This art icle was downloaded by: [ Quaid- i- azam Universit y] , [ Sayed Afzal Shah] On: 29 June 2014, At : 11: 41 Publisher: Taylor & Francis I nform a Lt d Regist ered in England and Wales Regist ered Num ber: 1072954 Regist ered office: Mort im er House, 37- 41 Mort im er St reet , London W1T 3JH, UK Palynology Publicat ion det ails, including inst ruct ions f or aut hors and subscript ion inf ormat ion: ht t p: / / www. t andf online. com/ loi/ t pal20 Reassessing palynological characters in the subfamilies Periplocoideae and Asclepiadoideae (Apocynaceae): taxonomic and evolutionary implications a Sayed Af zal Shah & Musht aq Ahmad a a Depart ment of Plant Sciences, Quaid-i-Azam Universit y, Islamabad, Pakist an Accept ed aut hor version post ed online: 11 Mar 2014. Published online: 17 Jun 2014. To cite this article: Sayed Af zal Shah & Musht aq Ahmad (2014): Reassessing palynological charact ers in t he subf amilies Periplocoideae and Asclepiadoideae (Apocynaceae): t axonomic and evolut ionary implicat ions, Palynology, DOI: 10. 1080/ 01916122. 2014. 893264 To link to this article: ht t p: / / dx. doi. org/ 10. 1080/ 01916122. 2014. 893264 PLEASE SCROLL DOWN FOR ARTI CLE Taylor & Francis m akes every effort t o ensure t he accuracy of all t he inform at ion ( t he “ Cont ent ” ) cont ained in t he publicat ions on our plat form . However, Taylor & Francis, our agent s, and our licensors m ake no represent at ions or warrant ies what soever as t o t he accuracy, com plet eness, or suit abilit y for any purpose of t he Cont ent . Any opinions and views expressed in t his publicat ion are t he opinions and views of t he aut hors, and are not t he views of or endorsed by Taylor & Francis. 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Term s & Condit ions of access and use can be found at ht t p: / / www.t andfonline.com / page/ t erm s- and- condit ions Palynology, 2014 http://dx.doi.org/10.1080/01916122.2014.893264 Reassessing palynological characters in the subfamilies Periplocoideae and Asclepiadoideae (Apocynaceae): taxonomic and evolutionary implications Sayed Afzal Shah* and Mushtaq Ahmad Downloaded by [Quaid-i-azam University], [Sayed Afzal Shah] at 11:41 29 June 2014 Department of Plant Sciences, Quaid-i-Azam University, Islamabad, Pakistan The palynological characters of selected species of Periplocoideae and Asclepiadoideae were assessed and a taxonomic key made. We observed that translators of Periplocoideae are taxonomically more important structures than pollen tetrads. Within Periplocoideae, the flatter translators of Periploceae are evolutionarily more primitive than the spatulate translators of Cryptolepideae. The quantitative characters of pollinia are important for the genusand species-level taxonomy in Asclepiadoideae. Pollinium length/width ratio was studied for the first time and it was observed to be a useful character for the delimitation of the tribes and sub-tribes of Asclepiadoideae. The position of attachment of caudicles to pollinia is an overlapping character amongst the tribes and sub-tribes and can be utilised only for the taxonomy of genera. True sterile margins of pollinia are present only in Ceropegieae and pseudo-sterile margins are rarely present in other tribes. Asclepiadoideae within Apocynaceae, and Ascelpiadeae within Asclepiadoideae are separate entities, and Secamonoideae is closer to Periplocoideae than Asclepiadoideae on evolutionary grounds. Keywords: Periplocoideae; Asclepiadoideae; Pakistan; taxonomy; evolution; pollinaria; translators 1. Introduction Palynology is the backbone of classification of Apocynaceae. Many authors have emphasised the palynological characters of Asclepiadoideae and allied taxa (e.g. Brown 1811; Endlicher 1838; Schumann 1895; Walker & Doyle 1975; Rosatti 1989; Swarupanandan et al. 1996; Civeyrel et al. 1998; Sultan et al. 2005; Khan et al. 2008; Sreenath et al. 2012). Tribal composition remains ambiguous in some parts of the family Apocynaceae. Clade structure is generally well understood within Apocynaceae. But the major challenges now lie in identifying characters that reflect and articulate these clades in a formal classification (Nazar et al. 2013). Asclepiadoideae (previously called Asclepiadaceae) and Periplocoideae (previously called Periplocaceae) are two of the five sub-families of Apocynaceae. Asclepiadaceae is represented by 347 genera and 2850 species worldwide (Mabberley 1993). In the flora of Pakistan, Asclepiadaceae (divided into Periplocoideae and Asclepiadoideae) is recognised as a separate family and is represented by 23 genera and 41 species (Ali 1983). In this paper, we follow the classification of Endress & Bruyns (2000). Asclepiadaceae was segregated from Apocynaceae by Brown (1810) based on the presence of pollinaria, and divided further into three sub-families (Periplocoideae, Secamonoideae and Asclepiadoideae). In the classifications of Bentham (1876) & Schumann *Corresponding author. Email: afzaltaxonomist@gmail.com Ó 2014 AASP – The Palynological Society (1895), two subfamilies were recognised within Asclepiadaceae, viz. Periplocoideae and Asclepiadoideae, whereas Secamonoideae was placed within Asclepiadoideae. Schlechter (1905) separated Periplocaceae from Asclepiadaceae because of the presence of pollen tetrads deposited on a spoon-like translator in the former. These dilemmas continued until the end of the twentieth century. Endress & Bruyns (2000) united all three families into one large family, namely Apocynaceae. According to this classification, Apocynaceae is now composed of five subfamilies, viz. Rauvolfioideae, Apocynoideae, Periplocoideae, Secamonoideae and Asclepiadoideae. Asclepiads can be distinguished from other angiosperms by the presence of milky latex, corona, gynostegium and pollinaria. The latter three characters are of particular interest due to their complexity and restricted distribution in other angiosperms (Fishbein 2001). Based on palynological characters, Periplocoideae is different from Asclepiadoideae in that it lacks pollinaria. Also, the pollen grains are present in the form of pollen tetrads which are shed on a spoon-like structure called a translator. A pollinarium is composed of three parts: a glandular part called a corpusculum, holding two arms called caudicles, and in turn each of them holds a sac-like structure called a pollinium (Plate 1). Pollinia contain the pollen. A typical asclepiad flower contains five pollinaria (10 pollinia per Downloaded by [Quaid-i-azam University], [Sayed Afzal Shah] at 11:41 29 June 2014 2 S. Afzal Shah and M. Ahmad flower). Similarly, members of Periplocoideae contain five translators per flower. Palynological characters such as the presence of tetrads, and the number and orientation of pollinia, are widely used in the classification of supra-generic ranks of Apocynaceae (Civeyrel et al. 1998). Previous palynological studies were based mostly on simple and few characters, and none of the abovementioned studies was consistent with the latest classification system of Endress and Brunys (2000). The main aim of this paper is to take into account the previously studied palynological characters in combination with some new ones, which may prove useful in the future for resolving the systematic problems of these two subfamilies. This overall assessment of palynological characters will allow us to check their systematic potential and the extent of their effectiveness in the systematics of these two subfamilies. Furthermore, this study will enhance our knowledge of the flora of Pakistan. 2. Materials and methods 2.1. Sampling This study is designed so that it represents all the tribes of Asclepiadoideae and Periplocoideae in the flora of Pakistan, their subtribes (except Glossonematineae of Asclepiadeae) and 61% of the genera. Four species belonging to three genera, namely Huernia, Orbea and Stapelia, are being studied for the first time in Pakistan because these genera have not been previously recorded from Pakistan. More than five fresh specimens were collected for each species from different areas of Pakistan, mainly the capital Islamabad, the district of Swat, Chitral, and Dera Ismail Khan and Hazara Divisions. Plants species were identified with help of Flora of Pakistan (Ali 1983). Voucher specimens were deposited in the herbarium of Quaid-i-Azam University, Islamabad, Pakistan. The botanical names of the plants along with authorities have been provided according to the International Plant Name Index (IPNI). A list of the taxa was made systematically according to the classifications of Endress & Bruyns (2000); Venter & Verhoeven (2001) and Rapini et al. (2003) (Table 1). 2.2. Methodology for pollinaria isolation and character observation For the isolation of pollinaria, flowers were boiled for 1 minute in water. Pollinaria were manually picked under a dissecting microscope using forceps and sharp needles. For the isolation of pollen tetrads, the dried flowers were invented on slides and gently hit with forceps. Glycerine jelly was applied to the slides where necessary, especially to those slides which contained the pollen tetrads. Both qualitative and quantitative characters of pollinaria and pollen tetrads (Table 2) were studied using a light microscope. Photographs were taken using a digital camera (Infinity 1–5 C-MEI, Canada) fitted on a Leica light microscope (model DM 1000). All qualitative and quantitative palynological characters studied in this paper were based on the pollen structures of at least five different plant specimens of each species. Mean values, standard error and range [e.g. 56.5  5.79 (40–75)] Table 1. Systematic order of the taxa included in this study and their accession numbers. Subfamily Periplocoideae Asclepiadoideae Tribe/subtribe Periploceae Cryptolepideae Marsdenieae Ceropegieae Asclepiadeae/ Aslepiadineae Metastelmatineae Astephanineae Plant name Periploca aphylla Decne. Periploca hydaspidis Falc. Cryptolepis buchananii Roem. & Schult. Hoya carnosa (L.) R.Br. Telosma cordata Merr. Wattakaka volubilis Stapf Leptadenia pyrotechnica Decne. Caralluma tuberculata N.E.Br Orbea decaisneana (Lem.) Bruyns Huernia macrocarpa N.E.Br. Huernia zebrina N.E.Br. Stapelia gigantea N.E.Br. Asclepias curassavica L. Calotropis procera (Aiton) W.T. Aiton Pergularia daemia (Forssk.) Blatt. & McCann Cynanchum auriculatum Buch.-Ham. ex Wight Cynanchum acutum L. Oxystelma esculentum (Roxb.) R.Br. Vincetoxicum arnottianum Wight Tylophora hirsuta (Wall) Wight Accession no. ISL-127415 ISL-127322 ISL-127323 ISL-127324 ISL-127325 ISL-127326 ISL-127328 ISL-127416 ISL-127417 ISL-127418 ISL-127419 ISL-127420 ISL-127329 ISL-127331 ISL-127332 ISL-127333 ISL-127334 ISL-127335 ISL-127337 ISL-127339 3 Palynology Table 2. Key to the palynological characters of translators and pollinaria. Structure Translators Pollen tetrads Downloaded by [Quaid-i-azam University], [Sayed Afzal Shah] at 11:41 29 June 2014 Pollinia Character Structure Type of translator Shape of tetrad Length and width Length/width ratio Exine thickness Orientation Shape Length and width Length/width ratio Exine thickness Number of surface cells per pollinium (1 surface) of each quantitative character given in Tables 3 and 5 were also based on five readings. Terminology for pollinaria was adapted from Wyatt (1978) & Newton (1984). 3. Results 3.1. 1. 2. 3. 4. Key to the Asclepiadoideae based on palynological characters þ Pollinia transverse, true sterile margins present____________________________________2 Pollinia pendent, true sterile margins absent___________________________________7 þ Corpuscular arms absent, pollinia orbicular_________________________________3 Corpuscular arms present_________________4 þ Sterile margins present apically, caudicles attached to base of the pollinium________________________Leptadenia pyrotechnica Sterile margins oblique in position, caudicles attached to inner side in middle position of the pollinium______________________________Caralluma tuberculata þ Pollinia oval shaped, up to 410 mm long, caudicle attached in sub-apical position to corpusculum______________________________5 Pollinia non-oval, greater than 510 mm in length, caudicle attached in apical position to corpusculum—6 Caudicles Corpusculum Character Shape of pollinial surface cells Size of pollinial surface cells Presence of sterile margin Orientation of sterile margins Length and width Attachment to pollinia Attachment to corpusculum Orientation Shape Length and width Presence of extra-corpuscular arms 5. þ Sterile margins present to outer side of pollinia, pollinium length/width ¼ 1.33____________Huernia zebrina Both sterile margins present on inner side of pollinium, pollinium length/width ¼ 1.42—H. macrocarpa 6. þ Pollinia reniform, up to 600 mm long-Orbea decaisneana Pollinia ovate, up to 950 mm long—Stapelia gigantea 7. þ Caudicle attached sub-basally, to the innerside of pollinia________________________________8 Caudicle attached to the base of pollinia__________________________________10 8. þ Pollinia ovate, exine up to 8.75 mm thick___________________________________9 Pollinia elliptic, up to 220 mm long, exine up to 5 mm thick__________________________Vincetoxicum arnottianum 9. þ Pollinium length/width ¼ 1.49, caudicles up to 170 mm long, corpusculum up to 310 mm long________________________________Cynanchum acutum Pollinium length/width ¼ 1.69, caudicles up to 100 mm long, corpusculum up to 240 mm long_____________________________________C. auriculatum 10. þ Pollinia obovate________________________11 Pollinia narrowly oblong________________12 Table 3. Qualitative and quantitative characters of Periplocoideae. Taxa Tetrad shape Tetrad length Tetrad width Periploca aphylla Oblong 56.5  5.79 (40–75) 25.3  1.6 (21.3–30) Periploca hydaspidis Oblong elliptical 44.3  1.92 (40–50) 27  1.66 (22.5–32.5) Cryptolepis buchananii Rhomboidal 25.3  0.83 (22.5–27.5) 15.8  0.75 (13.8–17.5) Length/width Exine thickness 2.23 1.63 1.6 1.65  0.13 (1.25–2) 24.3  2 (17.5–28.8) 12.3  0.83 (10–15) Downloaded by [Quaid-i-azam University], [Sayed Afzal Shah] at 11:41 29 June 2014 4 S. Afzal Shah and M. Ahmad Plate 1. Typical pollen translator of Periplocoideae (A) and pollinarium of Asclepiadoideae (B). 11. þ Pseudo-sterile margins present, caudicles short or inconspicuous__________________Pergularia daemia Pseudo-sterile margins absent, caudicles long_______________________________Calotropis procera 12. þ Caudicles attached to corpusculum in sub-apical position, up to 450 mm long_______Asclepias curassavica Caudicles attached to corpusculum in apical position, up to 458 mm long_________Oxystelma esculentum 13. þ Pollinia erect__________________________14 Pollinia transverse______________________15 14. þ Pollinium length/width ¼ 3.99, outer margins concave___________________________Telosma cordata Pollinium length/width ¼ 2.83, inner margins straighter to concave —————–Wattakaka volubilis 15. þ Pseudo-sterile margins present, pollinia oblong, caudicles attached to middle of corpusculum, pollinium length/width ¼ 2.93______________Hoya carnosa Pollinia elliptic, caudicles attached to apex of corpusculum, pollinium length/width ¼ 1.59______________________________Tylophora hirsuta 3.2. Morphology of translators and pollen tetrads of Periplocoideae Many pollen tetrads are present on the upper surface of translators (Plate 2, figure 2). In translators of Periploca, the spoon (spatula) is orbicular; margins are smooth, the stalk is prominent, thinner and arises from the middle of the adhesive disc whereas the adhesive disc is blunt at both ends and broader in the middle. Translators of Cryptolepis are different in that the spoon is narrow, its margins are curled to the inner side and become narrower towards the base, and the stalk is reduced whereas the adhesive disc is thinner and much shorter than the spoon (Plate 2, figures 2 and 4). Pollen tetrads are oblong in Periploca aphylla, oblong elliptic in Periploca hydaspidis and ovate or rhomboidal or irregular in Cryptolepis buchananii. The longest pollen tetrads within Periplocoideae are those of Periploca aphylla (56.5 mm) (Table 3). 3.3. Morphology of pollinia of Asclepiadoideae Seven different shapes of pollinia were observed in 17 species of Asclepiadoideae. Pollinia are oblong in Marsdenieae and oval or ovate to reniform in Ceropegieae. Within the Asclepiadeae, these are narrowly obovate in Asclepiadinae and elliptic in Astephaninae. True sterile margins of pollinia are present in all Ceropegieae. Sterile margins are variable in their position on the pollinia (Plate 2, figures 9–14). Downloaded by [Quaid-i-azam University], [Sayed Afzal Shah] at 11:41 29 June 2014 Palynology 5 Plate 2. Scale bars: 1, 3, 5 ¼ 25 mm; 2, 4, 6–9, 11–13, 17, 20 ¼ 250 mm; 10, 18, 19, 22, 23 ¼ 100 mm; 14–16 ¼ 1000 mm; 21 ¼ 50 mm. (1) and (2) Periploca hydaspidis, (3) P. aphylla, (4) and (5) Cryptolepis buchananii, (6) Hoya carnosa, (7) Telosma cordata, (8) Wattakaka volubilis, (9) Leptadenia pyrotechnica, (10) Caralluma tuberculata, (11) Orbea decaisneana, (12) Huernia macrocarpa, (13) H. zebrina, (14) Stapelia gigantea, (15) Asclepias curassavica, (16) Calotropis procera, (17) Pergularia daemia, (18) Cynanchum auriculatum, (19) C. acutum, (20) and (21) Oxystelma esculentum, (22) Vincetoxicum arnottianum, (23) Tylophora hirsuta. Cd ¼ caudicle, Cp ¼ corpuscular, CA ¼ corpuscular arm, P ¼ pollinium, PSC ¼ pollinium surface cells, SM ¼ sterile margins, Te ¼ tetrads, Tr ¼ translator. Pseudo-sterile margins were found in Hoya carnosa (present on inner side of the pollinium) and Pergularia daemia (present on the outer side of the pollinium) (Plate 2, figures 6 and 17). The size of pollinia was observed to be highly variable. The largest and smallest pollinia are those of Calotropis procera (1304  546 mm) and Tylophora hirsuta (173  109 mm) respectively. The upper and lower surfaces of pollinia are cellular, and these cells are observable under the microscope (Plate 2, figure 21). These cells have never been investigated before. The highest number of cells per surface 6 S. Afzal Shah and M. Ahmad Downloaded by [Quaid-i-azam University], [Sayed Afzal Shah] at 11:41 29 June 2014 Table 4. Qualitative characters of pollinaria. The characters of pollina, corpuscum and caudicles are given separately. Caudicle Orientation Caudicle attachment Corpuscular attachment to of sterile corpusculum arm margins to pollinium Taxa Pollinium orientation Pollinium shape Sterile margins Hoya carnosa Telosma cordata Wattakaka volubilis Leptadenia pyrotechnica Caralluma tuberculata Transverse Erect Erect Transverse Transverse Oblong Reniform Reniform Orbicular Orbicular Pseudo-sterile Absent Absent Present Present Outside – – Apical Oblique Orbea decaisneana Huernia zebrina Huernia macrocarpa Stapelia gigantea Asclepias curassavica Transverse Transverse Transverse Transverse Pendent Present Present Present Present Absent Inside Parallel Inside Inside – Absent Pseudo-sterile Absent – Parallel – Calotropis procera Pergularia daemia Cynanchum auriculatum Pendent Pendent Pendent Reniform Oval Oval Ovate Narrowly oblong Obovate Obovate Ovate Cynanchum acutum Pendent Ovate Absent – Oxystelma esculentum Vincetoxicum arnottianum Pendent Pendent Narrowly oblong Elliptical Absent Absent – – Transverse Elliptical Absent – Tylophora hirsuta of the pollinium was counted as 152 in Oxystelma esculentum, and the lowest as 22 in Vincetoxicum arnottianum. The size (length þ width) of pollinial surface cells is a less variable character. The largest and smallest cells are those of Calotropis procera (230  164 mm) and Tylophora hirsuta (29.5  20.5 mm) respectively. The cells next to the sterile margins are the longest cells of pollinia in all species of Ceropegieae. Exine thickness of pollinia was observed to be a variable character. The thickest and thinnest exines are those of Stapelia gigantea (15 mm) and Tylophora hirsuta (1.9 mm) respectively. Caudicles are attached to two different positions on the surface of the pollinia. In most of the species observed, the caudicles are attached to the base of the pollinium (most members of Marsdenieae, Ceropegieae and Asclepiadinae), while in some species these are attached to the bases of the inner margins of the pollinia (Table 5). 3.4. Morphology of caudicles Caudicles are significantly variable in size and shape (Tables 4 and 5). These are cellular, colourless and occasionally very thin. Caudicles of Pergularia daemia are reduced, and those of Tylophora hirsuta are very delicate, smaller and cylindrical. Some members of Ceropegieae possess extra-corpuscular arms. In these species, the caudicles are attached to the corpusculum through the extra-corpuscular arms (Plate 2, Basal Basal Basal Basal Middle, inside Basal Basal Basal Basal Basal Basal Basal Sub-basal, inside Sub-basal, inside Basal Sub-basal, inside Basal Absent Absent Absent Absent Absent Middle Apical Apical Apical Sub-apical Present Present Present Present Absent Apical Sub-apical Sub-apical Apical Sub-apical Absent Absent Absent Sub-apical Apical Apical Absent Apical Absent Absent Apical Apical Absent Apical figures 11–14). The width of the caudicles was measured at their broadest point. In the majority of the species, the caudicles are greater in length than width, except Huernia zebrina, Stapelia gigantea, Cynanchum acutum and Vincetoxicum arnottianum in which their width is greater than their length. The overall range of the length of caudicles amongst the species observed is 37.5 mm (Tylophora hirsuta) to 402 mm (Asclepias curassavica) (Table 5). 3.5. Morphology of the corpusculum The corpusculum is dark brown to black in all the species observed. It is erect in Marsdenieae (except Hoya carnosa) and Asclepiadeae whereas it is pendent in Ceropegieae. An elliptic corpusculum was observed in 50% of the species observed. Other shapes are oblong, tapering, rod-shaped and rectangular. In Calotropis procera and Oxystelma esculentum, the corpusculum possesses one extra-corpuscular wing on both of the two opposite sides (Plate 2, figures 16, 20). The point of attachment of caudicles to the corpusclum is an inconsistent character. Amongst the species observed, the caudicles were attached to the apex, sub-apex or middle of the corpusculum. Their length is always greater than their width. The longest and shortest corpuscula are those of Stapelia gigantea (461 mm) and Tylophora hirsuta (90 mm) respectively. The broadest corpusculum is that of Asclepias curassavica (262 mm) (Table 5; Plate 2, figure 15). Downloaded by [Quaid-i-azam University], [Sayed Afzal Shah] at 11:41 29 June 2014 Table 5. Quantitative characters of the different parts of the pollinaria. Taxa Hoya carnosa 765  9.75 261  9.27 (745–800) (230–280) 631 4 158  7.35 (620–640) (140–180) 453  9.13 160  2.24 (430–475) (153–165) 200 3.26 139.5  1.46 (190–207.5) (135–142.5) 365.5 14.78 273  2.89 (12.5–412.5) (265–282.50 584 6 422  3.74 (570–600) (410–430) 326 5.10 246  5.10 (310–340) (230–260) 400 4.47 282  3.74 (390–410) (270–290) 924 10.30 630  7.07 (900–950) (610–650) 922 34.41 326  9.80 (800–1000) (300–360) 1304 16.31 544  13.64 (1250–1350) (500–580) 588 5.83 274  2.45 (570–600) (270–280) 388 16.55 230  7.07 (340–430) (210–250) 324 21.59 217  7.68 (260–380) (190–230) 1150 3.16 274  2.45 (1140–1160) (270–280) 213 2.89 126  1.27 (205–220) (122.5–130) 173 2.78 109  3.76 (163–177.5) (100–122.5) 2.93 3.99 2.83 1.43 1.34 1.38 1.33 1.42 1.47 2.83 2.4 2.15 1.69 1.49 4.2 1.69 1.59 2.9  0.26 (2.5–3.75) 2  0.31 (1.25–2.5) 2.3  0.12 (2–2.5) 3.45  0.46 (2.5–5) 2.5  0.16 (2–3) 6.75  0.64 (5–8.750 4.25  (2.5–5) 4.25  1.09 (1.25–7.5) 15  2.24 (10–20) 6.25  1.58 (2.5–10) 9  0.73 (7.5–11.25) 2.75  0.25 (2.5–3.75) 5.75  1.09 (2.5–8.75) 5.75  1.09 (2.5–8.75) 4.25  0.50 (2.5–5) 4  0.47 (2.5–5) 1.9  0.28 (1.25–2.5) 68 2.80 (60–750) 62.6  0.68 (61–64) 52  1.25 (50–57) 25  0.84 (22–27) 32  1.69 (29–38) 72  1.69 (68–78) 68  0.95 (65–70) 45  1.72 (41–50) 121  2.52 (115–128) 84  1.59 (81–90) 141  2.52 (136–150) 27  1.14 (24–31) 31  2.31 (24–38) 59  4.14 (42–65) 152  2.87 (145–162) 22  31.22 (19–26) 29  1.34 (25–32) Pollinial cells length mm Pollinial cells width mm 59  6.83 (37.5–75) 42.5  5.76 (27.5–62.5) 63.5  16.37 (35–122.5) 41  4.51 (30–55) 96  24.76 (25–165) 134  25.81 (60–200) 130  15.17 (90–170) 54.5  6.44 (37.5–75) 80  10.49 (50–100) 222  34.89 (120–300) 230  42.07 (30–350) 66  8.09 (50–92.5) 55  6.52 (45–80) 46  5.95 (30–65) 43.5  3.12 (35–50) 32.5  3.45 (25–42.5) 29.5  3 (25–40) 28.75  1.25 (25–32.5) 27.5  3.06 (17.5–35) 36  5.16 (22.5–50) 29.5  3.48 (22.5–42.5) 44.5  5.67 (27.5–60) 48  5.83 (30–60) 82  14.63 (50–130) 34  2.57 (25–40) 42  8.46 (25–70) 168  24.7 (100–240) 164  20.15 (110–220) 51  4.65 (35–62.5) 39.5  2.55 (30–45) 27.5  3.79 (15–37.5) 29.5  3.48 (17.5–37.5) 23.5  1.5 (20–27.5) 20.5  3.10 (12.5–30) Caudicle length mm 135  5 (120–1500) 80  3.54 (70–90) 42.5  3.54 (35–55) 104  3.02 (95–112.5) 81.5  2.57 (75.87.5) 140  10.49 (110–160) 122  6.63 (100–140) 176  19.13 (100–200) 272  7.35 (260–300) 402  15.62 (360–450) 334  15.36 (300–800) 71.5  4.51 (62.5–82.5) 144  7.48 (130–170) 88  5.83 (70–100) 135.5  8.92 (107.5–157.5) 74.5  2.67 (67.5–82.5) 37.5  3.16 (27.5–47.5) Caudicle width mm Corpusculum length mm 110  10 312 5.83 (80–1400) (300–330) 70  2.50 304  5.10 (62.5–77.5) (290–320) 30.25  0.83 253  3.74 (27.5–32.5) (240–269) 59.5  2.15 96  5.28 (55–67.5) (80–107.5) 45.5  2 141.5  4.72 (40–50) (125–150) 218  2 285  5 (210–220) (270–300) 130  3.16 159  3.32 (120–140) (150–170) 140  3.16 194  5.10 (130–150) (180–210) 492  3.74 461  4 (480–500) (450–470) 96  5.10 414  16 (80–110) (360–450) 53  3.74 380  7.07 (40–60) (360–400) 48.5  1.70 226  3.22 (42.5–52.5) (217.5–237.5) 114  5.10 272  12.81 (100–130) (240–310) 96  6.78 214  10.77 (80–120) (180–240) 47.5  5.24 205  0.88 (37.5–67.5) (202.5–207.5) 102  1.46 225  4.61 (97.5–105) (212.5–237.5) 14  1.27 90  5 (10–17.5) (75–102.5) Corpusculum width mm 12180.72 (12.5–150) 10830.74 (100–120) 59.251.09 (56.25–62.5) 48.51.27 (45–52.5) 75.53.20 (65–85) 1472 (140–150) 13130.32 (120–140) 17020.74 (165–180) 17030.54 (160–180) 65.51.84 (60–70) 32.757.38 (3.75–42.5) 151.54.91 (137.5–167.5) 18219.60 (140–250) 19820.83 (130–260) 85.518.60 (11.25–107–5) 10110.35 (97.5–105) 59.55.27 (50–77.5) Palynology Telosma cordata Wattakaka volubilis Leptadenia pyrotechnica Caralluma tuberculata Orbea decaisneana Huernia zebrina Huernia macrocarpa Stapelia gigantea Asclepias curassavica Calotropis procera Pergularia daemia Cynanchum auriculatum Cynanchum acutum Oxystelma esculentum Vincetoxicum arnottianum Tylophora hirsuta Cells per Pollinaria Pollinaria Length/ Exine pollinial length (L) mm width (W) mm width thickness mm surface mm 7 8 S. Afzal Shah and M. Ahmad 4. Discussion Downloaded by [Quaid-i-azam University], [Sayed Afzal Shah] at 11:41 29 June 2014 4.1. Taxonomic implications of palynological characters in the Periplocoideae and Asclepiadoideae Palynology is of great significance in taxonomic and phylogenetic studies in all plant families (Walker & Doyle 1975). The separation of Asclepiadaceae from Apocynaceae by Brown (1810) was based on palynological characters such as the presence of pollen translators or pollinia, their number per flower and orientation. These characters are used today for the classification of Apocynaceae at major taxonomic ranks. Palynological characters of selected species of Periplocoideae and Asclepiadoideae revealed a high number of characteristics that can be utilised for resolving the taxonomic and evolutionary problems of these two groups. Periplocoideae can readily be distinguished by the presence of pollen tetrads on a spoon-like translator. There is little variation in the shape and size of pollen tetrads. Those of Cryptolepis buchananii can be differentiated from Periploca species by their rhomboidal shape and smaller size. However, pollen tetrads have a high level of similarity and are usually of little taxonomic value (Verhoeven & Venter 1993). Our observations are consistent with those of Schick (1982); Verhoeven et al. (1989); Venter et al. (1990); Kunze (1993); Verhoeven & Venter (1988, 1993, 1994a, 1994b, 1997, 1998) and Nilsson et al. (1993). The taxonomic and systematic values of translators have been highlighted by Verhoeven & Venter (1997). In this study we have also observed clear differences between the translators of Periploca and Cryptolepis. Two facts about the palynological characters of Periplocoideae are now obvious. Firstly, the morphological characters of translators are taxonomically more useful than pollen tetrads and, secondly, the translators can be used to delimit species, genera and tribes of Periplocoideae. Schill & J€ akel (1978), after an extensive study, revealed that palynological characteristics are useful in differentiating higher taxa of Asclepiadoideae. These findings have been supported by Nilsson (1986, 1990), Kunze (1993), Nilsson et al. (1993), Civeyrel (1994), Verhoeven & Venter (2001) and Verhoeven et al. (2003). Orientation of pollinia has been used as a diagnostic character for supra-generic taxa recognised within the Asclepiadoideae (Endlicher 1838; Swarupanandan et al. 1996; Civeyrel et al. 1998). Ceropegieae is often recognised to have an erect orientation of the pollinia, in addition to Marsdenieae (e.g. Liede & Albers 1994). According to our observations, pollinia are erect in Marsdenieae and more or less transverse in Ceropegieae. However, the corpusculum is erect in the former and pendent in the latter. Amongst the taxa observed, the orientation of the pollinia is mostly consistent within the tribes, but exceptional cases also exist within some tribes. For instance, in Hoya carnosa of the tribe Marsdenieae, the pollinia are transverse; similar to Tylophora hirsuta of the sub-tribe Astephaninae. In other members of Marsdenieae and Astephaninae, the pollinia are erect and pendent respectively. This problem has also been pointed out by Swarupanandan et al. (1996), Civeyral et al. (1998) and Endress & Bruyns (2000). It means that this character does not perfectly delimit the tribes of Asclepiadoideae. The structure of the pollinarium has been used as the most important and reliable character for the delimitation of major taxa (subfamilies and tribes) of Asclepiadaceae (Brown 1811; Endlicher 1838; Decaisne 1844; Schurnann 1895; Rosatti 1989; Swarupanandan et al. 1996; Civeyrel et al. 1998). This study revealed that the shape of the pollinia is an important character for the delimitation of genera and subtribes. Within the Astephaninae, for instance, both Vincetoxicum arnottianum and Tylophora hirsuta possess elliptic pollinia. Similarly, in Asclepiadinae, the pollinia are narrowly obovate. Pollinia of tribe Marsdenieae (except Hoya carnosa) and Asclepiadeae (except Pergularia daemia) lack sterile margins, whereas all members of Ceropegieae possess the true sterile margins in their pollinia. However, the sterile margins of Hoya carnosa and Pergularia daemia are pseudo-sterile margins. Within the Ceropegieae, species can be further distinguished by the position of sterile margins on the pollinia. However, pseudo-sterile margins are present rarely in some other tribes of Ascelpiadoideae, but true sterile margins are confined to the Ceropegieae (Plate 2, figures 9–14). Pollinia are variable in their length and width and these two characters are significant for the delimitation of the higher, and particularly the lower, ranks of the Asclepiadoideae. Another feature of the pollinium which seems to be more consistent within the tribes and subtribes of Asclepiadoideae is the length/width ratio. In this study, we have considered this character for the first time at the tribal level in Ascelpiadoideae. The range of the length/width ratio is 2.83–3.99 in Marsdenieae, 1.34–1.48 in Ceropegieae, 2.15–2.83 in Asclepiadinae and 1.59–16.9 in Astephaninae (Table 5). The surface of the pollinium is cellular. The number of cells per surface of the pollinium and the size of these cells is a variable character amongst the species observed that can be used as a supplementary character in the taxonomy of asclepiads. These characters of the pollinial surface have not been Downloaded by [Quaid-i-azam University], [Sayed Afzal Shah] at 11:41 29 June 2014 Palynology studied before. The number of cells per pollinium can be used as a diagnostic character for the delimitation of closely-related species. For instances Cynanchum auriculatum has 31 cells per surface of pollinium and Cynanchum acutum has 56. However, this character may not help to delimit supra-specific ranks. Furthermore, these pollinial surface cells vary in their size, which is a good supplementary character for species identification (Table 5). At the tribal level, Swarupanandan (1996) emphasised the orientation and attachment of caudicles to the pollinium. According to our observations, this is not a reliable character for the taxonomy of tribes and subtribes. We observed that species belonging to different tribes and subtribes of Asclepiadoideae have the same position of attachment of caudicles to the pollinia (Table 4). The shape, size and orientation of caudicles are important in the taxonomy of asclepiads (Sreenath et al. 2012). There is a high level of similarity regarding the shape of the corpusculum. However, the extra-corpuscular arms and wings are diagnostic features of some genera and species (Table 4, Plate 2, figure 16). Amongst the quantitative characters, the length of the corpusculum in particular is a better character for the taxonomy of species. 4.2. Evolutionary implications of palynological characters in Periplocoideae and Asclepiadoideae The evolution in the structure of pollinaria has been discussed previously by Safwat (1962), Kunze (1993, 1995), Sennblad & Bremer (1996), Swarupanandan et al. (1996), Civeyrel et al. (1998), Verhoeven & Venter (2001) & Sreenath et al. (2012). The translators within the Periplocoideae are obviously different in structure. In our opinion, the translators of Periploceae represent the evolutionarily primitive type whereas those of Cryptolepideae represent the advanced type of translators. Probably the flatter translators of Periploceae evolved into the more spatulate translators of Cryptolepideae through the curling process of the margins of the spatula to the upper side. The translators of Cryptolepideae are more advanced because they provide better protection to the pollen tetrads in the shape of a well-developed spoon. Asclepiadoideae evolved from Apocynaceae through Periplocoideae and Secamonoideae (Cronquist 1981; Rahman & Wilcock 1992; Omlor 1996). If Asclepiadoideae evolved from Periplocoideae through Secamonoideae then a single caudicle in Secamonoideae must be present which will represent a relationship between the two groups, as mentioned above that a single stalk (arm) is present in the basal taxa (Periploceae) of Periplocoideae. We have not included species of Secamonoideae in this study, but it is obvious from 9 the literature that Secamonoideae either possess a single caudicle in their pollinaria to which all the four pollinia are attached, or all four pollinia are sessile and are attached directly to the corpusculum (Endress & Bruyns 2000; Sreenath et al. 2012). The relationship between Periplocoideae and Secamonoideae is further strengthened by the presence of pollen tetrads in both groups. However, Secamonoideae and Asclepiadoideae both possess pollinaria as their pollen apparatus but there is one caudicle in the former and there are two in the latter. Also the Secamonoideae possess pollen tetrads, whereas the Asclpiadoideae possess simple pollen spores inside their pollinia. The transition of a single caudicle to two caudicles and the transition of pollen tetrads to pollen microspores do not make sense. Therefore, these contrasting characters prove Asclepiadoideae to be a separate entity from Periplocoideae and Secamonoideae. The orientation of translators is towards the gynostegium in Periplocoideae or, in other words, the apex of the spatula is directed upwards. A similar orientation of the pollinaria is found in Secamonoideae. This character also shows the close relationship between Periplocoideae and Secamonoideae. The erect orientation of the pollinia is also found in the basal tribes (Marsdenieae and Gonolobieae) of Asclepiadoideae (Fishbein 2001). Secamonoideae and Asclepiadoideae can be linked through this character, but Secamonoideae possess four pollinia per pollinarium whereas Asclepiadoideae possess two pollinia instead of four. Here, the number of pollinia per pollinarium makes Asclepiadoideae a separate entity from Secamonoideae. Within the Asclepiadoideae, the Marsdenieae possess completely erect pollinia, whereas Ceropegieae possess more or less transverse pollinia. However, Asclepiadeae possess completely pendent pollinia and this character is not found in any of the remaining tribes of the Asclepiadoideae. The transition of erect or transverse pollinia to pendent pollinia is incongruous. Therefore, on the basis of the orientation of the pollinia, Asclepiadeae is a separate entity within the Asclepiadoideae. 4.3. Concluding remarks Palynology is critical for the classification of major assemblages of Apocynaceae. Unlike some other angiosperms such as Poaceae, in which the pollen have high levels of similarity, Asclepiadoideae and Periplocoideae have sharply contrasting palynological characters which are rare outside these taxa. The character studies here represent the whole micro-morphology of pollen translators, pollen tetrads and pollinaria. All or most of these characters can be utilised to resolve the taxonomic problems within these groups. The high 10 S. Afzal Shah and M. Ahmad level of diversity in palynological characters of these groups can be utilised to carry out cladistic analysis and to make palyno-taxonomic keys in the floras. The new characters of taxonomic importance discovered here are: Downloaded by [Quaid-i-azam University], [Sayed Afzal Shah] at 11:41 29 June 2014 (1) Pollinium length/width ratio, (2) Number of cells per surface of pollinium, (3) Length and width of pollinium surface cells. Palynological characters are equally important for understanding the evolution within and between these two subfamilies. The following are the key structures and characters regarding the evolution in the taxa concerned: (1) Pollen translators (Periplocoideae) (2) Pollinia orientation (Asclepiadoideae) (3) Sterile margins of pollinia and extra-corpuscular arms (Ceropegieae) (4) Pollinia (Aslepiadineae and Astephanineae) (5) Caudicle morphology and their attachment to corpusculum and pollinia, and shape of pollinia (genera of Asclepiadoideae) We summarise the study of qualitative and quantitative palynological characters of Periplocoideae and Asclepiadoideae, and demonstrate that Asclepiadoideae within the Apocynaceae, and Ascelpiadeae within the Asclepiadoideae, are separate entities, and Secamonoideae is evolutionarily closer to Periplocoideae than Asclepiadoideae. Acknowledgements This paper is the outcome of an M. Phil thesis. The authors offer thanks to Dr Sarfaraz Khan Marwat for help in the collection of plants, Dr Mir Ajab Khan and Zahid Ullah for supportive guidance and Dr Muhammad Zafar for help with microscope techniques. We give our special thanks to the Higher Education Commission of Pakistan and the Herbarium of Quaid-i-Azam University Islamabad for support during the M. Phil research. Author biographies SAYED AFZAL SHAH obtained his Masters and MPhil degrees in botany from Quaid-i-Azam University, Islamabad, Pakistan. Afzal has researched the systematics of the Asclepiadaceae based on the micromorphology of the leaf epidermis and the pollen apparatus. His research interests include biodiversity, conservation, evolution, molecular systematics and taxonomy, especially in relation to the asclepiads (Apocynaceae). MUSHTAQ AHMAD obtained his PhD in biodiversity and plant systematics, and is an Assistant Professor in the Department of Plant Sciences at Quaidi-Azam University, Islamabad, Pakistan. 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