithelial tube morphogenesis (GO:0060562); epithelial cell development, migration, and proliferation (GO:0002064, GO:0010631, GO0050673); (2) gland‐related biological processes including gland morphogenesis (GO:0022612), gland development (GO:0048732), and mammary gland duct morphogenesis (GO:0060603); and (3) other growth and development related process, such as responses to growth factor (GO:0070848), tissue morphogenesis (GO:0048729), and tissue migration (GO0090130) (Figure 5C; Table S8). These related GO terms suggested that lncRNAs are highly relevant to the development of the eggshell gland.

Analyses investigated the expression of seven hub lncRNAs played in viviparous lizards. Four hub lncRNA orthologs were not found, and ortholog Vlalnc_ME‐0034 had no expression in P. vlangalii. Only two of seven hub lncRNA orthologs exhibited an expression pattern consistent with that observed in oviparity, demonstrating elevated expression levels in S1. Nevertheless, the trans‐regulatory network exhibited the absence and non‐expression of a large number of hub lncRNAs that affect the expression of important coding genes involved in eggshell gland development throughout the oviparity regulatory network. Ultimately, this led to phenotypic differences in viviparity.

The trans‐regulatory network of placental development focused on S3 in P. vlangalii. It involved co‐expression analyses for all 22 994 protein‐coding gene transcripts and 7863 lncRNAs from 30 oviduct tissue samples of six periods. The result identified 40 distinctly expressed modules of which a high correlation existed for 4949 mRNAs and 825 lncRNAs in MEturquoise at S3 (Figure S7; Figure 6A). Scatter plots showed GS significantly correlated with MM (Figure S8). A similarly constructed placental trait network for trans‐regulatory developmental traits contained 200 protein‐coding gene transcripts and 29 lncRNAs (Figure 6B; Table S7).

FIGURE 6.

FIGURE 6

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Trans‐regulation of placenta development traits by lncRNA. (A) Module–period relationships in Phrynocephalus vlangalii according to the WGCNA analysis. The color indicates the respective correlation coefficient, as shown in the scale bar. MEturquoise is the module with the highest correlation to S3. (B) Co‐expression network of hub genes and lncRNAs in P. vlangalii. The dots in the outer circle represent coding genes, and the genes in the top 30 of connectivity are highlighted. Triangles in the inner circle represent lncRNA. LncRNAs marked in red are absent in oviparity. (C) Enrichment GO term of genes in the co‐expression network. (D–F) Comparison of expression patterns of lncRNA Vlalnc_IG‐2095, Vlalnc_IT‐0133, and Vlalnc_IT‐0429 across the reproductive process in P. vlangalii and their orthologs in P. przewalskii.

The GO enrichment results for 200 target genes revealed that many functions directly or indirectly affected epithelial cell remodeling and embryo attachment, which is crucial in placental development. These included epithelial cell proliferation (GO:0050673), epithelial cell differentiation (GO:0030855), regulation of epithelial cell migration (GO:0010632), morphogenesis of an epithelium (GO:0002009), cell junction organization (GO:0034330), and uterus development (GO:0060065) (Figure 6C; Table S10). Among these, uterus development (GO:0060065) had the highest enrichment scores. Further, the enrichment of the TGF‐beta signaling pathway may have indicated the regulation of growth hormone in uterus remodeling, and the VEGFA‐VEGFR2 signaling pathway may have indicated angiogenesis and regulation in placental development (Table S11). Further, we observed the presence of ESR1, LRP6, IGF1R, and CELSR1 in the trans‐regulatory network of placental development. These genes have previously been identified as crucial for uterine remodeling and embryo adhesion during the placental development process (Brandley et al. 2012; Gao et al. 2019).

The analysis further examined how these 29 hub lncRNAs within the regulatory network of viviparous placental traits are presented in oviparous lizards. Results showed 25 hub lncRNA orthologs were absent, while the remaining lncRNA orthologs, Przlnc_IT‐0313, Przlnc_IT‐0284, and Przlnc_OE‐0008, had low expression during S3 in P. przewalskii (Figure 6D–F). Therefore, the large‐scale loss or low expression of central lncRNAs may lead to the lack of high expression of placental development‐related genes in oviparous species during this period, resulting in phenotypic differences from viviparous species.

4. Discussion

4.1. Characteristics and Regulation Modes of lncRNA

The characteristics of lncRNAs vary widely among vertebrates. In zebrafish, a significant proportion of newly identified lncRNAs (45%) ranged from 400 to 700 bp in length, while 16% exceeded 1000 bp (Banerjee et al. 2021). Similarly, in bullfrogs, lncRNAs varied in length from 200 bp to nearly 11 kb, with a median length of 973 bp (Hammond et al. 2017). In birds, a study on lncRNAs in chicken intramuscular preadipocytes found that their sequence lengths predominantly ranged between 200 and 400 bp, making them shorter than mRNAs (Zhang et al. 2017). Research on lncRNAs in mammals has been more extensive. In domesticated pigs, newly identified lncRNAs were generally shorter than mRNAs and contained fewer exons. However, their average exon length (451 bp) was longer than that of mRNAs (221 bp), and their overall expression levels were significantly lower (Zhou et al. 2014). Meanwhile, in yaks, most novel lncRNAs were located in intergenic regions, accounting for over 60% of the total (Wang et al. 2020). The lncRNAs newly identified in the two lizard species exhibited similar characteristics compared to those found in other vertebrates. About half of them had sequence lengths between 200 and 1000 bp, and over 60% were located in previously unannotated intergenic regions. These findings align with the common features of lncRNAs observed across vertebrates, including shorter sequence lengths, fewer exons, and lower expression levels than mRNAs. This consistency reflects differences in their biogenesis, processing, stability, and spatiotemporal expression patterns (Zhou et al. 2014).

Because lncRNAs usually act on target genes in cis or trans‐regulators (Kopp and Mendell 2018; Yan et al. 2017), we explore these underlying mechanisms of lncRNA to eggshell degeneration and placental emergence. Previous studies found that lncRNAs exert cis‐regulatory effects mainly on adjacent protein‐coding genes (Ma et al. 2013; Wang et al. 2011). In our lizards, lncRNAs significantly correlated with the expression of adjacent protein‐coding genes and similarly between adjacent protein‐coding genes, which is consistent with the study of (Zhou et al. 2014). Analysis of the correlation between the expression of lncRNAs and protein‐coding genes reveals a positive correlation between most lncRNA and protein‐coding gene pairs (65% pairs in P. przewalskii; 64% pairs in P. vlangalii). Thus, lncRNAs and protein‐coding genes share cis‐regulatory modules or chromatin domains, usually with positive associations.

4.2. Regulation of lncRNA on Eggshell Degeneration

Generally, oviparous lizards lay a calcified eggshell, which is composed of a layer of fibrous protein covered by calcium carbonate. Shortly following ovulation, the uterine eggshell glands secrete a substantial amount of fibrous protein, creating a thick, cushion‐like structure. Subsequently, calcium carbonate crystals envelop the surface of the fibrous protein, forming the eggshell (Packard and DeMarco 1991). Nearly all viviparous species exhibit loss or significant reduction in eggshell thickness (Guillette 1987), which is driven by degeneration or reduction of uterine eggshell glands (Albergotti and Guillette 2011). The eggshell glands of oviparous squamates exhibit seasonal changes influenced by the reproductive cycle, with their development primarily occurring during vitellogenesis, that is, the ovarian egg stage. They play a role shortly after the ovarian eggs are released into the oviduct and then gradually degenerate over time (Girling 2002; Heulin et al. 2005).

During the development of eggshell glands in S1, 107 cis‐regulated lncRNAs were located near 50 highly expressed genes (HEGs) in the oviparous lizard. In contrast, the viviparous lizard has a substantial absence of these lncRNAs, leading to the lack of a pattern where genes exhibit high expression levels during S1. Thus, these lncRNAs likely play an important positive regulatory role in the development of eggshell glands. For instance, Prz_IT‐0714 is located within the intronic region of the FGFRL1, and both exhibit a consistent, stage‐specific high expression in S1. FGFRL1 is involved in the FGF signaling pathway, which plays a crucial role in cell differentiation, migration, and tissue development. Notably, the FGF signaling pathway is essential for glandular development (Sumbal and Koledova 2019). Przlnc_IG‐1131 and Przlnc_IG‐3108 occur in the promoter regions of GALNT14 and SHISA2, respectively. Their high expression levels can promote transcriptional activation and elevated expression of their downstream cis‐target genes. Additionally, transcription of lncRNAs in the promoter region may induce chromatin remodeling, thereby activating the transcription of downstream protein‐coding genes. For instance, lncRNA at the promoter of FBP1 can activate its downstream transcription (Hirota et al. 2008). Meanwhile, cis‐regulated lncRNAs may also recruit transcription factors or chromatin modification complexes, contributing to the development of eggshell glands and the formation of eggshells in oviparous species. During the evolution of viviparity, lncRNAs near these protein‐coding genes appear to be lost gradually on a large scale, thus affecting the transcription of protein‐coding genes. Regardless, these ultimately drive the development of highly expressed genes in the eggshell glands to be inhibited, resulting in the disappearance of the eggshell in viviparity.

Trans‐regulation controls gene expression at a distance through various mechanisms that differ from cis‐regulation, which primarily influences the chromatin state and transcriptional activity of nearby genes. Thus, our trans‐regulatory network includes a greater number of genes than for cis‐translation, and the network for eggshell degeneration consists of 255 protein‐coding genes and 7 lncRNAs. Enrichment occurs in functions involved in epithelial cell differentiation and proliferation, as well as glandular development. The enriched signaling pathways involve several crucial growth and development processes (Table S9). For instance, the estrogen signaling pathway attracted our interest. Because estrogen plays a crucial role in oviduct growth and wall thickening. Previous experiments proved the number and size of eggshell glands significantly increased when the oviduct of reptiles was subjected to exogenous estrogen 17β‐estradiol (E2) (Albergotti and Guillette 2011; Girling 2002). The EGF/EGFR signaling pathway regulates several developmental processes, including proliferation, migration, differentiation, apoptosis, and intercellular communication during development (Wee and Wang 2017). Our analyses also detect enrichment in insulin receptor signaling pathways. The transformation of the expression patterns of estrogen receptor (ESR) and its downstream insulin‐like growth factor (IGF‐1) and epithelial growth factor (EGF) between oviparous and viviparous lizards was shown to drive the regression of the eggshell gland, which may contribute to the disappearance of the eggshell in P. vlangalii (Gao et al. 2019). Therefore, the functional enrichment of these genes is consistently associated with eggshell gland development.

Among the seven lncRNAs, only two still maintain high expression patterns similar to those in oviparous species, while the rest are either absent or expressed at low levels in viviparous species. This indicates that the large‐scale absence and non‐expression of core lncRNAs in the regulatory network affects gene expression involved in eggshell development, further leading to eggshell loss in viviparous species.

4.3. Regulation of lncRNA on Placenta Development

LncRNAs play a crucial role in placental development and functional regulation in mammals. Numerous studies have revealed their involvement in regulating gene expression at the embryo‐maternal interface, modulating trophoblast cell function, and maintaining placental homeostasis (Monteiro et al. 2021; McAninch et al. 2017). For example, H19, a highly conserved imprinted lncRNA, is highly expressed in the cytotrophoblasts and extravillous trophoblasts of the placenta and potentially controls trophoblast cell fate decisions during placenta development (Adu‐Gyamfi et al. 2024). Additionally, lncRNA MEG3 (maternally expressed gene 3) is expressed in the placenta and has been implicated in the proliferation and invasion of trophoblast cells (Liang et al. 2022).

Squamate placentas develop from extraembryonic membranes that grow and transform continuously throughout embryonic development to meet varying demands or developmental constraints. Consequently, different extraembryonic membranes give rise to different types of placentas at various locations (Stewart and Blackburn 1988; Stewart and Thompson 2003). The development of the placenta occurs during the mid‐stage of gestation when the embryo has reached approximately stages 30–31 (S3) (Adams et al. 2007).

Analyses detected 103 specific highly expressed protein‐coding genes at the placenta development (S3) were located in the 10‐kb upstream or downstream range of 191 lncRNAs in viviparous species. The functions of these highly expressed genes may be crucial for uterine development and embryo implantation in fetal lizards. Notably, among the lncRNA cis‐regulated target genes, we identified GLIS3, a zinc finger transcription factor that plays a crucial role in the development and function of various organs. Studies have shown that GLIS3 is highly expressed in the placenta and has been reported as a candidate imprinted gene in the human placenta (Barbaux et al. 2012).

The Wnt signaling pathway was reported to promote early placental trophoblast development, blastocyst activation, implantation, and chorion‐allantois fusion in mice (Sonderegger et al. 2010). In the estrogen signaling pathway, the placenta produces a major pregnancy hormone. It was found to play an important role in regulating the expression of vascular endothelial growth factor (VEGF) and angiogenesis in the uterus and placenta during the reproductive cycle (Albrecht and Pepe 2010; Park et al. 2018). The two genes encoding estrogen receptors, ESR1 and ESR2, along with downstream growth factors such as IGF1R, likely play critical roles in inducing uterine changes during placental formation (Gao et al. 2019). Our results indicate that ESR2 is cis‐regulated by lncRNA, while ESR1 is trans‐regulated by lncRNA, further highlighting the regulatory impact of lncRNAs on key genes in the placental formation process.

Further, functional enrichment of all target genes in the trans‐regulatory network involves epithelial tissue migration, morphology, and uterine development, among others. During the enrichment analysis, results show significant enrichment of the mTOR signaling pathway. A study of the human placenta found that mTOR‐mediated signaling plays an important role as a signaling sensor in trophoblastic cell proliferation and differentiation in placental development (Wen et al. 2005). Functional genes trans‐regulated by lncRNAs dominate during the development of the placenta. Many genes previously reported to be crucial for placental development, such as ESR1, LRP6, IGF1R, and CELSR1, occur in our trans‐regulatory network. Specifically, IGF1R is a key regulatory factor in mammalian placental development. By mediating the effects of IGF‐1, it promotes trophoblast proliferation and invasiveness, thereby supporting placental growth and development (Sferruzzi‐Perri et al. 2017; Bowman et al. 2010). LRP6 is widely expressed in placental tissue and plays a crucial role in the survival and invasion of trophoblast cells, which actively regulates maternal artery remodeling through vasculogenesis and trophoblast invasion (Li et al. 2019).

The regulatory networks of placental development reveal that the lncRNAs targeting functional genes exhibit a large number of deletions in the contrasting species. Most of the remaining lncRNAs also show low expression. The extensive trans‐regulation of lncRNAs can bind enhancers or promoters and some trans‐regulatory factors to affect gene expression. They can also play a role in scaffolding, histone protein complex exercise function, and subcellular structure, as well as in transcriptional activation of target genes, and promoting the expression of target genes (Kopp and Mendell 2018). When missing or have low expression, they inhibit target genes. Hub lncRNAs play an important regulatory role in the trans‐regulatory network for target genes, and their absence and low expression in the contrasting species leads to the downregulation of expression in target genes. In turn, lncRNAs play a decisive role in dominant traits related to the evolution of viviparity.

4.4. Limitations in this Study

A key limitation of this study is the lack of functional validation of the identified lncRNAs. While our findings suggest their regulatory roles in genes involved in eggshell gland development and placentation, experimental evidence is lacking due to the absence of genetic manipulation tools for reptiles, such as RNA interference or CRISPR‐based approaches. Additionally, the lack of in vitro models, such as cell lines or organoids, further restricts functional assays. As genetic tools for non‐model reptiles advance, functional assays such as lncRNA knockdown or overexpression in primary cells or organoids could provide direct validation in the future.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1 The gene expression heatmap shows high expression of genes regulated by lncRNA in cis at stage S1 in oviparous P. przewalskii (A), while the orthologs of these genes didn't exhibit high expression at stage S1 in the viviparous P. vlangalii (B).

Figure S2 The gene expression heatmap shows high expression of genes regulated by lncRNA in cis at stage S3 in viviparous P. vlangalii (A), while the orthologs of these genes do not exhibit high expression during stage S3 in the oviparous P. przewalskii (B).

Figure S3 The expression levels of the cis‐regulatory lncRNAs Przlnc_IG‐1131 (A) and Przlnc_IG‐3108 (B) for eggshell traits across stages S1‐S4, and Vlalnc_IG‐1300(C) and Vlalnc_IG‐2591 (D) for placental traits across stages S1‐S6 are shown. The Y‐axis represents the corrected log2 (FPKM + 1) values.

Figure S4 Immunoregulatory role of lncRNAs in viviparity.

Figure S5 Cluster Dendrogram of WGCNA trans‐regulatory network in P. przewalskii (A) and P. vlangalii (B)

Figure S6 In various stages of P. przewalskii, the module‐trait relationships across all samples show that during its S1 stage, MEblue exhibits the most significant relationship.

Figure S7 In various stages of P. vlangalii, the module‐trait relationships across all samples show that during its S3 stage, MEturquoise exhibits the most significant relationship.

Figure S8 The scatter plot represents the correlation between module membership and gene significance.

Table S1 Classification of novel lncRNAs in P. przewalskii

Table S2 Classification of novel lncRNAs in P. vlangalii

Table S3 GO terms of cis‐regulated genes in S1 of P. przewalskii

Table S4 GO terms of cis‐regulated genes in S3 of P. vlangalii

Table S5 Pathways of cis‐regulated genes in S3 of P. vlangalii

Table S6 Genes and lncRNAs in network of trans‐regulatory in P. przewalskii

Table S7 Genes and lncRNAs in network of trans‐regulatory in P. vlangalii

Table S8 GO terms of trans‐regulated genes in S1 of P. przewalskii

Table S9 Pathways of trans‐regulated genes in S1 of P. przewalskii

Table S10 GO terms of trans‐regulated genes in S3 of P. vlangalii

Table S11 Pathways of trans‐regulated genes in S3 of P. vlangalii

INZ2-21-1300-s001.pdf (1.4MB, pdf)

Acknowledgments

We thank Chuanxin Yu and Alex Plimo Karuno for their helpful comments. We thank for the support of Yunnan Provincial Science and Technology Project at Southwest United Graduate School (202302AO370006), the National Natural Science Foundation of China (32100341), the Second Tibetan Plateau Scientific Expedition and Research Program (STEP, Grant No. 2019QZKK0501), the Animal Branch of the Germplasm Bank of Wild Species, Chinese Academy of Sciences (the Large Research Infrastructure Funding), the Yunnan Revitalization Talent Support Program Yunling Scholar Project to Jing Che, and the Yunnan Revitalization Talent Support Program Young Talent Project to Wei Gao.

Feng, X. , Gao W., Dong W., et al. 2026. “Genome‐Wide Mining of lncRNAs Reveals Their Potential Regulatory Role in the Evolution of Viviparity.” Integrative Zoology 21, no. 5: 1300–1313. 10.1111/1749-4877.12992

Funding: This work was supported by Yunnan Provincial Science and Technology Project at Southwest United Graduate School (202302AO370006), the National Natural Science Foundation of China (32100341), the Second Tibetan Plateau Scientific Expedition and Research Program (STEP, Grant No. 2019QZKK0501), the Animal Branch of the Germplasm Bank of Wild Species, Chinese Academy of Sciences (the Large Research Infrastructure Funding), the Yunnan Revitalization Talent Support Program Yunling Scholar Project to Jing Che, and the Yunnan Revitalization Talent Support Program Young Talent Project to Wei Gao.

Data Availability Statement

Transcriptome data were obtained from the Genome Sequence Achieve (https://ngdc.cncb.ac.cn/gsa/) with accession No. CRA001096. The newly annotated lncRNA GTF files have been deposited in the Figshare database (https://figshare.com/projects/lncRNA_data_of_viviparity_project/212627).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1 The gene expression heatmap shows high expression of genes regulated by lncRNA in cis at stage S1 in oviparous P. przewalskii (A), while the orthologs of these genes didn't exhibit high expression at stage S1 in the viviparous P. vlangalii (B).

Figure S2 The gene expression heatmap shows high expression of genes regulated by lncRNA in cis at stage S3 in viviparous P. vlangalii (A), while the orthologs of these genes do not exhibit high expression during stage S3 in the oviparous P. przewalskii (B).

Figure S3 The expression levels of the cis‐regulatory lncRNAs Przlnc_IG‐1131 (A) and Przlnc_IG‐3108 (B) for eggshell traits across stages S1‐S4, and Vlalnc_IG‐1300(C) and Vlalnc_IG‐2591 (D) for placental traits across stages S1‐S6 are shown. The Y‐axis represents the corrected log2 (FPKM + 1) values.

Figure S4 Immunoregulatory role of lncRNAs in viviparity.

Figure S5 Cluster Dendrogram of WGCNA trans‐regulatory network in P. przewalskii (A) and P. vlangalii (B)

Figure S6 In various stages of P. przewalskii, the module‐trait relationships across all samples show that during its S1 stage, MEblue exhibits the most significant relationship.

Figure S7 In various stages of P. vlangalii, the module‐trait relationships across all samples show that during its S3 stage, MEturquoise exhibits the most significant relationship.

Figure S8 The scatter plot represents the correlation between module membership and gene significance.

Table S1 Classification of novel lncRNAs in P. przewalskii

Table S2 Classification of novel lncRNAs in P. vlangalii

Table S3 GO terms of cis‐regulated genes in S1 of P. przewalskii

Table S4 GO terms of cis‐regulated genes in S3 of P. vlangalii

Table S5 Pathways of cis‐regulated genes in S3 of P. vlangalii

Table S6 Genes and lncRNAs in network of trans‐regulatory in P. przewalskii

Table S7 Genes and lncRNAs in network of trans‐regulatory in P. vlangalii

Table S8 GO terms of trans‐regulated genes in S1 of P. przewalskii

Table S9 Pathways of trans‐regulated genes in S1 of P. przewalskii

Table S10 GO terms of trans‐regulated genes in S3 of P. vlangalii

Table S11 Pathways of trans‐regulated genes in S3 of P. vlangalii

INZ2-21-1300-s001.pdf (1.4MB, pdf)

Data Availability Statement

Transcriptome data were obtained from the Genome Sequence Achieve (https://ngdc.cncb.ac.cn/gsa/) with accession No. CRA001096. The newly annotated lncRNA GTF files have been deposited in the Figshare database (https://figshare.com/projects/lncRNA_data_of_viviparity_project/212627).

Articles from Integrative Zoology are provided here courtesy of Wiley

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