How different is similar – exploring development of Ceropegia sandersonii pitfall owers to elucidate convergent evolution of oral synorganization

Background: Lantern plants from the genus Ceropegia (Apocynaceae-Asclepiadoideae) have radially symmetric pitfall owers that are an outstanding example of functional oral complexity with high synorganization of specialized organs. The evolutionary origin and development of these complex owers is unclear, and the genetic background of oral organ formation is unknown. Flowers with similar deceptive pollination strategies and oral traits convergently evolved in non-related plant lineages. The partially bilaterally attened trap owers of pipevines are functionally similar to Ceropegia pitfall owers; many orchid taxa evolved complex fully bilaterally attened owers with specialized organs to trap pollinators. This study is the rst to investigate the genetic background of pitfall ower development in Ceropegia, and to explore (i) convergent evolution of extremely synorganized and complex owers as well as (ii) the homology of a highly specialized oral organ, the gynostegial corona. Methods: We obtained transcriptomes from C. sandersonii early oral buds and mature sepals, petals, and gynostegia, and analyzed differential expression of selected MADS-box genes in buds and mature oral organs using RT-PCR. In addition, we studied oral ontogeny and vascularization using SEM and 3D X-ray micro-CT scanning. Results: We identied ten phases of oral development from primordia to mature owers, and for the rst time visualized the vascular system of mature Ceropegia pitfall owers in a 3D-model. We identied 14 MADS-box gene homologs, representing all major MADS-box gene classes, in the oral transcriptomes of Ceropegia. Vascular bundle patterns, as revealed by 3D X-ray micro-CT scanning, in combination with high expression of GLOBOSA and AGAMOUS indicate a staminoid origin of this highly specialized oral organ which starts developing from stage seven onwards. Interestingly, AGAMOUS-LIKE6 was neither expressed in early oral buds nor in any mature oral organ, in line with the radial symmetry of all Ceropegia oral organs. Conclusion: We detected differential expression of MADS-box genes involved in Ceropegia oral organ identity and propose a new ABCDE-model for parachute owers. We compare this with current models of unrelated plants with similar oral traits but (partially) bilaterally attened owers, i.e. Aristolochia mbriata and Erycina pusilla. With this comparative approach we lay the foundation for understanding the genetic mechanisms driving convergent evolution of highly specialized deceptive trap owers. PCR. Actin as non-template control (NTC). CFX384 Touch Real-Time PCR system thermocycler (Biorad). a 1% agarose 1x TAE GeneRuler TM (Thermo Scientic)

osmophoric tissue as well as slippery surfaces promoting the trapping of pollinators. In many species the ower tip is decorated with movable hairs likely to support y attraction (see 10). The cylindrical ower tube with slippery surfaces and downward-pointing hairs on the inside make insects fall to be temporarily trapped inside the basal corolla in ation that encloses the gynostegium (see 10). In addition to various devices to catch, temporarily trap and nally release pollinators (10,11), the ultimate oral complexity is highlighted by pollen transfer via pollinaria, i.e. discrete pollen packages, which are mechanically clipped to the mouthparts of speci c ies. Successful pollination requires insertion of a pollinium (clipped to a y in a donor ower), into one of the ve lateral guide rails on the gynostegium of a receptive ower. For functioning of this elaborate pollinator trap, each oral organ had to evolve into a very speci c shape at a very particular position (2). The highly synorganized Ceropegia ower and the functionality of its oral organs, singly and joint, aroused the interest of naturalists more than a century ago (12,13). The most detailed and passionate descriptions of oral structure, functional oral parts and tissues, and their complex interaction with ies to achieve pollination were published by Vogel (9,10,14,15). Only recently, studies on chemical ecology of Ceropegia species (16)(17)(18)(19)(20) elucidated fascinating chemical mimicry strategies such as kleptomyiophily, i.e. mimicry of injured or dead insects as speci c food items of kleptoparasitic ies. Apparently, oral chemistry is the key for pollinator attraction in Ceropegia, while oral morphology is crucial for successful pollination. However, the molecular genetic processes controlling oral development of Ceropegia owers have not yet been studied and genes which are involved in the formation of speci c oral organs are unknown.
Additional novel organs, positioned between the oral whorls of petals and of stamens such as coronas/stelidia (involved in pollination), and pollinia (discrete pollen packages or clustered pollen) convergently evolved in Apocynaceae (Gentianales) and Orchidaceae (Asparagales). Furthermore, pollinator trap owers and gynostegia/gynostemia (fusion of male and female reproductive organs) evolved in a third unrelated plant family, pipevines (Aristolochiaceae, Piperales). The mechanisms and strategies to trap y pollinators also show similarities and are extremely divers within these three unrelated plant families. Many deceptive Orchidaceae attract their y pollinators through sexual or other chemical mimicry, and trap them with the help of cavern-like or motile labellum structures (e.g. Cypripedium, 21; Pterostylis, 22). In Aristolochiaceae, Aristolochia y pollinators are attracted by food source or oviposition site mimicry (23) and trapped in pitfall owers functionally comparable to Ceropegia owers, though the perianth in Aristolochia is sepal rather than petal derived (see 24). Such similarities in outstanding oral characters in three non-related plant families spread throughout the Angiosperms call for a comparative developmental study to reveal (dis)similarities in oral organ development and perianth identity.
In Aristolochia, genes involved in oral development and organ identity are continuously better understood (25)(26)(27), and in orchids, deceptive species such as Phalaenopsis equestris and Erycina pusilla are well studied with regard to the genetic background of oral development (28)(29)(30)(31)(32)(33)(34). Here we present the rst data on MADS-box gene expression during development of radially symmetric pitfall owers of Ceropegia sandersonii, the parachute plant; it is the rst oral transcriptome analyses for Ceropegia in general. We identi ed MADS-box genes expressed in early oral buds and mature owers, and carved out expression differences between early and late oral stages, as well as between different oral organs. To further explore the evolutionary origin of selected oral organs in Ceropegia, we visualized the vascular system of owers using 3D X-ray micro-computer tomography (micro-CT). Scanning electron microscopy (SEM) was applied to recognize distinct phases during corolla and gynostegium development. We compared our ndings to what is known from bilaterally attened deceptive orchids (E. pusilla, 33) and pipevines (A. mbriata, 26,27) to (i) explore convergent evolution of complex owers with extreme synorganization and (ii) investigate the homology of the highly specialized corona.

Early bud and gynostegium development
We roughly de ned ten phases during oral development of C. sandersonii with distinct changes in organ unfolding. Phase one (P1, Figure 1A) is the emergence of a oral primordium adaxially positioned to a oral bract. Phase two (P2, Figure 1B) is de ned by initiation of the ve sepal primordia. The third phase (P3, Figure 1C) de nes corolla initiation with ve petal primordia emerging in alternation to the sepals, and in penta-symmetrical order around the developing pollination apparatus. In phase four (P4, Figure   1D+E) the ve stamens initiate around the style head. In phase ve (P5, Figure 1F+G) the petals completely enclose the developing gynostegium with congenitally fused petal bases and postgenitally fused petal tips. In this level the stamens are clearly fused and the carpels are still separated. Colleters (glandular outgrowths of the sepals), start to develop as well in this phase. In phase six (P6, Figure 1H-J) the corolla is cylindrical and mainly formed by the postgenitally fused petal tips. Early stage ickering bodies are present on the edges of the petal tips and the "uvula" (see 9) is initiating. Stamens and style head are not yet postgenitally fused; the style head is still di-symmetric as both carpels are still distinct; colleters are prominent. In phase seven (P7, Figure 1K) the corona emerges, the guide rails become formed by anther wings, and corpuscula (parts of the pollinaria) are becoming visible. The carpels are postgenitally united and the style head has a pentagonal shape. In phase eight (P8, Figure 1N) the corona completely surrounds the gynostegium and is fused with the anthers; the nectar cavities are formed underneath the guide rails, and the corona lobe tips start to outgrow the style head. The corona outer surface becomes covered by long trichomes. In phase nine (P9, Figure 1O) the pollinia are formed and two pollinia from neighboring anthers become connected to the corpusculum to form a pollinarium; the corona lobe tips are at least as long as the gynostegium height. In the nal phase (P10, Figure 1P), the anthers back off, the pollen sacs release the pollinia, and the translator is hardened. Thus, gynostegium and pollinaria are fully developed, and the ower is mature.

Vascularization in mature owers
Vascular bundles were studied in three fully mature owers of Ceropegia sandersonii using micro-CT scanning. In the pedicel (Figure 2A, cross section 1), a vascular cylinder of ten (two times ve alternating) bundles could be discerned. One set of ve bundles forms the sepal supply ( Figure 2A+B, green) with one each becoming a sepal midrib bundle. The second alternating set of ve bundles ( Figure 2A+B

Differential gene expression-RT-PCR experiments
The expression of six isolated MADS-box gene homologs, i.e. CsanFUL2, CsanTM6, CsanGLO, CsanAG2, CsanAGL6, and CsanSEP1, was analyzed in early stage oral buds and in sepals, petals, gynostegia, and coronas of mature owers using semi-quantitative RT-PCR. Overall, these genes showed distinct expression patterns in time and space ( Figure 3). CsanFUL2 was found to be expressed in early oral buds, and in sepals and gynostegia of mature owers. CsanTM6 was only found to be active in mature sepals. CsanGLO was expressed in all tissue types analysed, CsanAG2 was active in early oral buds, and mature gynostegia and corona tissue. CsanSEP1 was expressed in mature petals and gynostegia, whereas CsanAGL6 was not expressed in any of the tissues analyzed.

Discussion
Our study is the rst comprehensive evolutionary developmental analysis of Ceropegia pitfall owers which belong to the most complicated owers within the Apocynaceae.
Although the basic oral Bauplan is similar in each species, the variety of oral shapes and colors is huge in Ceropegia. The Giant Ceropegia, C. sandersonii, has eye-catching parachute-like pitfall owers that are highly popular as ornamental plant. Besides being among the most charismatic and well-known Ceropegia species, it is also the only species thus far for which the pollination strategy has been fully elucidated: with its ower scent it mimics a defending honey bee attacked by a predator and thereby lures scavenger ies as unwilling pollinators (18).
We studied oral development in highly synorganized Ceropegia sandersonii pitfall owerscombining morphological (SEM and micro-CT) and molecular genetic (transcriptomics and RT-PCR) analyses. We, for the rst time, visualized and remodeled the vascular system in a Ceropegia pitfall owers. Comparing our results to what is known in other plant groups with similar oral traits and pollination strategies. i.e. Aristolochiaceae and Orchidaceae, provides new insights into the current picture of gene expression during development of specialized oral morphologies. Our comparative approach is a rst step to understand convergent evolution of trap owers that employ highly specialized oral organs (i.e. gynostegia/gynostemia, pollinia and corona/stelidia), and strategies (visual/olfactory deception) to ensure reproductive isolation and associated plant species diversi cation.

Morphological investigation of ower development
Though several morphological studies on ower development are published for Apocynaceae (e.g. 8,35,36), development of Ceropegia owers was never visualized from primordia to fully developed owers (but see 2, 10). We used scanning electron microscopy to circumscribe ower development of Ceropegia sandersonii from initiation of oral primordia to a mature ower. A SEM image sequence of successive ower development was created, and we could de ne ten developmental phases (P1-P10, Figure 1A-P) in which clear changes in organ formation take place. This framework lays the foundation for investigating gene expression during each of the de ned developmental phases to identify key genes responsible for consecutive changes. Flowers of C. sandersonii are special and different from many other species in ways (9). The ower tip forms a parachute-like structure with complex ontogenetic development (but see 9). This special morphology of the ower tip is not unique to C. sandersonii; petal tips fused in a parachute-like structure, though less marked than in C. sandersonii, is also a feature in a few other species, such as C. rendallii, C. mbriata, C. galeata, C. huberi, and C. connivens.
The development of the gynostegial corona only starts its development in phase P7 out of ten phases, which is relatively late. Late appearance of corona tissue has been described in other Ceropegia species (5) as well as in other Asclepiadoideae (see 35). The evolution of the gynostegial corona, as determined by previous morphological studies, was described to be of staminoid (androecial) origin for Asclepiadoideae (see 6). Coronas of corolline origin, and consequently with a distinct development, are known from several taxa spread over Apocynaceae (e.g. see 8).

Vascularization of Ceropegia pitfall owers
Novel 3D X-ray micro-computer tomography techniques (micro-CT) proved to be a powerful tool to study vascularization of orchid owers (e.g. Erycina pusilla, 33, Phalaenopsis equestris, 34). To the best of our knowledge this method has not yet been applied to any species in the Gentianales before. Usually, the positions and connections of vascular bundles are studied using microtome slicing and microscopic investigation of selected slices. The description of a vascular system is then based on a few selected slices and sketched sections. The disadvantage of this approach is that the entire vascular system hardly be visualized in its original three-dimensional structure. Studying vascularization with micro-CT provides a more comprehensive and informative insight into the evolutionary origin of different oral organs. The resulting 3-dimensional image of the entire vascular system can be rotated and studied from all possible perspectives to identify the origins of tissue speci c vascular bundles. These 3D data facilitate conclusions on the evolutionary origin of oral organs of unclear homologies.
We applied micro-CT scanning to study oral vascularization in mature Ceropegia sandersonii pitfall owers and our data allowed to re-model the vascular system of these complex owers which gave interesting new insights into how their different oral organs are connected via vascular bundles ( Figure  2). Each stamen is supported by a single vascular bundle which runs all the way through this oral organ without branching just as described for other Apocynaceae (37). We found that stamen bundles are formed by fusion of veins with separate origin, i.e. bundles that derive from two neighboring main bundles (Figure 2A, cross section 2; Figure 2B-4). The double origin of stamen bundles was already described and discussed in other plants (38,39). Also, vascular bundles running into the sepals were found to be of mixed origin, i.e. one main bundle is joined by secondary bundles branching off from an adjacent bundle ( Figure 2B-3). Carpels were found to be supplied by vascular bundles originating from stamen bundles (Figure 2A) which seems a logical consequence of the fusion event of androecium and gynoecium to form the gynostegium as a novel organ. We did not discern vascular bundles in the corona which con rms what was described for other Apocynaceae (37). Coronal structures generally seem to be non-vascular; the only bundles observed were marginal petal or staminal bundles (37). Absence of vascular bundles in the corona of Ceropegia sandersonii supports that this organ is an emergence of the stamens. It further suggests that the corona does not produce nectar via coronal nectaries as described for other species of Apocynaceae-Asclepiadoideae (35). The presence of nectar in C. sandersonii was never empirically veri ed. Thus, if present at all, the primary nectaries should be found beneath the guide rails, and the coronal nectar cups around the gynostegium only collect but not produce nectar (see 40). It would be very interesting to further explore whether in other species with coronal nectaries the coronas are not vascularized either.

MADS-box genes driving oral organ identity
We identi ed differentially expressed MADS-box genes involved in formation of highly synorganized Ceropegia pitfall owers. Based on our results and on what is generally known about MADS-box gene expression in owering plants (for review see 41), we propose a modi ed ABCDE model of oral organ identity for Ceropegia (Figure 4). According to this model, A-class genes are expressed in mature sepals and gynostegia. B-class genes are expressed in mature sepals, petals, corona, stamens, and the corona but not in the gynoecium (carpels and ovules). C-class genes are expressed in the mature corona, stamens, and carpels but not in the ovaries. D-class genes are only active in ripening ovules, and E-class genes are expressed in all oral organs.
In our Ceropegia sandersonii oral transcriptomes we could identify three A-class genes, i.e. two copies of FRUITFULL (FUL) and one copy of APETALA1 (AP1),, and our expression analyses indicated that A-class genes play a role in formation of mature sepals and gynostegia (see Additional le 6).
B-class genes generally control identity of petals and stamens. In our expression analyses we found that the GLOBOSA homolog CsanGLO is expressed in all investigated oral organs, which indicates a key function of this B-class gene in development of Ceropegia pitfall owers, in line with the oral quartet model. In the corona, a highly specialized organ situated in the oral whorl positioned between the whorls containing the stamens and corolla, and unique to Apocynaceae, CsanGLO was found to be expressed together with the C-class gene AGAMOUS (CsanAG2).. This, again, supports the idea that the corona in Asclepiadoideae is of staminal origin. Due to the exceptional diversity of corona morphology and development (petal or/and stamen derived, i.e. corolline or/and staminal) within Apocynaceae, the evolutionary origin and homology of this oral organ is not yet fully understood. Based on previous ontogenetic studies with traditional anatomical methods, the corona was assumed to be corolline in Rauvol oideae, Apocynoideae, and Periplocoideae, but typically of staminal origin in Asclepiadoideae and Secamonoideae (6, 8, but see 35). Our results further support this last hypothesis.
In orchids, combined MADS-box B-and C-class gene expression was also found in other highly specialized oral organs of staminodal origin in a oral whorl, situated between the petals and stamens, the stelidia. In the stelidia, however, expression of AGL6 was also detected, which was not found in the corona of C. sandersonii. Whether the presence of expression of AGL6 in orchid ower stelidia but absence in parachute ower coronas is correlated with the bilateral symmetry of the rst and radial symmetry of the latter organ remains to be investigated.
Interestingly, no AGL6 expression was detected in the early oral buds or any of the mature organs of C. sandersonii owers that are all radially symmetric. This nding is in contrast with previous ndings of Pabón-Mora et al. (26), who detected AGL6 expression in the bilaterally symmetric sepals and ovules of the pipevine Aristolochia mbriata and Dirks-Mulder et al. (33) and Pramanik et al. (34), who detected AGL6 expression in the bilaterally symmetric sepals, petals, stelidia and gynostemium in the rst, second, third and fourth whorl of the owers of the orchid species Erycina pusilla, Phaelonopsis equestris and P. pulcherrima.
No E-class gene expression was found in the mature corona of Ceropegia sandersonii. In the callus on the orchid labellum, also an organ of staminal identity (33,34), E-class gene expression was found. Expression was more pronounced in early than late developmental stages of the callus and thus, E-class gene expression may take place in the early stage Ceropegia corona as well. This aspect, however, could not be assessed because early stage coronas were too small to dissect for RNA extractions. Laser capture microdissection could be applied to further investigate this. E-class gene expression in Ceropegia was restricted to mature petals and gynostegia. This is different as compared with Aristolochia (26,42) and Erycina (33), where E-class gene expression was also found in early stage oral buds, but congruent with the nding for Coffea (43), where no E-class gene expression was detected in young oral buds either. The timing of E-class gene expression pattern found seems correlated with the positions of the species investigated in the Tree of Life for the Angiosperms (44): late stage E-class gene expression in Eudicots seems to have evolved from early and late stage expression in Monocots.
E-class gene expression was also detected in the mature gynostemia of Aristolochia (26,42), Erycina (33), and Phalaenopis (34) and we assume that these similarities are correlated to the fusion of male and female reproductive functions in a single organ.
E-class gene expression in mature petals was also found in Erycina (33). In Aristolochia, the pitfall owers are sepaloid and petals are lacking (24).
The MADS-box expression patterns found were summarized in Figure 4 in which we propose a rst ABCDE-model for parachute owers and compare this to previous models constructed for other deceptive owers in the orchid and pipevine families.

Conclusion
This study for the rst time investigated in comprehensive detail the development of highly complex and synorganized Ceropegia pitfall owers using the parachute plant C. sandersonii as model species. We combined micro-morphological (SEM and micro-CT) and molecular techniques (transcriptome and RT-PCR analysis) to unravel oral organ development and identity. In a SEM image series from oral primordia to fully mature owers we identi ed ten phases with distinct changes in oral organ development. We furthermore performed the rst transcriptome analyses of early buds and different tissues (sepals, petals, corona, gynostegium) of mature Ceropegia pitfall owers and determined MADSbox genes involved in oral organ identity. MADS-box genes expressed in the mature corona, a oral organ unique to Apocynaceae, revealed its staminal origin in C. sandersonii (Asclepiadoideae). This is the rst time that the origin of the corona was clari ed using molecular methods in a species of Apocynaceae. The corona emerges late during oral development and becomes rst distinct only in phase seven out of ten. Neither the corona nor any other tissue was found to express AGL6. In unrelated plants with similar oral traits but bilaterally attened owers, i.e. Aristolochia mbriata and Erycina pusilla, AGL6 controls symmetry and is also expressed in orchid stelidia, a oral organ likely homolog to the staminal corona in Apocynaceae. Its absence of expression in Ceropegia owers presumably leads to their radial symmetry. Based on our results, we propose an ABCDE model for parachute owers, and with our comparative approach to non-related plants with similar oral traits we take the rst step towards understanding the genetic mechanisms driving convergent evolution of highly specialized deceptive trap owers. 3D X-ray micro-computer tomography (micro-CT)

Plant material
Fresh mature owers were stained for 5 days with 1% phosphotungstic acid (PTA) in 70% ethanol as contrast agent whereby PTA was change daily. After staining, owers were washed twice with 70% ethanol and embedded in 1.5% low melting point agarose in a plastic container. Embedded owers were scanned using a Zeiss Xradia 510 Versa 3D X-ray equipped with a sealed transmission X-ray source (settings: voltage/power: 40 kV/3 W; source current: 75 μA; exposure time: 2 sec; camera binning 2; optical magni cation: 4x; pixel size: 3.5 μm; total exposure time: ~3-2 h). Single 2D images were stacked to build a 3D image which was processed using Avizo 3D software version 8.1 (Thermo Scienti c™).

RNA isolation
Early oral buds (<3 mm) and mature owers ( rst day of anthesis) were harvested from at least three different plant individuals. Mature owers were dissected to sepals, petals (tip, tube, base), gynostegium, and corona. Similar tissue types of different mature owers were pooled to reach the required amount of tissue needed for RNA isolation (~30-90 mg). All samples were snap-frozen in liquid nitrogen and stored at -80°C until RNA isolation. Plant tissue was ground in a 2.2 ml micro centrifuge tube with a 7 mm glass bead using TissueLyser II (QIAGEN). Total RNA was extracted using the RNeasy Plant Mini Kit (QIAGEN) and an adapted protocol which included a step to digest single-and double-stranded DNA (DNase I; Amp Grade, Invitrogen 1U/µl). The amount of RNA for RT-PCR was measured using a NanoDrop (ND-1000 Spectrophotometer, Marshall Scienti c). Samples used for RNA sequencing were further quality checked by determining the integrity (RNA Integrity Number; RIN) using the Plant RNA nano protocol on an Agilent 2100 Bioanalyzer (Agilent Technologies). Only samples with a RIN >9.5 were used for sequencing. To obtain a full petal sample, RNA extracted separately from petal tips, tubes and bases was pooled after quality control. RNA samples were sent to Beijing Genomics Institute (BGI) for NGS sequencing on an Illumina HiSeq platform.
All Ceropegia sandersonii gene sequences were blasted against a local database of Gentianales MADSbox gene homologs (Rubiaceae: Coffea arabica, Gardenia jasminoides; Gentianaceae: Gentiana scabra; Apocynaceae: Allamanda cathartica, Catharanthus roseus),, which was created by retrieving DNA sequences from NCBI GenBank. Sequences for Actin were also retrieved to identify the C. sandersonii actin homolog required as control gene for the RT-PCR experiments (see below). For identi ed C. sandersonii MADS-box gene homologs (see Additional le 2), expression differences between sample types and among sample replicates were visualized in a heatmap (based on the count table for the according sequences; Additional le 6) using an in house designed bioinformatic script (https://github.com/naturalis/orchid-transcriptome-pipeline/tree/master/Scripts). With this script, the number of matches between a speci c read in the transcriptomes and a reference gene was scored. In a separately generated 'Color Key and Histogram', the number of hits was translated to color codes. Color codes were based on the number of counts per gene and sample divided by the total number of counts, where cold colors correspond with a relative low number and war colors with a relative high number.
Additional differential expression analyses were carried out using DESeq in R to calculate the log2 fold change of expression of the genes investigated in the different oral organs and developmental phases. Six pairwise tests between the four sample types 'early buds', 'sepals', 'petals', and 'gynostegium' were performed (see Additional le 4). These tests identify those genes with signi cant differential expression (minimum log2 fold change of 0.25) between a given pair of sample types. All samples had >100 counts so that a cut-off for the analyses was not necessary. Reverse Transcription PCR (RT-PCR)

Phylogeny of MADS-box gene lineages
Semi-quantitative reverse transcription-PCR (RT-PCR) was performed for six selected MADS-box gene homologs, i.e. CsanFUL2, CsanTM6, CsanGLO, CsanAG2, CsanAGL6, and CsanSEP1. The thermal cycling regime used in the RT-PCR reaction was similar as for the gradient PCR (see above); however, the annealing temperature was set to 52°C as this temperature yielded the best results and most speci c products in the gradient PCR. Actin was ampli ed as a positive control; the negative control was a nontemplate control (NTC). All reactions were carried out in a CFX384 Touch Real-Time PCR system thermocycler (Biorad). The PCR products were run on a 1% agarose gel with 1x TAE and a 1 kb plus GeneRuler TM (Thermo Scienti c) as ladder. The gel was stained with ethidium bromide and digitally photographed using a gel doc (Ultima 10si, Isogen Life Science). Ethics approval and consent to participate Not applicable for present study.

Consent for publication
All authors have provided consent for publication.