Next Article in Journal
Liverworts of the South Kamchatka Nature Park: Survival in Active Volcanism Land
Previous Article in Journal
Biodiversity and Conservation of Vascular Flora: A Challenge for the Survival of Humans on Earth
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Diversity
Volume 14
Issue 9
10.3390/d14090721
diversity-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Composition Diversity and Expression Specificity of the TPS Gene Family among 24 Ficus Species

by
Peng Sun
1,
Xiaoyong Chen
2,
Bhanumas Chantarasuwan
3,
Xueying Zhu
1,
Xiaoxia Deng
4,
Ying Bao
1,* and
Hui Yu
1,4,*
1
School of Life Sciences, Qufu Normal University, Qufu 273165, China
2
School of Ecological and Environmental Sciences, Tiantong National Station for Forest Ecosystem Research, East China Normal University, Shanghai 200062, China
3
Thailand Natural History Museum, National Science Museum, Khlong Luang 12120, Ptthum Thani, Thailand
4
Key Laboratory of Plant Resource Conservation and Sustainable Utilization, South China Botanical Garden, The Chinese Academy of Sciences, Guangzhou 510650, China
*
Authors to whom correspondence should be addressed.
Diversity 2022, 14(9), 721; https://doi.org/10.3390/d14090721
Submission received: 8 August 2022 / Revised: 28 August 2022 / Accepted: 29 August 2022 / Published: 1 September 2022
Browse Figures
Review Reports Versions Notes

Abstract

:
Volatile organic compounds (VOCs) released by the receptive syconia of Ficus species play a vital role in attracting highly species-specific pollinating fig wasps. The components of VOCs vary considerably among Ficus species, but are generally dominated by a few common terpenoid compounds or specific proportions of several compounds. Terpene synthase (TPS) is the main source of specific and diverse terpenoids, but the evolution of the TPS gene family in Ficus and the potential functions of the TPS genes in species-specific pollination remain largely unelucidated. In this study, using transcriptomes of ostiole bracts of receptive male figs from 24 Ficus species collected from South China and Southeast Asia, we comprehensively scanned and investigated the composition and evolutionary characteristics of all TPS genes in all 24 species. We identified 248 TPS genes, including 33 orthologous genes and six singletons. Sequence and phylogenetic analysis showed that a majority of the 248 TPSs contained the DDXXD and DTE motifs, rather than the DXDD motif, and involved all subfamilies (TPS-a,b,c,e/f and g) known in other angiosperm genomes, suggesting a very diverse and complex composition of class I TPSs during the receptive phase. In addition, compared to TPS-a, which is generally the largest subfamily in some plants, the TPS-b subfamily contained the highest number of genes in Ficus species. Expression profile comparison showed that the distribution and expression levels of different TPSs among different Ficus species differed considerably, but a few TPS genes were common across most species. Positive selection analysis showed that the Ficus TPS genes were mainly under purifying selection, with only four genes having positive selection signals and two genes having positive selection sites, and two genes having relatively fast-evolving rates. The present study demonstrates the basic evolutionary characteristics of TPS genes in Ficus and reveals the roles of TPSs in shaping the diversity and specificity of the fig–fig wasp symbiotic relationship.

Graphical Abstract

1. Introduction

The interaction between plants and pollinating insects is considered to be an important driving force for the biodiversity and evolution of angiosperms in the tropics [1,2]. While the polymorphism of floral characteristics helps cater to diverse or specific flower visitors, the quantity and quality of pollen transmitted by flower visitors affect the reproductive success of plants. In nature, most of the interactions between plants and pollinators are generalized, except for a few highly obligate mutualisms, such as fig–fig wasp and yucca–yucca moth.
Ficus, which belongs to the Moraceae family, constitutes one of the largest genera of angiosperms with approximately 750 species, including trees, shrubs, epiphytic vines, and other life forms [3,4]. The most typical feature of Ficus is its unique enclosed inflorescence commonly known as a syconium or fig. A syconium contains hundreds or thousands of tiny flowers and connects to the external environment via a narrow ostiole at its top. The opening of the ostiole is controlled by bracts, which are the main parts of Ficus that emit volatile organic compounds (VOCs) in receptive-phase syconia to attract obligate pollinators [5,6]. Only morphologically specialized fig wasps can enter the syconium and pollinate the female flowers within [7,8]. A species of Ficus can only attract and drive one or a few fig wasp species to pollinate [9]. The key components that attract specific pollinating wasps are different and generally include one or a few common terpenoid compounds or specific proportions of several compounds [10]. These key components strongly attract specific pollinator wasps, but not the wasps that pollinate other Ficus species [11]. Even for the same Ficus species, the specific pollinating wasps attracted by syconia in the receptive phase may be rejected by those in other developmental stages. This “push and pull” mechanism maintains the specific symbiotic relationship between pollinating wasps and their host plants [12,13,14].
VOCs include terpenoids, fatty acids derivatives, phenylpropanol/benzene ring compounds, and amino acid derivatives. Terpenoids represent the largest and most diverse class of chemicals, among the myriad of compounds produced by plants [15]. In Ficus, terpenoids provide key olfactory signals that attract pollinating wasps [10,16]. Terpenoids can be classified into monoterpenes (including 10 carbons), sesquiterpenes (including 15 carbons), diterpenes (including 20 carbons), and triterpenes (including 30 carbons). Most monoterpenes and sesquiterpenes are oily liquids that become volatile at room temperature and have special odors [17]. The synthesis of plant terpenoids generally occurs in three stages: (1) isopentenyl diphosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) are synthesized through the mevalonate (MVA) pathway in the cytoplasm and the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway in the plastid, respectively; (2) under the action of allyl transferase, geranyl diphosphate (GPP), farnesyl diphosphate (FPP), geranylgeranyl diphosphate (GGPP), and intermediates with various molecular weights are formed; (3) GPP, FPP, and GGPP, under the action of various terpene synthases (TPSs), generate monoterpenes, sesquiterpenes, diterpenes, and their derivatives (Figure S1). Finally, the modified enzymes catalyze hydroxylation, methylation, isomerization, and reduction to further produce terpene alcohols, acids, esters, and other derivatives [15,18].
The TPS gene family exists widely in plant genomes, most of which are involved in the production of secondary metabolites [19,20]. At the protein level, each TPS enzyme has two conserved domains, Pfam ID PF01397 and PF03936, distributed at the N- and C-terminals. On the basis of their related catalytic motifs, TPSs can be divided into classes I and II [21,22,23]. Class I TPSs, including all monoterpenes, all sesquiterpenes, and some diterpenes, contain two conserved DDXXD and NSE/DTE motifs. Class II TPSs, which only include diterpenes, contain only a conserved DXDD motif. In addition, the N-terminal domain of plant TPSs usually has a conserved R(R)X8W motif [21]. The second R of the motif R(R)X8W is not conserved, and some cases even feature the absence of the R(R)X8W motif [24].
At present, genome-wide comparative analysis of the evolutionary characteristics of the TPS gene family has been performed in more than 50 species [22,25]. Meanwhile, the number of plant species for which TPS gene family information has been obtained, on the basis of individual genome screening, is increasing [9,23,26,27,28]. Previous studies showed that the plant TPS gene family can be divided into seven subfamilies, namely, TPS-a, TPS-b, TPS-c, TPS-d, TPS-e/f, TPS-g, and TPS-h [21,22]. These subfamilies are specific for angiosperms, gymnosperms, land plants, vascular plants, and Selaginella moellendorffii Hieron, respectively [21]. TPS-a, TPS-b, and TPS-g encode proteins that produce sesquiterpenes and monoterpenes, respectively.
Owing to the importance of terpenoids in plant growth, chemical interactions, and protection in the environment [15], the cloning, expression, and functional characterization of TPS genes have also been completed in many plant species [16,19,26,28,29,30]. For example, He et al. [28] demonstrated the dynamic regulation of TPS genes in the emission of herbivore-induced plant volatiles after studying the expression and promoter activity of cucumber TPS genes in response to different herbivores. Recently, eight TPS genes from F. carica L. were isolated and confirmed to play a key role in their defense against pests during syconium development by triggering diverse and dynamic changes in volatile emissions [16].
Although numerous studies have provided an important basis for understanding the functions and contributions of TPS genes, existing studies only focused on a few species and paid more attention to plant protection. In this study, to reveal the TPS diversity of Ficus and the evolutionary characteristics of the special gene family, we investigated the TPS genes in 24 Ficus species using RNA-seq. We aimed to (1) identify the potential TPS genes that function in Ficus pollination attraction, (2) determine the gene structures, phylogenetic relationships, and expression divergence of the TPS genes among different Ficus species, and (3) evaluate the probable effects of positive selection on the Ficus TPS genes. The findings of this study could lead to an improved understanding of the production mechanism of VOCs in Ficus and, most importantly, may shed light on the evolution of the TPS gene family across the genus.

2. Results

2.1. Characteristics of the TPS Genes in the Receptive Phase of Male Figs of 24 Ficus Species

After RNA-sequencing, we obtained 41.52–56.03 million raw reads from the ostiole bract transcriptomes of 24 Ficus species. For different species, we achieved 40.17–55.09 million clean reads after adapter clipping and quality control. Using the Trinity program, the clean reads of each species were assembled into 34,454–90,120 transcripts. The high-quality transcripts of the 24 species were subjected to cluster and assembly analyses, resulting in coding of 18,344–68,322 unigenes, N50 lengths of 877–1970 bp, and GC contents of 43.77–52.24%. All assembly statistics are summarized in Table S1.
Using blast scanning, a total of 248 TPS genes with two conserved N-terminal and C-terminal domains (PF01397 and PF03936) were identified from the unigenes of the 24 ostiole bract transcriptomes (Table S2). Among the genes, 180 TPSs were found to have complete coding sequences, and 68 were found to have incomplete coding regions (Table S2). All TPS genes containing a typical Terpene_Synth_C (or Terpene_cyclase_plant_C1) conserved domain were confirmed after revalidation by the NCBI Online Batch CD-Search. At the protein level, those complete TPS genes showed an obvious sequence length polymorphism ranging from 405 aa in F. variolosa to 850 aa in F. microcarpa (Table S2). Different numbers of TPS genes were found to be expressed during the receptive phase of male figs. Among the 24 Ficus species. F. altissima showed the most TPS genes (19), while F. fistulosa and F. montana showed the fewest (3) (Table 1).
A total of 10 motifs were extracted from the 248 TPS genes, but three (DDXXD, DTE, and RXR) showed the highest degree of conservation. Generally, these TPS genes contained DDXXD and DTE motifs at the C-terminus (Figure 1A).
We classified 248 TPSs into 33 orthologous genes (FTPS1–FTPS33) and six singletons (FTPS34–FTPS39) using OrthoMCL clustering analysis and by manually checking the sequence similarity (Figure 1B, Table S2). Subsequently, we annotated these genes using the nonredundant protein database (Table S3). Consequently, we found that these FTPSs were probably matched to 18 known terpene synthases and their analogs, including frequently occurring myrcene synthase, (−)-germacrene D synthase, linalool synthase, and E-beta-caryophyllene synthase. Generally, these expressed FTPSs tended to encode acyclic monoterpenes (12 acyclic and five cyclic) and cyclic sesquiterpenes (five acyclic and 15 cyclic), indicating the complex configuration of the compounds. In addition, 10 pair paralogous TPSs were found in eight FTPS genes of 10 species (Table S4).

2.2. Subfamily Analysis of the FTPS Genes

A phylogenetic tree for 248 FTPS genes and 33 Arabidopsis TPS genes was constructed in RAxML using the ML method (Figure 1B). In the ML tree, the FTPS genes were classified into four separate clades. According to the classification of the Arabidopsis TPSs in each clade, the four clades corresponded to four TPS subfamilies, namely, a, b, g, and c and e/f. Most FTPSs were clustered into the TPS-b (114), TPS-a (94), and TPS-g subfamilies (29). In contrast, the number of genes in TPS-e/f and TPS-c subfamilies was greatly reduced. Two TPS-c genes were mixed into the nine TPS-e/f genes.

2.3. Expression Analysis of the FTPS Genes

To check the expression differences of the FTPSs, we compared the TPM values of the 39 FTPSs (Figure 1C, Table S5). For those paralogous pairs (Table S4), only the higher expression values were used. Consequently, we found that the expression of FTPS genes varied among the 24 species. Although most FTPSs were expressed in a limited number of species, a few genes were common across multiple species. For example, six genes (FTPS1–FTPS5, FTPS7, and FTPS8) were expressed in more than half of the 24 Ficus species. Remarkably, FTPS1 was expressed in 21 species. In contrast, six singletons were highly species-specific, and only one species (F. rumphii) had two singletons (Figure 1C, Table S4). Nevertheless, each species usually has a specific FTPS expression profile. However, two distantly related species, F. pyrifomis and F. semicordata, shared very similar FTPS expression profiles (Figure 1C).

2.4. Positive Selection Analysis

The ML tree of the 24 species of Ficus constructed from the concatenated TPS genes (Figure S2) conformed to the taxonomy of the species [31]. Using the ML tree as a guide, we performed a positive selection analysis for 20 of the 33 FTPS genes, distributed across >4 species.
Using one ratio model calculation, the ω range of 20 FTPS genes was found to be 0.250 (FTPS15) to 0.758 (FTPS16) (Table 2). In order to better detect the probable selective constraint on each FTPS gene in the evolutionary process, the free ratio model, which allows varied ω ratios in each branch, was implemented by the likelihood ratio test (LRT). Two genes, FTPS10 and FTPS12 showed significant selection signals (Table 2), suggesting divergent evolutionary rates of the genes among different Ficus species.
In the branch-site model, we identified three genes (FTPS3, FTPS16, and FTPS20) with significant statistics values (p < 0.01 or p < 0.05) in four species-specific (F. montana or F. pyrifomis, F. fistulosa, and F. formosa) clades (Table 3), suggesting a divergent evolutionary rate of the genes in these species. In addition, we also found that three and one positive selection sites in FTPS4 and FTPS8 in F. hirta–F. triloba and F. heteromorpha clades, respectively (Table 3).

3. Discussion

TPS is an important enzyme that is primarily responsible for catalyzing the synthesis of various terpenoids [23]. In the present study, using ostiole bract transcriptomes, we compared the genic characteristics of expressed TPSs among 24 Ficus species. The number, distribution, and expression level of the TPS genes among different Ficus species were obviously inconsistent, suggesting a diverse composition of terpenes in Ficus species.

3.1. Numbers of TPS Genes in 24 Ostiole Bract Transcriptomes

The TPS gene family is a mid-size family of angiosperms [21]. The number of TPSs in angiosperms varies widely, ranging from two TPS in Zostera marina [22] to 152 in Vitis vinifera [21,22,24]. In particular, TPS genes typically follow a lineage-specific evolutionary pattern, resulting in diverse terpenoid products [32,33]. In addition, given that the TPSs are expressed in a spatiotemporal differential and organ-specific manner, the number of TPS genes identified from the transcriptomes is probably smaller than the number localized in plant genomes. In this study, within Ficus, using tissues at the same development stage, we also found that the number of TPS genes was quite different among the 24 species. For example, there were only two TPS in F. fistulosa, but 19 in F. altissima (Table 1). In addition, for species within the same subgenus, even within the same section of Ficus, we did not find any similarities in the distribution pattern of the TPS genes. To further estimate the occurrence frequency of the TPS genes during the receptive stage of male syconia, we scanned two known genomes of F. microcarpa and F. hispida and compared their TPS number at a genome-wide level to the number identified in our study. We identified 33 and 59 TPS genes in the two genomes and found that only 39% (13) and 13% (eight) TPS genes of two species occurred during the receptive stage (Table 1). This result suggests that the number and occurrence frequency of TPS genes in the receptive stage of male syconia largely depend on the species in Ficus. This situation is consistent with the hypothesis that different species of Ficus release specific volatiles to attract specific pollinating wasps [34].

3.2. Sequence Characteristics and Subfamily Categories of the FTPS Genes

According to the amino acid motifs and catalytic mechanism, TPS enzymes can be divided into two classes: class I and II [21,22,23]. Class I TPSs contain two conserved motifs, DDXXD and (N,D)DXX(S,T,G)XXX(E,D) (also named DTE), which facilitate ionization of an isoprenoid diphosphate moiety and generate a reactive carbocation intermediate. In contrast, Class II TPSs typically contain only a DXDD motif, which protonates substrates, catalyzes scaffold rearrangements without cleavage of the diphosphate ester bond, and produces initial carbocation intermediates [35]. In this study, at the protein level, the DXDD motif was only found in one sequence of FTPS25 (fihir39312). Phylogenetic analysis showed there was another paralog of the sequences in F. hirta (fihir17484). However, owing to the low expression levels of the two sequences (Table S3), we could not detect sufficient reads to assemble complete coding sequences, including the DXDD domain in the second sequence. Because the DXDD motif is the characteristic of Class II TPS gene, the FTPSs identified in our transcriptomes should be dominated by Class I TPS enzymes [36]. This suggestion was also supported by the subsequent phylogenetic analysis.
On the basis of genome-wide comparisons of multiple plants, the TPS family has been split into seven subfamilies, i.e., TPS-a, TPS-b, TPS-c, TPS-d, TPS-e/f, TPS-g, and TPS-h [21,22,37]. Among the subfamilies, TPS-d and TPS-h are distributed only in Gymnosperms and Selaginella moellena, respectively [21]. Notably, in this study, we identified all of the other five TPS subfamilies that can be distributed in angiosperms in the 24 Ficus species, suggesting a diverse and complicated composition of terpenes in the receptive stage of male syconia. In addition, compared with other plants such as A. thaliana [38], grapevine [24], and tomato [30], in which TPS-a genes are the most abundant genes, here, we found that a majority of FTPSs were categorized into TPS-b (46%) rather than the TPS-a (38%) subfamily (Figure 1B). Previous studies have shown that the TPS-b subfamily is mainly composed of angiosperm monoterpene synthases genes [39,40], except a sesquiterpene, (E,E)-α-farnesene synthase gene, found in apple [41], poplar [42], and soybean [43]. Here, we also identified an orthologous gene encoding (E,E)-α-farnesene synthase (FTPS8) in 12 Ficus species (Table S3). More interestingly, our study revealed that most FTPSs protein sequences in the TPS-b subfamily (63%) lack the second R of the R(R)X8W motif, i.e., lacking the RR domain (Figure 2). Previous authors confirmed that the RR domain was functionally important to isomerize GPP to a cyclizable intermediate [44]. However, the RR domain may be absent in the monoterpene synthases producing only acyclic compounds, which do not require isomerization [45]. Therefore, our study suggests that a majority of the Ficus TPS-b genes likely encode acyclic terpene synthases. This suggestion is supported by our current annotations that 70% of monoterpenes were acyclic (Table S3). However, the result is inconsistent with previous findings that many of the enzymes in the plant TPS-b group produce cyclic monoterpenes [21]. Therefore, the production of rich highly volatile acyclic monoterpenes may be a distinguishing characteristic for the Ficus TPS-b enzymes.
The TPS-a subfamily, which included 14 FTPS genes (Figure 1B), is the second largest TPS subfamily in the Ficus species. Similar to other plants, most TPSs in the TPS-a clade were sesquiterpene synthase genes [21,38]. A total of 13 out the 14 FTPS genes identified are responsible for encoding sesquiterpenes. Only FTPS13, which encodes a cadinene synthase, is an acyclic monoterpene.
The third largest TPS subfamily in the 24 Ficus species is TPS-g. This subfamily includes 29 sequences (Figure 1B). TPS-g genes are often believed to show prevalence in catalyzing acyclic products [21,46]. Here, these TPS-g genes were categorized into three genes (FTPS5, FTPS10, and FTPS21) and two singletons (ficha23769 and fiful14859). According to annotation by the nonredundant protein database, all five genes were matched to two acyclic monoterpenes, linalool synthase and (3S, 6E)-nerolidol synthase 1.
TPS-e/f and TPS-c are small subfamilies in the Ficus species. The FTPS-e/f genes are present in six species and involved in only two orthologous genes (FTPS15 and FTPS32). The genes encode a diterpene (ent-kaur-16-ene synthase) and a monoterpene S-linalool synthase, respectively. The ent-kaurene synthases often serve as precursors to plant growth regulators, and they exhibit a variety of useful pharmacological properties [47]. In other words, the role of the compound is more focused on pest defense. However, in sunflowers, Morris et al. [48] showed that kaurane diterpenes can be used as oviposition stimulants of insects. By contrast, S-linalool is often used as an attractive volatile that some flowers emit to please pollinators [49]. Despite their variability in function, in the fig–fig wasp symbiosis, terpenes are always considered to be chemical attractants to fig wasps. Therefore, the exact functions of the two TPSs in Ficus deserve further exploration. In the TPS-c subfamily, we only found two paralogous sequences that encoded for FTPS25. The two sequences were annotated as ent-copalyl diphosphate synthases and were highly specifically expressed in F. hirta. This obvious gene expansion event implies that the FTPS25 may function during the receptive phase of male syconia of F. hirta. In addition, the presence of the classic DXDD motif in the gene suggests they function as Class II TPS.

3.3. Expression Profiles of the FTPS Genes

By examining the expression profiles of the FTPSs, we determined the diversity and complexity of FTPSs expression status in 24 Ficus species. Here, each species had a unique expression profile indicating a highly species-specific expression array of the FTPS genes. However, although we identified six singletons for five species, we showed that the other 33 orthologous FTPSs were shared by at least two species (Figure 1C). This result suggests that the odors emitted by each Ficus species blend compounds of multiple common terpenoids, rather than entirely species-specific volatiles. By comparing the blends of VOCs released from receptive figs of 20 Ficus species, Grison-Pige et al. [50] found that a few major compounds such as β-ocimene, germacrene D, and linalool are common among floral fragrances, while the rare compounds are usually present in a very low proportion in the blends. In addition, Proffit and Johnson [11] showed that (E)-β-ocimene was responsible for 41.75% of VOCs in receptive syconia of F. sycomorus. In this study, we found that at least 10 FTPSs were annotated as myrcene synthases (Table S3) and were highly expressed in multiple species (Figure 1C). For example, FTPS1 and FTPS2 that encode different myrcene synthases were found in 21 and 19 species, respectively. Given that myrcene is an isomer of ocimenes, the myrcene/ocimene synthases should also produce common chemical compounds during the repetitive phase of male syconia for the Ficus species. In addition, we also found six FTPSs encoding germacrene D synthases were simultaneously expressed in more than 16 species (Figure 1C). In plants, germacrene D, comprising some 300 isomers (C15H24), has evolved ecological roles in the interaction of the plant with insects [51,52]. This plant sesquiterpene can specifically activate a major type of antennal receptor neuron of the tobacco budworm moth Heliothis virescens [53]. However, whether germacrene D is involved in the interaction of figs and fig wasps remains unclear.
In addition, it is worth noting that the distribution and expression intensity of the FTPSs across species do not always correlate with the phylogenetic relationship of the species. Recently, Yu et al. [54] investigated the volatiles emitted by receptive male figs of F. hirta and F. triloba, two phylogenetic related species, and found that the two species share 48 VOCs, but the most abundant volatiles differ. Here, we identified 11 homologous FTPS pairs between the two species, but the expression level of each FTPS pair was very different (Figure 1C). These results suggest that the proportion of compounds is more important than the composition itself.
However, interestingly, we also found that two distantly related species (F. pyrifomis and F. semicordata) had highly similar expression profiles of FTPSs (Figure 1C). Within the genus of Ficus, species F. pyrifomis and F. semicordata belong to different subgenera, Ficus and Sycomorus, respectively. In this study, the single relevance between the two species is that they were all collected from Thailand. Given that the two species have distinguished morphologies from each other and from the other fig species, sampling and identification errors can be ruled out. We also checked the other genes in the transcriptomes of the two species and found different gene lists and expression profiles, suggesting that the two RNA libraries were not misused or mixed. Previous studies confirm that similar volatile blends of fig species can trigger their evolutionary convergence to attract particular wasp species [55,56,57]. In turn, regionally pollinator sharing can produce gene flow between fig species [54,58]. In Ficus, hybridization and introgression can occur not only among fig taxa from the same section but also among taxa from different sections [58]. Therefore, the current deviant may be a special case of hybridization and introgression of distantly related species. Further confirmation research is very necessary. In addition, small doses of compounds produced by lowly expressed genes may also play some key roles in attracting special fig wasp in different symbiosis systems.

3.4. Positive Selection of FTPSs

In this study, the branch model and branch-site model methods were used to check for the positive selection of homologous TPS genes. Except for five genes (FTPS10 and FTPS12 in the branch model test and FTPS3, FTPS16, and FTPS20 in the branch-site model test) that showed significantly positive selection signals in some specific species (Table 2 and Table 3), a majority of the FTPSs were still highly conserved among species and under purification selective pressure, suggesting functional stability of the TPS genes. The five FTPS genes encode four chemical compounds, linalool synthase, myrcene synthase, elemene synthase, and E-beta-caryophyllene synthase (Table S3). Notably, these compounds are all common in Ficus species rather than species-specific ones [50,54]. Grison-Pige et al. [50] confirmed that beta-caryophyllene, linalool, myrcene, and beta-elemene can be identified from fig odors of 18, 10, six, and five Ficus species, respectively. Yu et al. [54] revealed that (E)-caryophyllene and beta-element were all highly abundant compounds released by both F. hirta and F. triloba. These results imply that the FTPS evolution may prefer to select those common genes shared by several Ficus species rather than recruit some specific or rare new genes. In addition, in this study, two genes (FTPS4 and FTPS8) with three and one positive selection sites in species of F. hirta and F. triloba, and F. erecta var. beecheyana were also identified under branch-site model, respectively. Although these sites were not located in the conserved protein domains of plants TPS genes, these hidden interspecies divergences may prompt the diversification and complexity on the structure and function of the FTPS proteins, and then enforce VOC specificity. Further in-depth functional verification should be performed.

4. Materials and Methods

4.1. Sample Collection and RNA Sequencing

Twenty-four Ficus species belonging to four subgenera, with five sections and eight subsections [3], were collected from South China and Southeast Asia (Table 1). To simplify species names during data analysis, abbreviated names of the 24 Ficus species were used (Table 1). The ostiole bracts of three receptive male syconia from three plants of each of the 24 species were dissected and placed in RNAlater™ Stabilization Solution (00936134, Takara).
Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The quantity of RNA was evaluated using 1% agarose gel electrophoresis and a NanoDrop 2000 spectrophotometer (NanoDrop, Wilmington, DE, USA). A total of 1.5 μg of RNA per species was used as the input material for an RNA library constructed using an Illumina TruSeq™ RNA Sample Preparation Kit (Illumina, San Diego, CA, USA). Paired-end high-throughput sequencing was performed by Novogene Bioinformatics Technology Co. Ltd. (Beijing, China) on an Illumina HiSeq 4000 platform.

4.2. RNA-Seq Assembly and Identification of TPS Genes

Data obtained by the RNA-seq of 24 Ficus species were assembled de novo using Trinity v2.8.5 [59] under the De Bruijn algorithm to construct unique consensus sequences. TransDecoder v5.5.0 (Haas et al., 2013) was used to predict the coding sequence (CDS) for each isoform of a gene. The raw sequence data were deposited in the Genome Sequence Archive (GSA) in the National Genomics Data Center, Chinese Academy of Sciences (https://ngdc.cncb.ac.cn/gsa/; accessed on 31 July 2022), under the accession number PRJCA009602.
To facilitate subsequent analysis, we processed all sequences with an in-house Perl script and converted the nucleotide sequence of each unigene to protein sequence. Finally, we obtained a protein database for the 24 Ficus species.
We selected amino acid sequences of the known TPS genes of Arabidopsis thaliana (L.) Heynh. (https://www.uniprot.org/; accessed on 31 October 2021) as query sequences, and then identified the TPS genes from the above constructed protein databases for the 24 Ficus species using a BlastP search (E-value < 10−5). PF01397 (N-terminal) and PF03936 (C-terminal) are the query models derived from the hidden Markov model of the TPS genes in the PFAM database (https://pfam.xfam.org/; accessed on 31 July 2022). Using these two HMM models as the query, we identified the TPS genes from the protein database of 24 Ficus species using HMMER 3.2.1 [60]. To ensure data accuracy, the conserved domains of the TPS proteins obtained using the above two methods were reconfirmed in the Conserved Domain Database (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi; accessed on 31 July 2022). The validated TPS genes were annotated using the online tool eggNOG-mapper v2 (http://eggnog-mapper.embl.de/; accessed on 31 March 2022) with default parameter [61] and the nonredundant protein database (https://www.ncbi.nlm.nih.gov/; accessed on 31 March 2022) using BLASTP with the E-value cutoffs ≤ 10−5.

4.3. Phylogenetic Relationship, Subfamily Classification, and Motif Analysis of the Ficus TPS Genes

All identified Ficus TPS genes and 33 TPS genes from the A. thaliana genome (https://phytozome-next.jgi.doe.gov/; accessed on 1 July 2022) were combined to build a data matrix. MAFFT v.7407 software [62] was used to perform multiple sequence alignments. RAxML v8.2.12 software [63] was used to construct a maximum likelihood (ML) phylogenetic tree based on full-length amino acid sequences with the executed command “raxmlHPC-HYBRID-SSE3 -f a -x 12345 -p 12345 -# 1000 -m GTRGAMMA”. The multiple Em-for-Motif-Elicitation web server (https://meme-suite.org/meme/; accessed on 21 May 2022) [64] was used for the motif analysis of the Ficus TPS genes using the parameters -nostatus -mod anr -minw 10 -maxw 50 -nmotifs 10.

4.4. Clustering, Annotation, and Expression Analysis of the Ficus TPS Genes

The OrthoMCL v2.0.9 [65] with a highly strict threshold of the following parameters: —evalue 1 × 10−12 —identity 0.5 —CIP 0.6 —CALP 0.6 [66] was used to cluster the identified TPS genes, and in-house Perl scripts were used to screen the clustering results to obtain different TPS orthologs.
The value of transcripts per kilobase per million mapped reads (TPM) of each TPS gene calculated by RSEM [67] was used to evaluate transcription abundance. To facilitate data comparison, for the TPS with duplicated paralogs within the same species, we retained only the TPS with the highest expression level for further analysis. After standardization using the trimmed mean of M values (TMM), we obtained the original data matrix of gene expression. Heat maps of the orthologous TPS genes were illustrated using TBtools v1.09876 (https://github.com/CJ-Chen/TBtools/; accessed on 29 June 2022) [68], and a color gradient was displayed as the log2(TPM + 1) transformed expression levels of each gene.

4.5. Positive Selection Analysis

We performed gene positive selection analyses using the branch-site model and branch model in the PAML package version 4 [69]. A concatenated TPS gene topology phylogenetic tree was constructed as the guide tree using RAxML v8.2.12 software [63] under the parameters “raxml HPC-HYBRID-SSE3 -f a -x 12345 -p 12345 -# 1000 -m GTRGAMMA”. Species Morus notabilis C. K. Schneider was used as the outgroup. Because the PAML documentation recommends that the absolute minimum be four or five if the sequence divergence is optimal, the TPS genes that occurred in fewer than four species were ignored from further processing.
In the branch model, the one-ratio model was the null model, which means that all branches had the same ω value, while the free-ratio model considered that each branch had a different ω value. We compared the likelihood ratio of the two models and calculated the p-value using the LRT test.
In order to find genes that potentially experienced positive selection, the branch-site model (model = 2 and NSsites = 2) of the PAML package was used, with each branch specified as the foreground. To avoid false positive results in the branch-site model, the following rigorous criteria were used: a dN/dS ratio (ω) greater than 1 on the foreground branch, and positively selected sites with a posterior probability calculated by the Bayes empirical Bayes (BEB) method greater than 0.95 and a p-value ≤ 0.05 in the likelihood ratio test [70].

5. Conclusions

This study identified 248 TPS genes in 24 Ficus species, and then comprehensively analyzed their sequence characteristics, phylogenetic relationships, expression profiles, and selection divergences. Studies revealed that the FTPSs are dominated by class I synthase genes, and the terpenoids encoded by the FTPSs may involve sesquiterpenes, monoterpenes, and diterpenes. The 248 FTPSs can be classified into 33 homologous genes with different expression strengths and expression profiles in 24 Ficus species. Complex combination and divergent expression of the multiple terpenoid synthase genes may be an important factor responsible for the diversity and specificity of Ficus terpenoids. In short, this study deepens our understanding of the diversity of the fig TPS gene family.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/d14090721/s1: Figure S1: The biosynthesis pathway of plant terpenoids and classification of terpene synthase (TPS); Figure S2: A concatenated FTPS phylogenetic tree for positive selection analysis; Table S1: Summary of assembly results of transcriptome data for 24 Ficus species; Table S2: Amino acid sequences of 248 TPS genes in the 24 Ficus species; Table S3: Annotation of Ficus TPS genes by the nonredundant protein database; Table S4: Expression of different FTPS paralogous pairs in six species; Table S5: TPM values of FTPSs in the 24 Ficus species.

Author Contributions

P.S., Y.B. and H.Y. designed the work; P.S. and Y.B. drafted the manuscript; P.S., Y.B., X.C., B.C., X.Z. and X.D. analyzed the data. All authors have read and agreed to the published version of the manuscript.

Funding

This work was funded by the National Natural Science Foundation of China (grant numbers 32070246, 31971568, and 32150410364). This work was also supported by the High-Performance Computing Center of Qufu Normal University.

Data Availability Statement

The raw sequence data are available in the Genome Sequence Archive (GSA) at the National Genomics Data Center, Chinese Academy of Science (https://bigd.big.ac.cn/gsa; accessed on 31 July 2022), under accession number PRJCA009602.

Acknowledgments

The authors would like to thank all those involved in the implementation of this study for their assistance, guidance, and support.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Ehrlich, P.R.; Raven, P.H. Butterflies and Plants: A Study in Coevolution. Evolution 1964, 18, 586–608. [Google Scholar] [CrossRef]
  2. Oku, H.; Inafuku, M.; Ishikawa, T.; Takamine, T.; Ishmael, M.; Fukuta, M. Molecular cloning and biochemical characterization of isoprene synthases from the tropical trees Ficus virgata, Ficus septica, and Casuarina equisetifolia. J. Plant Res. 2015, 128, 849–861. [Google Scholar] [CrossRef] [PubMed]
  3. Berg, C.C. Classification and distribution of Ficus. Experientia 1989, 45, 605–611. [Google Scholar] [CrossRef]
  4. Herre, E.A.; Jander, K.C.; Machado, C.A. Evolutionary Ecology of Figs and Their Associates: Recent Progress and Outstanding Puzzles. Annu. Rev. Ecol. Evol. Syst. 2008, 39, 439–458. [Google Scholar] [CrossRef]
  5. Souza, C.D.; Pereira, R.A.S.; Marinho, C.R.; Kjellberg, F.; Teixeira, S.P. Diversity of fig glands is associated with nursery mutualism in fig trees. Am. J. Bot. 2015, 102, 1564–1577. [Google Scholar] [CrossRef]
  6. Hu, R.; Sun, P.; Yu, H.; Cheng, Y.F.; Wang, R.; Chen, X.Y.; Kjellberg, F. Similitudes and differences between two closely related Ficus species in the synthesis by the ostiole of odors attracting their host-specific pollinators: A transcriptomic based investigation. Acta Oecologica-Int. J. Ecol. 2020, 105, 103554. [Google Scholar] [CrossRef]
  7. Galil, J. Fig biology. Endeavour 1977, 1, 52–56. [Google Scholar] [CrossRef]
  8. Weiblen, G.D. How to be a fig wasp. Annu. Rev. Entomol. 2002, 47, 299–330. [Google Scholar] [CrossRef]
  9. Zhang, X.; Wang, G.; Zhang, S.; Chen, S.; Wang, Y.; Wen, P.; Ma, X.; Shi, Y.; Qi, R.; Yang, Y.; et al. Genomes of the Banyan Tree and Pollinator Wasp Provide Insights into Fig-Wasp Coevolution. Cell 2020, 183, 875–889. [Google Scholar] [CrossRef]
  10. Hossaert-McKey, M.; Soler, C.; Schatz, B.; Proffit, M. Floral scents: Their roles in nursery pollination mutualisms. Chemoecology 2010, 20, 75–88. [Google Scholar] [CrossRef]
  11. Proffit, M.; Johnson, S.D. Specificity of the signal emitted by figs to attract their pollinating wasps: Comparison of volatile organic compounds emitted by receptive syconia of Ficus sur and F. sycomorus in Southern Africa. S. Afr. J. Bot. 2009, 75, 771–777. [Google Scholar] [CrossRef]
  12. Gu, D.; Compton, S.G.; Peng, Y.; Yang, D. ‘Push’ and ‘pull’ responses by fig wasps to volatiles released by their host figs. Chemoecology 2012, 22, 217–227. [Google Scholar] [CrossRef]
  13. Borges, R.M.; Bessiere, J.M.; Ranganathan, Y. Diel variation in fig volatiles across syconium development: Making sense of scents. J. Chem. Ecol. 2013, 39, 630–642. [Google Scholar] [CrossRef] [PubMed]
  14. Yu, Z.; Zhao, C.; Zhang, G.; Teixeira da Silva, J.A.; Duan, J. Genome-Wide Identification and Expression Profile of TPS Gene Family in Dendrobium officinale and the Role of DoTPS10 in Linalool Biosynthesis. Int. J. Mol. Sci. 2020, 21, 5419. [Google Scholar] [CrossRef] [PubMed]
  15. Tholl, D. Biosynthesis and Biological Functions of Terpenoids in Plants. Adv. Biochem. Eng. Biotechnol. 2015, 148, 63–106. [Google Scholar] [CrossRef]
  16. Nawade, B.; Shaltiel-Harpaz, L.; Yahyaa, M.; Kabaha, A.; Kedoshim, R.; Bosamia, T.C.; Ibdah, M. Characterization of terpene synthase genes potentially involved in black fig fly (Silba adipata) interactions with Ficus carica. Plant Sci. 2020, 298, 110549. [Google Scholar] [CrossRef]
  17. Dhifi, W.; Bellili, S.; Jazi, S.; Bahloul, N.; Mnif, W. Essential Oils’ Chemical Characterization and Investigation of Some Biological Activities: A Critical Review. Medicines 2016, 3, 25. [Google Scholar] [CrossRef]
  18. Zhou, H.C.; Shamala, L.F.; Yi, X.K.; Yan, Z.; Wei, S. Analysis of Terpene Synthase Family Genes in Camellia sinensis with an Emphasis on Abiotic Stress Conditions. Sci. Rep. 2020, 10, 933. [Google Scholar] [CrossRef]
  19. Gao, F.; Liu, B.; Li, M.; Gao, X.; Fang, Q.; Liu, C.; Ding, H.; Wang, L.; Gao, X. Identification and characterization of terpene synthase genes accounting for volatile terpene emissions in flowers of Freesia x hybrida. J. Exp. Bot. 2018, 69, 4249–4265. [Google Scholar] [CrossRef]
  20. Zhou, S.S.; Xing, Z.; Liu, H.; Hu, X.G.; Gao, Q.; Xu, J.; Jiao, S.Q.; Jia, K.H.; Jin, Y.Q.; Zhao, W.; et al. In-depth transcriptome characterization uncovers distinct gene family expansions for Cupressus gigantea important to this long-lived species’ adaptability to environmental cues. BMC Genom. 2019, 20, 213. [Google Scholar] [CrossRef]
  21. Chen, F.; Tholl, D.; Bohlmann, J.; Pichersky, E. The family of terpene synthases in plants: A mid-size family of genes for specialized metabolism that is highly diversified throughout the kingdom. Plant J. 2011, 66, 212–229. [Google Scholar] [CrossRef] [PubMed]
  22. Jiang, S.Y.; Jin, J.; Sarojam, R.; Ramachandran, S. A Comprehensive Survey on the Terpene Synthase Gene Family Provides New Insight into Its Evolutionary Patterns. Genome Biol. Evol. 2019, 11, 2078–2098. [Google Scholar] [CrossRef] [PubMed]
  23. Alicandri, E.; Paolacci, A.R.; Osadolor, S.; Sorgona, A.; Badiani, M.; Ciaffi, M. On the Evolution and Functional Diversity of Terpene Synthases in the Pinus Species: A Review. J. Mol. Evol. 2020, 88, 253–283. [Google Scholar] [CrossRef] [PubMed]
  24. Martin, D.M.; Aubourg, S.; Schouwey, M.B.; Daviet, L.; Schalk, M.; Toub, O.; Lund, S.T.; Bohlmann, J. Functional annotation, genome organization and phylogeny of the grapevine (Vitis vinifera) terpene synthase gene family based on genome assembly, FLcDNA cloning, and enzyme assays. BMC Plant Biol. 2010, 10, 226. [Google Scholar] [CrossRef]
  25. Hattan, J.; Shindo, K.; Sasaki, T.; Misawa, N. Isolation and Functional Characterization of New Terpene Synthase Genes from Traditional Edible Plants. J. Oleo Sci. 2018, 67, 1235–1246. [Google Scholar] [CrossRef]
  26. Booth, J.K.; Yuen, M.M.S.; Jancsik, S.; Madilao, L.L.; Page, J.E.; Bohlmann, J. Terpene Synthases and Terpene Variation in Cannabis sativa. Plant Physiol. 2020, 184, 130–147. [Google Scholar] [CrossRef]
  27. Huang, L.M.; Huang, H.; Chuang, Y.C.; Chen, W.H.; Wang, C.N.; Chen, H.H. Evolution of Terpene Synthases in Orchidaceae. Int. J. Mol. Sci. 2021, 22, 6947. [Google Scholar] [CrossRef]
  28. He, J.; Verstappen, F.; Jiao, A.; Dicke, M.; Bouwmeester, H.J.; Kappers, I.F. Terpene synthases in cucumber (Cucumis sativus) and their contribution to herbivore-induced volatile terpenoid emission. New Phytol. 2022, 233, 862–877. [Google Scholar] [CrossRef]
  29. Huang, X.Z.; Xiao, Y.T.; Kollner, T.G.; Jing, W.X.; Kou, J.F.; Chen, J.Y.; Liu, D.F.; Gu, S.H.; Wu, J.X.; Zhang, Y.J.; et al. The terpene synthase gene family in Gossypium hirsutum harbors a linalool synthase GhTPS12 implicated in direct defence responses against herbivores. Plant Cell Environ. 2018, 41, 261–274. [Google Scholar] [CrossRef]
  30. Zhou, F.; Pichersky, E. The complete functional characterisation of the terpene synthase family in tomato. New Phytol. 2020, 226, 1341–1360. [Google Scholar] [CrossRef] [Green Version]
  31. Berg, C.C.; Wiebes, J.T. African Fig Trees and Fig Wasps; KNAW: Amsterdam, The Netherlands; New York, NY, USA, 1992; p. 298. [Google Scholar]
  32. Chen, F.; Ro, D.K.; Petri, J.; Gershenzon, J.; Bohlmann, J.; Pichersky, E.; Tholl, D. Characterization of a root-specific Arabidopsis terpene synthase responsible for the formation of the volatile monoterpene 1,8-cineole. Plant Physiol. 2004, 135, 1956–1966. [Google Scholar] [CrossRef] [PubMed]
  33. Allen, K.D.; McKernan, K.; Pauli, C.; Roe, J.; Torres, A.; Gaudino, R. Genomic characterization of the complete terpene synthase gene family from Cannabis sativa. PLoS ONE 2019, 14, e0222363. [Google Scholar] [CrossRef] [PubMed]
  34. Souto-Vilaros, D.; Machac, A.; Michalek, J.; Darwell, C.T.; Sisol, M.; Kuyaiva, T.; Isua, B.; Weiblen, G.D.; Novotny, V.; Segar, S.T. Faster speciation of fig-wasps than their host figs leads to decoupled speciation dynamics: Snapshots across the speciation continuum. Mol. Ecol. 2019, 28, 3958–3976. [Google Scholar] [CrossRef] [PubMed]
  35. Liu, W.; Feng, X.; Zheng, Y.; Huang, C.H.; Nakano, C.; Hoshino, T.; Bogue, S.; Ko, T.P.; Chen, C.C.; Cui, Y.; et al. Structure, function and inhibition of ent-kaurene synthase from Bradyrhizobium japonicum. Sci. Rep. 2014, 4, 6214. [Google Scholar] [CrossRef]
  36. Kruppa, J.; Jo, W.K.; van der Vries, E.; Ludlow, M.; Osterhaus, A.; Baumgaertner, W.; Jung, K. Virus detection in high-throughput sequencing data without a reference genome of the host. Infect. Genet. Evol. 2018, 66, 180–187. [Google Scholar] [CrossRef]
  37. Jia, Q.; Brown, R.; Kollner, T.G.; Fu, J.; Chen, X.; Wong, G.K.; Gershenzon, J.; Peters, R.J.; Chen, F. Origin and early evolution of the plant terpene synthase family. Proc. Natl. Acad. Sci. USA 2022, 119, e2100361119. [Google Scholar] [CrossRef]
  38. Aubourg, S.; Lecharny, A.; Bohlmann, J. Genomic analysis of the terpenoid synthase ( AtTPS) gene family of Arabidopsis thaliana. Mol. Genet. Genom. 2002, 267, 730–745. [Google Scholar] [CrossRef]
  39. Luck, K.; Chen, X.L.; Norris, A.M.; Chen, F.; Gershenzon, J.; Kollner, T.G. The reconstruction and biochemical characterization of ancestral genes furnish insights into the evolution of terpene synthase function in the Poaceae. Plant Mol. Biol. 2020, 104, 203–215. [Google Scholar] [CrossRef]
  40. Dudareva, N.; Klempien, A.; Muhlemann, J.K.; Kaplan, I. Biosynthesis, function and metabolic engineering of plant volatile organic compounds. New Phytol. 2013, 198, 16–32. [Google Scholar] [CrossRef]
  41. Green, S.; Friel, E.N.; Matich, A.; Beuning, L.L.; Cooney, J.M.; Rowan, D.D.; MacRae, E. Unusual features of a recombinant apple alpha-farnesene synthase. Phytochemistry 2007, 68, 176–188. [Google Scholar] [CrossRef]
  42. Danner, H.; Boeckler, G.A.; Irmisch, S.; Yuan, J.S.; Chen, F.; Gershenzon, J.; Unsicker, S.B.; Kollner, T.G. Four terpene synthases produce major compounds of the gypsy moth feeding-induced volatile blend of Populus trichocarpa. Phytochemistry 2011, 72, 897–908. [Google Scholar] [CrossRef] [PubMed]
  43. Lin, J.; Wang, D.; Chen, X.; Kollner, T.G.; Mazarei, M.; Guo, H.; Pantalone, V.R.; Arelli, P.; Stewart, C.N., Jr.; Wang, N.; et al. An (E,E)-alpha-farnesene synthase gene of soybean has a role in defence against nematodes and is involved in synthesizing insect-induced volatiles. Plant Biotechnol. J. 2017, 15, 510–519. [Google Scholar] [CrossRef] [PubMed]
  44. Williams, D.C.; McGarvey, D.J.; Katahira, E.J.; Croteau, R. Truncation of limonene synthase preprotein provides a fully active ‘pseudomature’ form of this monoterpene cyclase and reveals the function of the amino-terminal arginine pair. Biochemistry 1998, 37, 12213–12220. [Google Scholar] [CrossRef] [PubMed]
  45. Degenhardt, J.; Kollner, T.G.; Gershenzon, J. Monoterpene and sesquiterpene synthases and the origin of terpene skeletal diversity in plants. Phytochemistry 2009, 70, 1621–1637. [Google Scholar] [CrossRef]
  46. Haas, B.J.; Papanicolaou, A.; Yassour, M.; Grabherr, M.; Blood, P.D.; Bowden, J.; Couger, M.B.; Eccles, D.; Li, B.; Lieber, M.; et al. De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nat. Protoc. 2013, 8, 1494–1512. [Google Scholar] [CrossRef]
  47. García, P.A.; de Oliveira, A.B.; Batista, R. Occurrence, Biological Activities and Synthesis of Kaurane Diterpenes and their Glycosides. Molecules 2007, 12, 455–483. [Google Scholar] [CrossRef]
  48. Morris, B.D.; Foster, S.P.; Grugel, S.R.; Charlet, L.D. Isolation of the diterpenoids, ent-kauran-16alpha-ol and ent-atisan-16alpha-ol, from sunflowers, as oviposition stimulants for the banded sunflower moth, Cochylis hospes. J. Chem. Ecol. 2005, 31, 89–102. [Google Scholar] [CrossRef]
  49. Pichersky, E.; Lewinsohn, E.; Croteau, R. Purification and characterization of S-linalool synthase, an enzyme involved in the production of floral scent in Clarkia breweri. Arch. Biochem. Biophys. 1995, 316, 803–807. [Google Scholar] [CrossRef]
  50. Grison-Pige, L.; Hossaert-McKey, M.; Greeff, J.M.; Bessiere, J.M. Fig volatile compounds--a first comparative study. Phytochemistry 2002, 61, 61–71. [Google Scholar] [CrossRef]
  51. Prosser, I.; Altug, I.G.; Phillips, A.L.; Konig, W.A.; Bouwmeester, H.J.; Beale, M.H. Enantiospecific (+)- and (-)-germacrene D synthases, cloned from goldenrod, reveal a functionally active variant of the universal isoprenoid-biosynthesis aspartate-rich motif. Arch. Biochem. Biophys. 2004, 432, 136–144. [Google Scholar] [CrossRef]
  52. Stranden, M.; Borg-Karlson, A.K.; Mustaparta, H. Receptor neuron discrimination of the germacrene D enantiomers in the moth Helicoverpa armigera. Chem. Senses 2002, 27, 143–152. [Google Scholar] [CrossRef] [PubMed]
  53. Rostelien, T.; Borg-Karlson, A.K.; Faldt, J.; Jacobsson, U.; Mustaparta, H. The plant sesquiterpene germacrene D specifically activates a major type of antennal receptor neuron of the tobacco budworm moth Heliothis virescens. Chem. Senses 2000, 25, 141–148. [Google Scholar] [CrossRef] [PubMed]
  54. Yu, H.; Zhang, Z.; Liu, L.; Cheng, Y.; Deng, X.; Segar, S.T.; Compton, S.G. Asymmetric sharing of pollinator fig wasps between two sympatric dioecious fig trees: A reflection of supply and demand or differences in the size of their figs? Bot. Stud. 2022, 63, 7. [Google Scholar] [CrossRef] [PubMed]
  55. Cornille, A.; Underhill, J.G.; Cruaud, A.; Hossaert-McKey, M.; Johnson, S.D.; Tolley, K.A.; Kjellberg, F.; van Noort, S.; Proffit, M. Floral volatiles, pollinator sharing and diversification in the fig-wasp mutualism: Insights from Ficus natalensis, and its two wasp pollinators (South Africa). Proc. Biol. Sci. 2012, 279, 1731–1739. [Google Scholar] [CrossRef] [PubMed]
  56. Su, Z.H.; Sasaki, A.; Kusumi, J.; Chou, P.A.; Tzeng, H.Y.; Li, H.Q.; Yu, H. Pollinator sharing, copollination, and speciation by host shifting among six closely related dioecious fig species. Commun. Biol. 2022, 5, 284. [Google Scholar] [CrossRef]
  57. Wang, G.; Compton, S.G.; Chen, J. The mechanism of pollinator specificity between two sympatric fig varieties: A combination of olfactory signals and contact cues. Ann. Bot. 2013, 111, 173–181. [Google Scholar] [CrossRef]
  58. Wang, G.; Cannon, C.H.; Chen, J. Pollinator sharing and gene flow among closely related sympatric dioecious fig taxa. Proc. Biol. Sci. 2016, 283, 20152963. [Google Scholar] [CrossRef]
  59. Grabherr, M.G.; Haas, B.J.; Yassour, M.; Levin, J.Z.; Thompson, D.A.; Amit, I.; Adiconis, X.; Fan, L.; Raychowdhury, R.; Zeng, Q.D.; et al. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat. Biotechnol. 2011, 29, 644-U130. [Google Scholar] [CrossRef]
  60. Wheeler, T.J.; Eddy, S.R. nhmmer: DNA homology search with profile HMMs. Bioinformatics 2013, 29, 2487–2489. [Google Scholar] [CrossRef]
  61. Cantalapiedra, C.P.; Hernandez-Plaza, A.; Letunic, I.; Bork, P.; Huerta-Cepas, J. eggNOG-mapper v2: Functional Annotation, Orthology Assignments, and Domain Prediction at the Metagenomic Scale. Mol. Biol. Evol. 2021, 38, 5825–5829. [Google Scholar] [CrossRef]
  62. Katoh, K.; Kuma, K.; Toh, H.; Miyata, T. MAFFT version 5: Improvement in accuracy of multiple sequence alignment. Nucleic Acids Res. 2005, 33, 511–518. [Google Scholar] [CrossRef] [PubMed]
  63. Stamatakis, A. RAxML version 8: A tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 2014, 30, 1312–1313. [Google Scholar] [CrossRef] [PubMed]
  64. Bailey, T.L.; Elkan, C. Fitting a mixture model by expectation maximization to discover motifs in biopolymers. Proc. Int. Conf. Intell. Syst. Mol. Biol. 1994, 2, 28–36. [Google Scholar] [PubMed]
  65. Fischer, S.; Brunk, B.P.; Chen, F.; Gao, X.; Harb, O.S.; Iodice, J.B.; Shanmugam, D.; Roos, D.S.; Stoeckert, C.J., Jr. Using OrthoMCL to assign proteins to OrthoMCL-DB groups or to cluster proteomes into new ortholog groups. Curr. Protoc. Bioinform. 2011, 12, 11–19. [Google Scholar] [CrossRef]
  66. Mahmoudinia, S.; Niapour, A.; Ghasemi Hamidabadi, H.; Mazani, M. 2,4-D causes oxidative stress induction and apoptosis in human dental pulp stem cells (hDPSCs). Environ. Sci. Pollut. Res. 2019, 26, 26170–26183. [Google Scholar] [CrossRef]
  67. Borodina, T.; Adjaye, J.; Sultan, M. Chapter five—A Strand-Specific Library Preparation Protocol for RNA Sequencing. In Methods in Enzymology; Jameson, D., Verma, M., Westerhoff, H.V., Eds.; Academic Press: San Diego, CA, USA, 2011; Volume 500, pp. 79–98. [Google Scholar]
  68. Chen, C.; Chen, H.; Zhang, Y.; Thomas, H.R.; Frank, M.H.; He, Y.; Xia, R. TBtools: An Integrative Toolkit Developed for Interactive Analyses of Big Biological Data. Mol. Plant 2020, 13, 1194–1202. [Google Scholar] [CrossRef]
  69. Yang, Z. Statistical properties of a DNA sample under the finite-sites model. Genetics 1996, 144, 1941–1950. [Google Scholar] [CrossRef]
  70. Jovanovic, V.M.; Sarfert, M.; Reyna-Blanco, C.S.; Indrischek, H.; Valdivia, D.I.; Shelest, E.; Nowick, K. Positive Selection in Gene Regulatory Factors Suggests Adaptive Pleiotropic Changes During Human Evolution. Front. Genet. 2021, 12, 662239. [Google Scholar] [CrossRef]
Diversity 14 00721 g001 550
Figure 1. Sequence characteristic, phylogeny, subfamily classification, and expression profile of 248 FTPS genes. (A) Three conserved motif logos of the 248 Ficus genes, showing DDXXD, DTE, and RXR motif, respectively. (B) A maximum likelihood tree based on amino acid sequences of 248 FTPS genes and 33 A. thaliana genes, showing that these FTPSs are classified into five subfamilies and 33 orthologous genes (A. thaliana genes are colored in red). (C) Expression heatmap of 248 FTPS genes across 24 species. * Genes with positive sites.
Figure 1. Sequence characteristic, phylogeny, subfamily classification, and expression profile of 248 FTPS genes. (A) Three conserved motif logos of the 248 Ficus genes, showing DDXXD, DTE, and RXR motif, respectively. (B) A maximum likelihood tree based on amino acid sequences of 248 FTPS genes and 33 A. thaliana genes, showing that these FTPSs are classified into five subfamilies and 33 orthologous genes (A. thaliana genes are colored in red). (C) Expression heatmap of 248 FTPS genes across 24 species. * Genes with positive sites.
Diversity 14 00721 g001
Diversity 14 00721 g002 550
Figure 2. The R(R)X8W motif of the FTPS genes in the TPS-b subfamily. (A) The R(R)X8W motif logo of all FTPS-b subfamily genes. (B) Some representative sequences of FTPS-b subfamily genes, showing the R(R)X8W motif without the tandem RR domain.
Figure 2. The R(R)X8W motif of the FTPS genes in the TPS-b subfamily. (A) The R(R)X8W motif logo of all FTPS-b subfamily genes. (B) Some representative sequences of FTPS-b subfamily genes, showing the R(R)X8W motif without the tandem RR domain.
Diversity 14 00721 g002
Table
Table 1. Twenty-four Ficus species used in this study.
Table 1. Twenty-four Ficus species used in this study.
SubgenusSectionSubsectionSpeciesName AbbreviationsTPS No.SiteLatitude
(N)		Longitude
(E)
FicusEriosyceaEriosyceaF. chartaceaficha8China—Guangdong8.77699.724
F. fulvafiful14China—Guangdong8.77699.724
F. grossularoidesfigro3Thailand—Narathiwat5.799101.762
F. hirtafihir16China—SCBG23.171113.349
F. langkokensisfilan11China—Guangdong24.227112.006
F. ruficaulis var. antaoensisfiruf4Taiwan21.962120.811
F. trilobafitri13China—SCBG23.180112.537
FicusFrutescentiaeF. abelifiabe12China—Guangdong23.636113.780
F. erecta var. beecheyanafiere11China—Guangdong23.765113.915
F. formosafifor6China—Guangdong23.623113.811
F. heteromorphafihet15China—Guangdong24.918113.033
F. ischnopodafiisc11Thailand—Chiang Mai18.50498.665
F. panduratafipan13China—Guangdong24.252112.036
F. pyrifomisfipyr12Thailand—Chiang Mai18.50498.665
F. variolosafivar13China—Guangdong23.624113.797
SycidiumSycidiumSycidiumF. montanafimon3Thailand7.55799.776
SycomorusSycomorusSycocarpusF. fistulosafifis3China—Guangdong23.156112.511
F. hispidafihis8China—SCBG23.180113.350
NeomorpheF. variegatafivae9China—Guangdong23.176112.538
HemicardiaF. semicordatafisem11Thailand—Chiang Mai19.36298.922
UrostigmaUrostigmaConosyceaF. altissimafialt19China—SCBG23.188113.363
F. benjaminafiben7China—SCBG23.186113.358
F. microcarpafimic13China—SCBG23.178113.352
UrostigmaF. rumphiifirum13Myanmar21.96696.069
Table
Table 2. Positive selection analysis of FTPS genes occurred in more than four species under branch model test.
Table 2. Positive selection analysis of FTPS genes occurred in more than four species under branch model test.
GeneModelnpMates of ParameterlnLdf2ΔlnLp-Value a
FTPS1Free-ratio79Variable ω−4424.4693835.0580.606
One-ratio410.522−4441.998
FTPS2Free-ratio67Variable ω−5413.2963233.8350.379
One-ratio350.449−5430.214
FTPS3Free-ratio67Variable ω−5872.8223237.5590.229
One-ratio350.679−5891.602
FTPS4Free-ratio59Variable ω−5024.7472821.2980.813
One-ratio310.496−5035.396
FTPS5Free-ratio51Variable ω−3504.0332419.1380.745
One-ratio270.345−3513.602
FTPS6Free-ratio23Variable ω−3252.649203.6130.999
One-ratio130.465−3254.456
FTPS7Free-ratio43Variable ω−3883.7142019.8830.465
One-ratio230.503−3893.656
FTPS8Free-ratio43Variable ω−6311.5602014.6850.794
One-ratio230.504−6318.902
FTPS9Free-ratio23Variable ω−3433.342108.1370.615
One-ratio130.479−3437.411
FTPS10Free-ratio35Variable ω−4081.2201626.3370.049 *
One-ratio190.426−4094.389
FTPS11Free-ratio27Variable ω−3358.7801218.8820.091
One-ratio150.499−3368.221
FTPS12Free-ratio27Variable ω−3715.0701223.2050.026 *
One-ratio150.517−3726.673
FTPS13Free-ratio23Variable ω−4886.043106.8410.740
One-ratio130.382−4889.464
FTPS14Free-ratio15Variable ω−3614.771610.3290.111
One-ratio90.637−3619.935
FTPS15Free-ratio15Variable ω−3086.01264.5300.605
One-ratio90.250−3088.278
FTPS16Free-ratio15Variable ω−3125.85966.1150.410
One-ratio90.758−3128.916
FTPS17Free-ratio19Variable ω−3127.50183.0090.934
One-ratio110.447−3129.006
FTPS19Free-ratio15Variable ω−2834.75364.8010.570
One-ratio90.407−2837.153
FTPS20Free-ratio23Variable ω−3453.67273.6390.820
one-ratio130.548−3455.491
FTPS26Free-ratio23Variable ω−5711.069814.1550.078
one-ratio130.489−5718.147
a Significantly different values according to the χ2 value with specific k degrees of freedom at a significance level of 0.05 (*).
Table
Table 3. Positive selection analysis of FTPS genes occurred in more than four species under branch site model test.
Table 3. Positive selection analysis of FTPS genes occurred in more than four species under branch site model test.
GeneCladeModels Comparednpp-Value alnLω ValuesPositive Sites (BEB) b
FTPS3fimonModel A381.490 × 10−2 *−5595.802p0 = 0.023 ω1 = 1.000 ω2 = 44.817
Model A null37 −5598.162p0 = 0.023 ω1 = 1.000 ω2 = 1.000
fipyrModel A386.105 × 10−4 **−5490.893p0 = 0.015 ω1 = 1.000 ω2 = 315.194
Model A null37 −5496.038p0 = 0.018 ω1 = 1.000 ω2 = 1.000
FTPS4fihir-fitriModel A340.051−4860.385p0 = 0.000 ω1 = 1.000 ω2 = 92.201258S * 345Y * 424M *
Model A null33 −4861.730p0 = 0.000 ω1 = 1.000 ω2 = 1.000
FTPS8fihetModel A260.059−5773.442p0 = 0.047 ω1 = 1.000 ω2 = 28.837217H **
Model A null25 −5776.607p0 = 0.047 ω1 = 1.000 ω2 = 1.000
FTPS16fifisModel A131.129 × 10−3 **−3118.162p0 = 0.417 ω1 = 1.000 ω2 = 314.584
Model A null12 −3122.826p0 = 0.408 ω1 = 1.000 ω2 = 1.000
FTPS20fiforModel A166.005 × 10−7 **−3399.251p0 = 0.588 ω1 = 1.000 ω2 = 246.640
Model A null15 −3427.039p0 = 0.626 ω1 = 1.000 ω2 = 1.000
a Significantly different values according to the χ2 value with specific k degrees of freedom at a significance level of 0.01 (**) or 0.05 (*). b Significantly different values with p > 95% (*) and p > 99% (**).
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Sun, P.; Chen, X.; Chantarasuwan, B.; Zhu, X.; Deng, X.; Bao, Y.; Yu, H. Composition Diversity and Expression Specificity of the TPS Gene Family among 24 Ficus Species. Diversity 2022, 14, 721. https://doi.org/10.3390/d14090721

AMA Style

Sun P, Chen X, Chantarasuwan B, Zhu X, Deng X, Bao Y, Yu H. Composition Diversity and Expression Specificity of the TPS Gene Family among 24 Ficus Species. Diversity. 2022; 14(9):721. https://doi.org/10.3390/d14090721

Chicago/Turabian Style

Sun, Peng, Xiaoyong Chen, Bhanumas Chantarasuwan, Xueying Zhu, Xiaoxia Deng, Ying Bao, and Hui Yu. 2022. "Composition Diversity and Expression Specificity of the TPS Gene Family among 24 Ficus Species" Diversity 14, no. 9: 721. https://doi.org/10.3390/d14090721

APA Style

Sun, P., Chen, X., Chantarasuwan, B., Zhu, X., Deng, X., Bao, Y., & Yu, H. (2022). Composition Diversity and Expression Specificity of the TPS Gene Family among 24 Ficus Species. Diversity, 14(9), 721. https://doi.org/10.3390/d14090721

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop