The calcium‐dependent protein kinase (CDPK) and CDPK‐related kinase gene families in Hevea brasiliensis—comparison with five other plant species in structure, evolution, and expression

Calcium‐dependent protein kinases (CDPKs or CPKs) play important roles in various physiological processes of plants, including growth and development, stress responses and hormone signaling. Although the CDPK gene family has been characterized in several model plants, little is known about this gene family in Hevea brasiliensis (the Para rubber tree). Here, we characterize the entire H. brasiliensis CDPK and CDPK‐related kinase (CRK) gene families comprising 30 CDPK genes (HbCPK1 to 30) and nine CRK genes (HbCRK1 to 9). Structure and phylogeny analyses of these CDPK and CRK genes demonstrate evolutionary conservation in these gene families across H. brasiliensis and other plant species. The expression of HbCPK and HbCRK genes was investigated via Solexa sequencing in a range of experimental conditions (different tissues, phases of leaf development, ethylene treatment, and various abiotic stresses). The results suggest that HbCPK and HbCRK genes are important components in growth, development, and stress responses of H. brasiliensis. Parallel studies on the CDPK and CRK gene families were also extended to five other plant species (Arabidopsis thaliana, Oryza sativa, Populus trichocarpa, Manihot esculenta, and Ricinus communis). The CDPK and CRK genes from different plant species that exhibit similar expression patterns tend to cluster together, suggesting a coevolution of gene structure and expression behavior in higher plants. The results serve as a foundation to further functional studies of these gene families in H. brasiliensis as well as in the whole plant kingdom.

Calcium-dependent protein kinases (CDPKs or CPKs) play important roles in various physiological processes of plants, including growth and development, stress responses and hormone signaling. Although the CDPK gene family has been characterized in several model plants, little is known about this gene family in Hevea brasiliensis (the Para rubber tree). Here, we characterize the entire H. brasiliensis CDPK and CDPK-related kinase (CRK) gene families comprising 30 CDPK genes (HbCPK1 to 30) and nine CRK genes (HbCRK1 to 9). Structure and phylogeny analyses of these CDPK and CRK genes demonstrate evolutionary conservation in these gene families across H. brasiliensis and other plant species. The expression of HbCPK and HbCRK genes was investigated via Solexa sequencing in a range of experimental conditions (different tissues, phases of leaf development, ethylene treatment, and various abiotic stresses). The results suggest that HbCPK and HbCRK genes are important components in growth, development, and stress responses of H. brasiliensis. Parallel studies on the CDPK and CRK gene families were also extended to five other plant species (Arabidopsis thaliana, Oryza sativa, Populus trichocarpa, Manihot esculenta, and Ricinus communis). The CDPK and CRK genes from different plant species that exhibit similar expression patterns tend to cluster together, suggesting a coevolution of gene structure and expression behavior in higher plants.
The results serve as a foundation to further functional studies of these gene families in H. brasiliensis as well as in the whole plant kingdom.
Plants have evolved a series of survival mechanisms to adapt to diverse environmental challenges, including drought, salinity, wounding, and low temperatures [1][2][3][4]. Calcium (Ca 2+ ), functioning as a second messenger of plant cells, plays an essential role in various signaling transduction pathways [5,6]. The changes in Ca 2+ concentration are sensed by several Ca 2+ sensors or Ca 2+ -binding proteins. To date, three major classes of Ca 2+ -binding proteins, including calcium-dependent protein kinases (CDPKs or CPKs), calmodulins (CaMs), and CaM-like proteins (CMLs) and calcineurin B-like proteins (CBLs), have been characterized in higher plants [7,8]. The CDPKs constitute one of the largest calcium-sensing subfamilies of serine/ threonine protein kinases that have been identified throughout the plant kingdom, from algae to angiosperms as well as in some protozoans, but not in animals [9]. The CDPK proteins have four characterized domains: an N-terminal variable region, a Ser/Thr kinase catalytic domain, an autoregulatory/autoinhibitory domain, and a calmodulin-like domain containing EF-hands for Ca 2+ binding [10][11][12].
Accumulating evidence indicates that CDPKs participate in plant responses to a variety of abiotic and biotic stresses, as well as in plant development [13][14][15]. In Arabidopsis thaliana, CPK10 participates in abscisic acid (ABA) and Ca 2+ -mediated stomatal regulation in response to drought stress [16]. Two homologs, AtCPK4 and AtCPK11, acting as positive regulators in ABA signaling pathways, are involved in seed germination, seedling growth, stomatal movement, and salt stress tolerance [17]. Another CDPK gene, AtCPK12, has been characterized as a negative ABA-signaling pathway regulator [18], while AtCPK6 demonstrates crucial roles in responses to drought and salt stresses [19]. In Oryza sativa, transgenic plants overexpressing OsCDPK7/OsCPK13 show enhanced resistance to cold, drought, and salt stress [20]. OsCDPK13/ OsCPK7 and OsCPK21 are involved in responses to cold and salt stress, respectively [21,22]. In Nicotiana tabacum, two CDPK genes, NtCDPK2 and NtCDPK3, play an essential role in the defense response, and NtCDPK2 functions together with the stress-induced MAPKs (mitogen-activated protein kinases) to control response specificity to abiotic and biotic stresses [23,24]. In addition, genome-wide expression patterns of CDPK genes have been characterized in O. sativa, Zea mays, Populus trichocarpa, and Brassica napus, and they point to important roles in the regulation of abiotic stresses, hormones, and the intrinsic developmental program in plant growth and development [25][26][27][28].
Natural rubber (cis-1,4-polyisoprene, NR) is an important industrial raw material, and the sole commercial source of NR is Hevea brasiliensis (the Para rubber tree), a perennial tropical tree species [29]. NR is synthesized and stored in the laticifer cells, which are differentiated from the cambium and arranged in concentric rings in the phloem region [29,30]. The bark of rubber tree is incised every 2-3 days to sever the laticifer rings and collect the latex, and this process is called tapping [29]. At each tapping, several tens to a few hundred milliliters of latex per tree are expelled from the laticifers and harvested for sustainable rubber production. Application of ethylene gas or ethephon (2-chloroethylphosphonic acid, an ethylene generator) to the trunk bark of the rubber tree increases rubber yield. However, the underlying mechanisms in ethylene stimulation are yet to be elucidated, although ethylene signaling and response are assumed to play critical roles [29]. In a previous report, one CDPK gene (HbCDPK1) was shown to be induced by ethephon in H. brasiliensis [31]. Both environmental and harvesting stresses affect rubber yield. For example in tapping, the moderate stresses generate a positive stimulatory effect on latex production in virgin (previously untapped) rubber trees, bringing the rubber yield from a meager exudation at the first tapping to more than a hundred milliliters after 7-10 consecutive tappings [32]. However, excessive environmental and harvesting stresses, especially a combination of overtapping and overstimulation by ethylene or ethephon, lead to the occurrence of tapping panel dryness (TPD), a physiological disorder that can result in complete stoppage of latex flow in the rubber tree [29]. In view of the importance of CDPK genes in stress responses and hormone signaling in other plants [6], it is worthwhile investigating the CDPK gene family in the rubber tree in relation to the regulation of latex production.
In this study, we performed a genome-wide analysis of the CDPK and CRK gene families in H. brasiliensis and compared the results with those from five other plant species, i.e. Manihot esculenta, Ricinus communis, A. thaliana, O. sativa, and P. trichocarpa. The study encompassed a total of 161 CDPK and 45 CDPKrelated kinase (CRK) genes, the expression patterns of which were analyzed in different plant tissues in response to various treatments, and at several phases of tissue development. In addition, the gene structure and phylogeny of these genes were also compared, and all the results obtained will help further understanding of the roles of CDPK and CRK genes in the regulation of latex production and regeneration. Data on CDPK and CRK genes and their expression in six plant species would also contribute to the understanding of the structure of these gene families and the functions of their members in the plant kingdom.

Results and Discussion
Genome-wide identification of CDPK and CRK gene families in H. brasiliensis and five other plant species We identified all CDPK and CRK gene family members in six plant species (H. brasiliensis, A. thaliana, O. sativa, P. trichocarpa, M. esculenta, and R. communis) from their published genome sequences [33][34][35][36][37][38][39]. The latest genome and protein sequences of these species were downloaded from Phytozome v10. Local blast searches of the genomes were performed by using the published CDPK and CRK sequences of three model plants of A. thaliana, O. sativa, and P. trichocarpa as queries [12,26,28,40]. This analysis identified a total of 161 CDPK genes and 45 CRK genes in the six plant species, including 39 H. brasiliensis CDPK and CRK genes (HbCPK1 to 30, and HbCRK1 to 9,  Table S1). The molecular weights of the predicted 206 CDPK and CRK proteins range from 48.5 to 72.3 kDa, while their isoelectric points (pI) fall between 5.02 and 9.36 (Table S1).

Phylogenetic analysis of the CDPK and CRK gene families
In order to establish the phylogenetic relationships in the CDPK and CRK gene families among H. brasiliensis and the five other plant species, we aligned the 206 plant CDPK and CRK protein sequences and constructed a phylogenetic tree as shown in Fig. 1. The plant CDPK and CRK proteins that clustered into five major groups with high bootstrap values are named CPKI, CPKII, CPKIII, CPKIV, and CRK. The proteins in three CPK groups (I-III) and the CRK group are further classified into a number of distinct subgroups consisting of both dicot and monocot proteins. The 39 H. brasiliensis genes are scattered among the five groups (CPKI, CPKII, CPKIII, CPKIV, and CRK) with 11,8,9,2, and 9 isoforms, respectively. Similarly, the CDPK and CRK family genes in the five other species are also clustered into different groups (CRKI, II, III, and IV, and CRK): 10, 13, 8, 3, and 8 isoforms, respectively, in A. thaliana; 11, 8, 8, 2, and 5 in O. sativa; 11, 8, 9, 2, and 9 in P. trichocarpa; 8, 5, 7, 2, and 9 in M. esculenta; 5, 5, 5, 1, and 5 in R. communis (Table S1, Fig. 1). Unlike the five other plant species, A. thaliana has a smaller number of genes in the CPK I group than in the CPK II group, which is apparently due to the striking expansion of the subgroup of CPK IIe in A. thaliana (Fig. 1). In all six plant species, CPK IV has the smallest number of genes among the five CPK and CRK groups, and is most closely related to the CRK group rather than the other three CPK groups in the phylogenetic tree ( Fig. 1), suggesting the divergence of CPK IV and CRK from a common ancestor comparatively recently. Generally, the HbCPK isoforms are more closely related to their putative CDPK orthologs in M. esculenta and R. communis, which are in the same family of Euphorbiaceae with H. brasiliensis, reflecting consistency in the evolution of CDPK isoforms and plant lineages.

Structural organization of CDPK and CRK genes
The exon-intron structures of the 205 CDPK and CRK genes in six plant species were determined based on the predicted sequences. As shown in Fig. 3A, most H. brasiliensis CDPK members within the same groups share very similar gene structures in terms of intron number, domain localization, and exon length. Although the lengths vary, introns are inserted into nearly the same locations of the gene ORFs. There are 6, 7-8, 6-9, 10, and 11 introns in the CPK I, CPK II, CPK III, CPK IV, and CRK groups, respectively. The number of introns in different CPK and CRK groups is broadly similar in the five other plants examined (Fig. 3B-F), containing mainly 5-8 introns in CPK I to III groups, and 11-12 introns in CPK IV and CRK groups. The similar exon-intron structure shared by CPK IV and CRK genes reflects their close phylogenetic relationship. Most CDPK members contain two EF-hand pairs, except for a few members, such as AtCPK25, which contains only one EF-hand pair. It is noted that the EF-pair domain in group CRK is a degenerative form of the EF-hand domain that could be predicted with Gene3D but not with Pfam and ProSiteProfiles as used for the identification of the EF-hand pairs in CPKs. In addition, the lengths of most AtCPK genes are shorter than those of the other plant CDPK genes, suggesting a correlation of the CDPK gene length with the genome size of its source species.

Expression analysis of CDPK and CRK genes in six plant species
Increasing evidence indicates that CDPKs are involved in various physiological adaptations [17,19,42]. Such functions are indicated by the expression of CDPK genes in response to various stimuli, for example, hormones, salt, cold, drought, heat, and wounding. To understand the potential functions of specific members of CDPKs and CRKs in the six plant species, their expression patterns in different tissues, developmental stages, and under different stress treatments were analyzed. The Solexa sequencing data available at the NCBI Sequence Read Archive (SRA) database were used for the expression analysis of CDPK and CRK genes in five species, i.e. M. esculenta, R. communis A. thaliana, P. trichocarpa, and O. sativa. In the case of H. brasiliensis, two sets of Solexa sequencing data were used, one from the NCBI SRA database (http:// www.ncbi.nlm.nih.gov/nuccore/448814761) and the other from our own data [38]. As shown in Fig. 4A, transcripts of nine HbCPK genes (HbCPK1, 2, 3, 7, 14, 17, 22, 25, 26, and 28) and five HbCRK genes (HbCRK4, 5, 6, 7, and 8) were barely detectable in almost all the tissues and all the treatments examined. Such genes comprise a large portion (~1/3) of the total HbCPK and HbCRK gene family. This character seems to be shared by the CDPK and CRK gene families in other plant species. For example, similar expression patterns were observed for eight of 31 CDPK and CRK genes in M. esculenta (Fig. 4B), seven of 21 in R. communis (Fig. 4C), 15 of 42 in A. thaliana (Fig. 4D), 13 of 39 in P. trichocarpa (Fig. 4E), and 13 of 35 in O. sativa (Fig. 4F). This result suggests that the CDPK and CRK gene families in higher plants may have experienced an event of gene expansion followed by nonfunctionalization in the course of evolution [43]. Due to the data limitation of the SRA database, some analyses may not be so accurate and need further verification. For example, AtCPK16 and AtCPK20 are highly expressed but only in pollen which has not been analyzed here [44]. Among the expressed H. brasiliensis genes, HbCPK24 and HbCRK9 were special for their highly specific expression, which showed abundant expression in the leaf when exposed to low temperature and in the seed, respectively, but their expression were otherwise hardly detected in nearly all the tissues and treatments (Fig. 4A). In comparison, the remaining HbCPK and HbCRK genes were expressed in a wide range of tissues, showing dissimilar but partially overlapping patterns of expression. In the bark, where the rubberproducing laticifers are located, 16 HbCPK genes were expressed more abundantly than in the other tissues (Table S1-1, Fig. 4A), and these genes may contain the key CDPK member s involved in signaling transduction in the rubber tree bark. HbCPK13 and 27 were abundantly expressed in latex, essentially the cytoplasm of laticifers, whereas HbCPK10 was mainly expressed in leaves (Table S1-1, Fig. 4A). To obtain further information on the HbCPK and HbCRK genes in leaf development, we examined their expression levels by RNA-seq at four progressive stages of leaf development (bronze, color change, pale-green, and mature). As shown in Fig. 4A and Table S1-1, most of the HbCPK and HbCRK genes were obviously down-regulated during leaf development, and their expression was mainly restricted to the first three stages (bronze, color change, and pale-green). This result is in agreement with the assay of CDPK activity in maize, in which expanding tissues such as rapidly growing tips, leaves, and coleoptiles have particularly high levels of CDPK activity, while mature leaves have much less activity [45]. The involvement of CDPK genes in early developmental processes such as embryogenesis, seed development, and germination has been reported in sandalwood [46]. Here, we reveal that one R. communis CDPK gene in subgroup CPK IIc, RcCPK12, was mainly expressed in the early stages of embryo development and the male developing flowers, suggesting a similar function. It is interesting that the other members of this subgroup were either expressed at a low level or they showed no expression. Ethephon, an ethylene generator, is widely used in H. brasiliensis to stimulate rubber production, but the yield-stimulating mechanisms are still poorly understood [29,31]. Ethylene-mediated cross-talk has been observed between CDPK and MAPK, indicative of signaling that controls stress responses in plants [23]. In this study, we examined the expression levels of HbCPK and HbCRK genes in latex under ethephon treatment. As shown in Fig. 4A and Table S1-1, the expressions of two CPK genes, HbCPK9 and 15, were obviously up-regulated by ethephon treatment, implicating them in the ethylene-simulated latex production. Further studies on these genes would help to understand the underlying molecular mechanism. Although the expression of one HbCPK gene, HbCDPK1 (HbCPK30 in this study) has been reported to be markedly induced by ethephon [31], it was expressed mainly in the leaf and only marginally in latex in the present study (Fig. 4A). In P. trichocarpa, most of the CDPK and CRK genes were associated with ABA activity, but several genes were also affected by MeJa (methyl jasmonate), which apparently downregulated the expression of PtCDPK1, 4, and 11 (Fig. 4E, Table S1). In O. sativa, many CDPK genes were up-regulated by treatment with ABA, GA (gibberellic acid), or ABA plus GA. In a previous study, AtCPK4 and AtCPK11 have been demonstrated to be involved in ABA signaling-mediated regulation of seed germination, seedling growth, stomatal movement, and salt stress tolerance [17]. Together, these results suggest that the CDPK and CRK gene families are  implicated in the regulation of plant growth and development through participation in the signaling pathways of various plant hormones, especially ABA.
As low temperature is the major obstacle to the expansion of rubber planting areas in the subtropics, the studies of the low-temperature response and adaptation  in this species would be helpful in breeding rubber trees for cold tolerance. Recently, Ma et al. [47] found that low temperature sensing in rice was related to changes in Ca 2+ influx during cold treatment, and the change in Ca 2+ influx was controlled by COLD1, a regulator of Gprotein signaling. In this study, the expression levels of HbCPK and HbCRK genes (Ca 2+ sensors) were examined under cold stress. Many HbCPK and HbCRK genes are regulated by cold stress, and most of them showed depressed expression after low-temperature treatment (Fig. 4A, Table S1-1). In three of the other plants examined (A. thaliana, O. sativa, and P. trichocarpa), the expression of many CDPK and CRK genes was also regulated by low temperatures, but different expression patterns were displayed by respective genes (Fig. 4D-F; Table S1-4, 1-5 and 1-6). In addition, the expression data used in this study also show some discrepancies with other published studies. For example in rice, OsCPK9 and OsCPK21 have been reported to be induced by salt stress and ABA [22,48], which is not observed in Fig. 4F. These differences may be due to different approaches, kinetics, or studied organs/ tissues. The expression levels of CDPK and CRK genes in H. brasiliensis, A. thaliana, O. sativa, and P. trichocarpa were also examined under other kinds of stresses, including fungus infection, drought, and salt. The results showed that these treatments could also regulate the expression of many CDPK genes (Fig. 4A, D-F, Table S1). Under these stresses, the H. brasiliensis and P. trichocarpa CDPK and CRK genes were mainly down-regulated, while the A. thaliana and O. sativa genes were mainly up-regulated (Table S1). To verify the expression patterns of the RNA-seq analyses, quantitative reverse transcriptase PCR (qPCR) expressional analyses were performed on HbCPK genes, which are among the isoforms somewhat regulated by ethephon treatment and leaf development, or tissue-specific as revealed by RNA-seq data ( Fig. 4A; Table S1-1). First, qPCR expressional analyses were performed with the RNA samples from the same cultivated varieties as Solexa sequencing (our own data). As shown in Fig. 5, the results from sequencing-based expression analyses were broadly similar to the qPCR results. Second, new RNA samples were used to further verify the expression patterns of HbCPK genes which are regulated by the ethephon treatment, the new RNA samples were different from the samples of Solexa sequencing and have three biological replicates. As shown in Fig. 6, the results from sequencing-based expression analyses were broadly similar to the qPCR results at least one time point post treatment. The RNA-seq data were also verified by our published analyses [38,49]. Above all, we can verify that the results of the RNA-seq analyses on HbCPK genes are authentic.

Expression analysis of CDPK and CRK genes based on structure and evolution
The two closely related genes in each of the CDPK and CRK paralogous gene pairs mentioned earlier may have similar cellular localization and similar roles, as reported for the A. thaliana AtCPK4 and AtCPK11 [17]. However, when the expression patterns of the H. brasiliensis paralogous gene pairs were analyzed (Fig. 4A), the two genes in the same gene pair displayed four different expression patterns. First, in the case of three gene pairs (HbCPK1/25, HbCPK2/7, and HbCPK22/26), both genes were expressed either at a low level or not at all in all the tissues and treatments examined. Second, in the gene pair of HbCPK5/12 and HbCPK18/19, both genes exhibited a similar pattern of universal expression across all tissues. Third, for the gene pair of HbCRK3/6, the two genes displayed dissimilar patterns of expression, with HbCRK3 showing universal expression but HbCRK6 being expressed at very low levels in all the tissues or treatments. Fourth, for the gene pair of HbCPK24/28, HbCPK28 was barely expressed in all the tissues or treatments, but the expression of HbCPK24 was only restricted in leaf when exposed to the low-temperature treatment. We also investigated the expression patterns of the genes pairs or clusters that are closely located on chromosomes of A. thaliana, O. sativa, and P. trichocarpa (Fig. 2). The results showed that the two paralogous genes in most of the gene pairs shared similar expression except AtCPK5 and 6 ( Fig. 4D-F). AtCPK5 was expressed at very high levels in all the samples examined, while AtCPK6 was mainly specifically expressed in the root. In the paralogous gene cluster of AtCPKs in subgroup CPK IIe, only AtCPK21 was expressed in all the tissues and treatments, whereas the other members (AtCPK22, 23, 27, and 31) were expressed at low levels or not at all in all the samples examined. Interestingly, the CDPK and CRK genes from different plant species that exhibited low or no expressions in nearly all the samples examined tended to cluster in the same subgroups, for example, CPK Ib and c, CPK IIa and c, CPK IIIa and CRK a (Figs 1 and 4). This kind of phenomenon can be extended to the universally expressed CDPK and CRK genes from different species. These findings suggest that the structures of some CDPK and CRK gene families coevolved with their expression patterns in higher plants.

Conclusion
In this study, we pioneered a genome-wide analysis of two protein kinase subfamilies (CDPK and CRK) in H. brasiliensis, M. esculenta, and R. communis. In silico analysis of the H. brasiliensis genome database allowed the identification of 30 HbCPK and 9 HbCRK genes. The phylogenetic analysis of CDPKs and CRKs from H. brasiliensis and five other plant species (A. thaliana, O. sativa, P. trichocarpa, M. esculenta, and R. communis) showed the classification of these genes into five major groups. Members within each group might have recent common evolutionary origins, as they share common protein motifs and exon-intron structures. Solexa sequencing analyses revealed that most H. brasiliensis CDPK and CRK genes exhibit different patterns of expression under a number of experiments, suggesting their distinct roles in developmental and stress responses. Relevant studies of gene evolution and expression have been extended to the five other plant species. The results presented here provide a foundation for further functional investigation of the CDPK and CRK gene families in H. brasiliensis as well as the whole plant kingdom.

Materials and methods
Database search for CDPK and CRK genes in H. brasiliensis and five other plant species Sequences of A. thaliana, P. trichocarpa, and O. sativa CDPKs and their closely related CRK genes were downloaded from the A. thaliana Information Resource (http:// www.Arabidopsis.org/), the rice genome annotation database (http://rice.plantbiology.msu.edu/) and GenBank (http://www.ncbi.nlm.nih.gov/genbank), respectively. The genome and protein sequences of A. thaliana, O. sativa, P. trichocarpa, M. esculenta, and R. communis were downloaded from Phytozome v10 (http://www.phytozome.net/). The H. brasiliensis genome and transcriptome data were obtained from GenBank (http://www.ncbi.nlm.nih.gov/nucc ore/448814761) and our own data [38]. Local BLAST alignment was performed using published CDPK and CRK sequences form A. thaliana, P. trichocarpa, and O. sativa as queries to search against the deduced proteome of each species for the candidate CDPKs and CRKs from H. brasiliensis, A. thaliana, O. sativa, P. trichocarpa, M. esculenta, and R. communis. All putative candidates were manually verified with the InterProScan server (http://www.ebi.ac.uk/Tools/ pfa/iprscan/) to confirm the presence of protein kinase and EF-hand domains.

Phylogenetic and gene structure analyses
Multiple alignments of the amino acid sequences of CDPKs and CRKs from six species were performed using the CLUS-TAL X (version 1.83, http://www.clustal.org/) program. The phylogenetic tree was constructed with MEGA6.0 (http:// www.megasoftware.net/) by employing the Neighbor-Joining (NJ) method with a bootstrap test for 1000 replicates. Exon-intron structures of the six species of CDPK and CRK genes were analyzed by comparing the cDNA and their genomic DNA sequences through the web server GSDS 2.0 (http://gsds.cbi.pku.edu.cn/).

Supporting information
Additional Supporting Information may be found online in the supporting information tab for this article: Table S1. Characteristics of CDPK and CRK genes in six plant species (H. brasiliensis, A. thaliana, P. trichocarpa, and O. sativa). Table S2. Basic information for the Solexa sequencing data of Hevea brasiliensis and five other plant species.