doi: 10.1186/s13227-020-00160-z. eCollection 2020.
Evolution and development of three highly specialized floral structures of bee-pollinatedPhalaenopsis species
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- PMID:32793330
- PMCID: PMC7418404
- DOI: 10.1186/s13227-020-00160-z
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Evolution and development of three highly specialized floral structures of bee-pollinatedPhalaenopsis species
Dewi Pramanik et al. Evodevo..
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Abstract
Background: Variation in shape and size of many floral organs is related to pollinators. Evolution of such organs is driven by duplication and modification of MADS-box and MYB transcription factors. We applied a combination of micro-morphological (SEM and micro 3D-CT scanning) and molecular techniques (transcriptome and RT-PCR analysis) to understand the evolution and development of the callus, stelidia and mentum, three highly specialized floral structures of orchids involved in pollination. Early stage and mature tissues were collected from flowers of the bee-pollinatedPhalaenopsis equestris andPhalaenopsis pulcherrima, two species that differ in floral morphology:P. equestris has a large callus but short stelidia and no mentum, whereasP. pulcherrima has a small callus, but long stelidia and a pronounced mentum.
Results: Our results show the stelidia develop from early primordial stages, whereas the callus and mentum develop later. In combination, the micro 3D-CT scan analysis and gene expression analyses show that the callus is of mixed petaloid-staminodial origin, the stelidia of staminodial origin, and the mentum of mixed sepaloid-petaloid-staminodial origin.SEP clade 1 copies are expressed in the larger callus ofP. equestris, whereasAP3 clade 1 andAGL6 clade 1 copies are expressed in the pronounced mentum and long stelidia ofP. pulcherrima. AP3 clade 4,PI-,AGL6 clade 2 andPCF clade 1 copies might have a balancing role in callus and gynostemium development. There appears to be a trade-off betweenDIV clade 2 expression withSEP clade 1 expression in the callus, on the one hand, and withAP3 clade 1 andAGL6 clade 1 expression in the stelidia and mentum on the other.
Conclusions: We detected differential growth and expression of MADS boxAP3/PI-like,AGL6-like andSEP-like, and MYBDIV-like gene copies in the callus, stelidia and mentum of two species ofPhalaenopsis, of which these floral structures are very differently shaped and sized. Our study provides a first glimpse of the evolutionary developmental mechanisms driving adaptation ofPhalaenopsis flowers to different pollinators by providing combined micro-morphological and molecular evidence for a possible sepaloid-petaloid-staminodial origin of the orchid mentum.
Keywords: Callus; Mentum; RT-PCR; Stelidia; Transcriptomics.
© The Author(s) 2020.
Conflict of interest statement
Competing interestsThe authors declare that they have no competing interests.
Figures
Variation in shape and size of floral callus, mentum, stelidia and corresponding orchid pollinators.a AnHoplitis adunca male bee is pollinating aSerapias cordigera flower in France, its front legs grab the callus on the labellum, while the pollinia are attached to its forehead;b anAndrena sp. male bee receives pollinia on its proboscis while pseudocopulating with a flower ofOphrys splendida in France;c aDrosophila sp. fly is trapped against the anther, while pollinating a flower ofSpecklinia spectabilis in Costa Rica;d anAmegilla nigritar bee bears a pollinarium on its head while approaching a flower of Phalaenopsis pulcherrima in China.Ca callus,Me mentum,St stelidia. Scale bars: 1 mm. Photographs by Jean Claessens (a,b), Adam Karremans (c) and Jin Xiaohua (d)
Variation in shape and size of floral callus, mentum and stelidia in the orchid genusPhalaenopsis.a.Phalaenopsis amabilis;b.P. bellina;c.P. celebensis;d.P. equestris;e.P. pulcherima;f.P. pulcherrima. Abbreviations: Ca = callus; Me = mentum; St = stelidia. Scale bar: 100 µm. Photographs by Rogier van Vugt (a) and Dewi Pramanik (b-f)
Different floral developmental stages ofPhalaenopsis equestris andP. pulcherrima.a.P. equestris;b.P. pulcherrima. Abbreviations: Ca = callus; L = labellum; Me = mentum; St = stelidia. Scale bar: 1 cm. Photographs by Dewi Pramanik
Floral ontogeny ofPhalaenopsis equestris.a Undifferentiated oval-shaped early stage primordia covered by bracts, ×50 magnitude;b The transversally stretched bulges of early floral development with the initiation of sepals, ×350 magnitude;c–e further differentiation and development of sepals, petals and labellum of a single flower, ×200–×150 magnification;e–f an almost fully developed labellum and initiation of anther, ×150–×130 magnification;g further differentiation of median carpel, anther and staminodes, ×100 magnitude;h almost full-grown gynostemium, ×70 magnification;i halfway developed callus on the labellum, ×45 magnification. Scale bar: 100 µm.*F primordial floral bud;A1 anther,a1–a2 staminodes,Br bracts,Ca callus,Gm gynostemium,L labellum,mC median carpel,P1–P2 abaxial petals,S1–S2 abaxial sepals,S3 median sepal,St stelidia. Scale bar: 100 µm. Photographs by Dewi Pramanik
Floral ontogeny ofPhalaenopsis pulcherrima.a Early stage gynostemium in which the staminodes are still visible as separate organs;b early formation of the callus on the labellum;c,d late stage floral bud in which the size of the anther cap, stelidia, rostellum, callus and labellum further increases;e almost fully developed apical part of the gynostemium, the rostellum pokes out from underneath the anther cap and the stelidia are almost fully merged with the gynostemium;f early stage labellum and almost fully developed callus;g late stage apical part of the gynostemium, from which the anther cap and pollinia were removed, showing the fully developed rostellum; h. fully developed labellum and callus. Images were made under various magnifications ranging from ×37 to ×130 magnification.A1 anther,a1–a2 staminodes,Ac anther cap,Ca callus,L labellum,lC lateral carpel,mC median carpel,St stelidia,Ro rostellum. Scale bar: 100 µm. Photographs by Dewi Pramanik
Reconstruction of vascular bundle patterns in a maturePhalaenopsis flower based on μCT scanning.a–dP. equestris;e–h.P. pulcherrima.a Frontal view;b successive clockwise turn of 45 degrees;c successive clockwise turn of 90 degrees;d successive clockwise turn of 135 degrees;e frontal view;f successive clockwise turn of 45 degrees;g successive clockwise turn of 90 degrees;h. successive clockwise turn of 135 degrees. Color codes: purple = vascular bundles in ovary; yellow = vascular bundles in stamen and staminodes; green = vascular bundles in sepals; red = vascular bundles in labellum and petals. Scale bar: 100 μm
Heat map representation of number of counts of MADS-box gene expression in transcriptomes of early floral bud, mature callus and gynostemium ofP. equestris andP. pulcherrima. Each sample had three biological replications. The scale of the heat map is based on the number of counts obtained from the transcriptome data.Bud early stage floral bud,Ca mature callus,Gm mature gynostemium
Heat map representation of number of counts ofMYB andTCP gene expression in transcriptomes of early floral bud, mature callus and gynostemium ofP. equestris andP. pulcherrima. Each sample has three biological replications. The scale of the heat map is based on the number of counts obtained from the transcriptome data.Bud early stage floral bud,Ca mature callus,Gm mature gynostemium
MADS-box, MYB and TCP genes expression in floral organs ofP. equestris andP. pulcherrima.a Expression of isolatedAP3,PI,SEP,AGL6, DIV andPCF genes in dissected floral organs ofP. equestris;b expression of isolatedAP3,PI,SEP,AGL6, DIV andPCF genes in dissected floral organs ofP. pulcherrima. Bud floral bud stage 1,Ca callus,Gm gynostemium,Gm + St + Me gynostemium including stelidia and mentum,–C PCR amplification reaction without template (NTC)
Reconstruction of vascular bundle patterns in a mature flower ofP. equestris based on μCT scanning. Figures a-h shown in sequence from base to apex.a Section through the base of the ovary, showing six vascular bundles indicated in purple;b base of receptacle, also showing the vascular bundle in the base of the labellum (L) derived from staminode (Sm) a3 that is feeding the callus and the vascular bundles feeding the lateral sepals (Ls);c position where one of the two lateral sepals (Ls) is connecting to the base of the labellum (L);d position where the dorsal sepal (Ds), lateral petals (Lp), and labellum (L) connect, showing the first visible staminodal vascular bundle (St/A2);e position showing more staminodal (St/A2-A3) vascular bundles, and red vascular bundles feeding the lateral petals (Lp) and labellum (L);f position showing the first visible vascular bundles of the fertile stamen (Fs/A1) and four staminodal vascular bundles feeding the stelidia (St/a1-A3 and St/a2-A2); it also shows the first development of the callus (Ca);g transversal position of gynostemium showing the vascular bundle running to the fertile stamen (St/A1), and four vascular bundles running to the stelidia (St/a1-A3 and St/a2-A2);h position of the labellum with callus (Ca) showing it is supported by a staminodal vascular bundle (Sm/a3).Ds dorsal sepal,Ls lateral sepal,Lp lateral petal,L labellum,Me mentum,Ca callus,Fs fertile stamen,Ss sterile stamen,St stelidia,A1–A3 staminodes in outer floral whorl,a1–a3 staminodes in inner floral whorl. Color codes: green = vascular bundles in sepals; red = vascular bundles in petals; yellow = vascular bundles in androecium; purple = vascular bundles in gynoecium. Scale bars:a = 0.5 mm; (b–h) = 100 μm
Reconstruction of vascular bundle patterns in a mature flower ofP. pulcherrima based on μCT scanning. Figures a–i shown in sequence from base to apex.a. Section through the base of the ovary, showing six vascular bundles indicated in purple;b. base of the receptacle, also showing the vascular bundles in the base of the mentum (Me) derived from the lateral sepals indicated in green, lateral petals indicated in red and staminode a3 indicated in yellow;c. position where the labellum connects to the receptacle, showing the vascular bundles that are feeding the fertile stamen (Fs/A1) and stelidia (St/A2-A3) indicated in yellow, and two red vascular bundles feeding the lateral petals (Lp) and labellum (L);d. further development of fertile stamen (Fs) and staminodal (Sm/a3) vascular bundles;e–f. position where dorsal sepal (Ds), lateral petals (Lp) and labellum (L) are merging with the receptacle.g. Position where the first vascular bundles that feed the stelidia (St/A2-a2 and St/A3) become visible in the gynostemium;h. transversal section through the gynostemium showing the vascular bundles running towards the dorsal sepal (Ds), fertile stamen (Fs), and the stelidia (St/A2-a2 and St/A3-a1);i. section through the labellum showing that the callus is supported by the vascular bundle running towards staminode a3 (Sm/a3).Ds dorsal sepal,Ls lateral sepal,Lp lateral petal,L labellum,Me mentum,Ca callus,Fs fertile stamen,Ss sterile stamen,St stelidia,A1–A3 staminodes in outer floral whorl,a1–a3 staminodes in inner floral whorl. Color codes: green = vascular bundles in sepals; red = vascular bundles in labellum and petals; yellow = vascular bundles in androecium; purple = vascular bundles in gynoecium. Scale bars:a = 0.5 mm;b–i = 100 μm
Summary of expression of MADS-box (black), MYB (blue), and TCP (red) genes involved in the differentiation of callus, stelidia and mentum (all indicated in yellow) of P. equestris andP. pulcherrima. a Expression of SEP clade 1 genes is high (indicated on the left side of the white triangle) in the large callus ofP. equestris (left side) but absent (indicated on the right side of the white triangle) in the small callus ofP.pulcherrima (right side). Expression ofAP3 clade 1,AP3 clade 2,AGL6 clade 1,AGL6 clade 3,SEP clade 2,SEP clade 3,SEP clade 4, andDIV clade 2 genes is high (indicated on the right side of the black triangle) in the small callus ofP. pulcherrima (right side) but absent (indicated on the left side of the black triangle) in the large callus ofP. equestris (left side). Expression ofAP3 clade 4,PI clade 1,AGL6 clade 2, andPCF clade 1 genes is high (indicated by the grey box) in the callus of bothPhalaenopsis species.b Expression ofSEP clade 1 andDIV clade 2 genes is high (indicated on the left side of the white triangle) in the short stelidia and absent mentum ofP. equestris (left side) but absent (indicated on the right side of the white triangle) in the long stelidia and pronounced mentum ofP. pulcherrima (right side). Expression ofAP3 clade 1, andAGL6 clade 1 genes is high (indicated on the right side of the black triangle) in the long stelidia and pronounced mentum ofP. pulcherrima (right side) but absent (indicated on the left side of the black triangle) in the short stelidia and absent mentum ofP. equestris (left side). Expression ofAP3 clade 2,AP3 clade 4,PI clade 1,AGL6 clade 2,AGL6 clade 3,SEP clade 2,SEP clade 3,SEP clade 4, andPCF clade 1 genes is high (indicated by the grey box) in the stelidia and mentum of bothPhalaenopsis species. Illustrations by Richa Kusuma Wati
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