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Article • Acta Bot. Bras. 34 (1) • Jan-Mar 2020 • https://doi.org/10.1590/0102-33062019abb0276   copy

Taxonomic relevance of leaf anatomy in Banisteriopsis C.B. Rob. (Malpighiaceae)

Authorship SCIMAGO INSTITUTIONS RANKINGS

ABSTRACT

We describe the leaf anatomy of 42 of the 60 species of the genus Banisteriopsis, one of the largest and most diverse genera of Malpighiaceae in the Cerrado domain. Our aim was to identify anatomical leaf characters useful for the taxonomy and systematics of this morphological diverse genus. Expanded leaves from herbarium specimens were rehydrated and processed according to usual anatomical methods for analysis under light microscopy. All characters were compared using the Unweighted Pair Group Method with Arithmetic Mean algorithm and expressed in dendrograms. The most useful leaf anatomical characters were petiole contour, presence/absence of vascular bundles in the petiole, the position of the vascular bundles in the petiole, shape of the midrib, presence/absence of sheath extension, mesophyll type, and both the presence/absence and shape of leaf glands. Our study indicates that the morphological groups proposed in the literature for Banisteriopsis are natural groups.

Keywords:
leaf glands; Malpighiales; multivariate analysis; plant anatomy; stigmaphylloid clade

Introduction

Banisteriopsis is one of the most diversified genera of Malpighiaceae in Neotropical savannas, comprising ca. 60 species of shrubs and vines confined to the Americas (Gates 1982Gates B. 1982. Banisteriopsis, Diplopterys (Malpighiaceae). Flora Neotropica 30: 1-237.; Davis & Anderson 2010Davis CC, Anderson WR. 2010. A complete generic phylogeny of Malpighiaceae inferred from nucleotide sequence data and morphology. American Journal of Botany 97: 2031-2048.). The genus is easily recognized by its leaves bearing extra-floral nectaries on the leaf blades (Fig. 1), and by its greatly expanded glandular connectives (Gates 1982Gates B. 1982. Banisteriopsis, Diplopterys (Malpighiaceae). Flora Neotropica 30: 1-237.). Even though Banisteriopsis was taxonomically revised in the early 1980’s (Gates 1982Gates B. 1982. Banisteriopsis, Diplopterys (Malpighiaceae). Flora Neotropica 30: 1-237.), it was evidenced to be polyphyletic in the first phylogenetic studies for Malpighiaceae (Cameron et al. 2001Cameron KM, Chase MW, Anderson WR. 2001. Molecular systematics of Malpighiaceae: evidence from plastid rbcL e matK sequences. American Journal of Botany 88: 1847-1862.; Davis et al. 2001Davis CC, Anderson WR, Donoghue MJ. 2001. Phylogeny of Malpighiaceae: Evidence from chloroplast ndhF and trnl-F nucleotide sequences. American Journal of Botany 88: 1830-1846.; Davis & Anderson 2010Davis CC, Anderson WR. 2010. A complete generic phylogeny of Malpighiaceae inferred from nucleotide sequence data and morphology. American Journal of Botany 97: 2031-2048.), leading to the segregation of some species to Diplopterys or to the newly described Bronwenia (Anderson & Davis 2006Anderson WR, Davis CC. 2006. Expansion of Diplopterys at the expense of Banisteriopsis (Malpighiaceae). Harvard Papers in Botany 11:1-16.). After rendering Banisteriopsis monophyletic, these authors failed to morphologically re-circumscribe Banisteriopsis sensu stricto. The morphological characters currently used to distinguish the species of Banisteriopsis are mostly restricted to inflorescence architecture, flower, and fruit morphology (Gates 1982Gates B. 1982. Banisteriopsis, Diplopterys (Malpighiaceae). Flora Neotropica 30: 1-237.; Anderson & Davis 2006Anderson WR, Davis CC. 2006. Expansion of Diplopterys at the expense of Banisteriopsis (Malpighiaceae). Harvard Papers in Botany 11:1-16.). As several floral characters are not retained in fruiting specimens (e.g., color and floral display), correctly assigning newly collected specimens of Malpighiaceae to Banisteriopsis became one of the main challenges for general botanists.

Figure 1
Leaves of some analyzed species of Banisteriopsis: A. stipitate leaf gland of B. adenopoda, B. sessile petiole glands of B. membranifolia, C. leaf of B. adenopoda in abaxial view, D. leaf of B. angustifolia in adaxial view, E. leaf of B. argyrophylla in abaxial view, F. leaf of B. anisandra in abaxial view, G. leaf of B. harleyi in abaxial view, H. leaf of B. laevifolia in abaxial view, I. leaf of B. membranifolia in abaxial view, J. leaf of B. parviglandula in abaxial view, K. leaf of B. schizoptera in adaxial view, L. leaf of B. stellaris in abaxial view, M. flowering branch of B. variabilis showing leaves, inflorescences and flowers in anthesis (photographs by R.F.Almeida).

Leaf anatomy applied to taxonomic purposes has historically been used as a useful tool for exploring additional morphological characters in taxonomically convoluted groups (Metcalfe & Chalk 1950Metcalfe CR, Chalk L. 1950. Anatomy of the dicotyledons. Vol. 2. Oxford, Oxford Claredon Press.; 1979Metcalfe CR, Chalk L. 1979. Anatomy of the dicotyledons. Vol. 1. Systematic anatomy of the leaf and stem . 2nd. edn. Oxford, Oxford Claredon Press .). This approach makes it possible to correctly identify fragmented or sterile plant specimens, besides pointing out evolutionary trends in groups (Metcalfe & Chalk 1950Metcalfe CR, Chalk L. 1950. Anatomy of the dicotyledons. Vol. 2. Oxford, Oxford Claredon Press.; 1979Metcalfe CR, Chalk L. 1979. Anatomy of the dicotyledons. Vol. 1. Systematic anatomy of the leaf and stem . 2nd. edn. Oxford, Oxford Claredon Press .). Nonetheless, few studies exploring the anatomy of specific genera in Malpighiaceae are available in the literature, such as Mamede (1993Mamede MCH. 1993. Anatomia dos órgãos vegetativos de Camarea (Malpighiaceae). Acta Botanica Brasilica 7: 3-19.), Araújo et al. (2010Araújo JS, Azevedo AA, Silva LC, Meira RMSA. 2010. Leaf anatomy as an additional taxonomy tool for 16 species of Malpighiaceae found in the Cerrado area (Brazil). Plant Systematics and Evolution 286: 117-131.), Possobom et al. (2015Possobom CCF, Guimarães E, Machado SR. 2015. Structure and secretion mechanisms of floral glands in Diplopterys pubipetala (Malpighiaceae), a neotropical species. Flora 211: 26-39.), Araújo & Meira (2016Araújo JS, Meira RMSA. 2016. Comparative anatomy of calyx and foliar glands of Banisteriopsis C. B. Rob. (Malpighiaceae). Acta Botanica Brasilica 30: 112-123.), and Mello et al. (2019Mello ACMP, Almeida RF, Amorim AMA, Oliveira DMT. 2019. Leaf structure in Amorimia and closely related Neotropical genera and implications for their systematics and leaf evolution in Malpighiaceae. Botanical Journal of the Linnean Society 190: 1-26.).

In order to search for additional leaf characters that might contribute to specific and generic delimitation in Banisteriopsis, we present a thorough survey of the leaf anatomy of 42 species of this genus. Additionally, we provide a multivariate analysis of all the analyzed species to compare the dendrograms obtained with the most recent phylogenetic hypothesis and current infrageneric classification for Banisteriopsis.

Material and methods

We analyzed leaves of 42 species of Banisteriopsis C.R. Rob , as well as leaves of closely related genera based on Davis & Anderson (2010Davis CC, Anderson WR. 2010. A complete generic phylogeny of Malpighiaceae inferred from nucleotide sequence data and morphology. American Journal of Botany 97: 2031-2048.) (Tab. 1, Fig. 1A-M). All leaves were obtained from exsiccates deposited in Brazilian herbaria (CEPEC, HUEFS, INPA, MBM, RB, SP, SPF, UB, UPCB, and VIC; acronyms according to Thiers 2019Thiers B. 2019. Index Herbariorum: A global directory of public herbaria and associated staff. New York, New York Botanical Garden's Virtual Herbarium. http://sweetgum.nybg.org/ih/
http://sweetgum.nybg.org/ih/... ). Fully expanded and well-preserved leaves were sampled from three specimens for each taxon, except for rare species (i.e., Banisteriopsis martiniana (A. Juss.) Cuatrec, and B. quadriglandula B. Gates). The leaves were rehydrated in boiling distilled water until complete submersion (within five to fifteen minutes, in general), embedded in KOH 2 % for full distention (two hours in general), washed and dehydrated in ethyl alcohol series and stored in 70 % ethanol. All specimens were hand free sectioned using a shaving blade to obtain transversal and longitudinal sections from the leaf blade (base, middle, margin, and apex) and petiole. We adopted the median region for anatomical descriptions since there were not significative differences among all sectioned portions of the leaf blade. All sections were clarified in 20 % sodium hypochlorite and stained in basic fuchsine - Astra blue, and the slides were mounted in glycerin jelly and sealed with colorless varnish (Kraus & Arduin 1997Kraus JE, Arduin M. 1997. Manual básico de métodos em morfologia vegetal. Rio de Janeiro, Editora da Universidade Federal Rural do Rio de Janeiro.).

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Table 1
List of Banisteriopsis species and outgroups analyzed, and specimens examined from Brazilian herbaria (acronyms according to Thiers 2019Thiers B. 2019. Index Herbariorum: A global directory of public herbaria and associated staff. New York, New York Botanical Garden's Virtual Herbarium. http://sweetgum.nybg.org/ih/
http://sweetgum.nybg.org/ih/... ).

Part of the samples was diaphanized according to a modified Foster (1950Foster AS. 1950. Practical plant anatomy. New York, D. Van Nostrand Co.) protocol. Samples measuring ca. 20 mm2 were kept in a solution of sodium hydroxide for two hours, washed in distilled water several times, and then kept in 20 % sodium hypochlorite for 18 hours or until the tissue became translucid. After an additional wash, samples were dehydrated in a 50 % ethyl alcohol series overnight, and the slides were mounted in glycerin jelly and sealed with colorless varnish.

Vascular bundles and vein patterns were classified according to Howard (1979Howard RA. 1979. The petiole. In: Metcalfe CR, Chalk L. (eds.) Anatomy of the dicotyledons.Vol. 1. Systematic anatomy of the leaf and stem. 2nd. edn. Oxford, Claredon Press Oxford. p. 88-96.) and to Ellis et al. (2009Ellis B, Daly DC, Hickey LJ, et al. 2009. Manual of leaf architecture. New York, Cornell University Press.), respectively. Hair types were classified according to Theobald et al. (1979Theobald WL, Krahulik JL, Rollins RC. 1979. Trichome description, and classification. In: Metcalfe CR, Chalk L. (eds.) Anatomy of the dicotyledons .Vol. 1. Systematic anatomy of the leaf and stem. 2nd. edn. Oxford, Oxford Claredon Press . p. 40-53. ). Macromorphological data were obtained from Gates (1982Gates B. 1982. Banisteriopsis, Diplopterys (Malpighiaceae). Flora Neotropica 30: 1-237.) for elaborating a binary matrix (Tab. 2). All analyses and picture records were performed using a photomicroscope (Olympus AX70TRF, Olympus Optical, Tokyo, Japan) with a U-Photo system with a digital camera coupled (AxioCam HRc; Zeiss, Göttingen, Germany).

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Table 2
Morphological matrix of binary characters (absence/presence) including 42 species of Banisteriopsis and four outgroups: 1. White petals from flowers in anthesis, 2. yellow petals from flowers in anthesis, 3. pink petals from flowers in anthesis, 4. rounded, concave and sessile leaf glands, 5. rounded, concave and stalked leaf glands, 6. rounded, not concave, sessile leaf glands, 7. concave-convex petiole in transversal section, 8. plane-convex petiole in transversal section, 9. cylindrical petiole in transversal section, 10. vascular system of petioles disposed in open arch, 11. vascular system of petiole arranged in open arc with convoluted extremities, 12. accessory bundles, 13. sclereid sheath surrounding petiole’ bundle, 14. concave midrib in adaxial view, 15. plane midrib in adaxial view, 16. convex midrib in adaxial view, 17. unistratified epidermis on both surfaces, 18. bistratified epidermis in some regions of the adaxial surface, 19. bistratified epidermis in some areas of the abaxial surface, 20. isobilateral mesophyll, 21. dorsiventral mesophyll, 22. sclereid groups throughout the mesophyll, 23. sheath extension, 24. quaternary veins, 25. quintenary veins, 26. Areoles, 27. veinlets ending freely.

The identification key was elaborated using the data from the matrix (Tab. 2) for all analyzed species, and a reduced matrix comprising all 18 species sampled by Davis & Anderson (2010Davis CC, Anderson WR. 2010. A complete generic phylogeny of Malpighiaceae inferred from nucleotide sequence data and morphology. American Journal of Botany 97: 2031-2048.) in the most recent molecular phylogeny for Malpighiaceae. Leaf and floral characters for all species were compared among each other by converting the binary matrix into a symmetric matrix for Sorensen’s similarity. The Unweighted Pair Group Method with Arithmetic Mean (UPGMA) was used in NTSYS-pc 2.2 software (Rohlf 2000Rohlf FJ. 2000. NTSYS-pc: numerical taxonomy and multivariate analysis system, version 2.1. New York, Exeter Software: Setauket.; Sokal & Rohlf 1995Sokal RR, Rohlf FJ. 1995. Biometry: the principles of statistics in biological research. New York, Freeman.) to generate dendrograms based on the Jaccard index, and using the species of Diplopterys A.Juss. , Peixotoa and Stigmaphyllon as outgroups. Results obtained from this analysis were also compared to the most recent molecular phylogeny study for Malpighiaceae (Davis & Anderson 2010Davis CC, Anderson WR. 2010. A complete generic phylogeny of Malpighiaceae inferred from nucleotide sequence data and morphology. American Journal of Botany 97: 2031-2048.).

Results

Leaf anatomy of Banisteriopsis

Petiole contour in transversal section displayed significant morphological variance (Fig. 2). In most species (29 species), the contour is plane-convex (Fig. 2A), while in 11 species (B. adenopoda, B. goiana, B. megaphylla, B. membranifolia, B. muricata, B. nummifera, B. oxyclada, B. prancei, B. paraguariensis, B. parviflora and B. quadriglandula) it is concave-convex (Fig. 2C). In the remaining two species (B. acerosa and B. scutellata), the contour is circular (Fig. 2B). Two types of configurations of vascular bundles were observed in petioles: an open arch with convolute extremities, found in B. gardneriana and B. martiniana (Fig. 2E), and an open arch in the remaining species (Fig. 2A-D, F). The occurrence of accessory bundles (Fig. 2A, C-F) was observed in most analyzed species, except in B. acerosa (Fig. 2B) and B. stellaris, but the number of vascular bundles has also varied within the same species.

Figure 2
Shape and disposition of vascular bundles in petioles of Banisteriopsis species: A. plane-convex in B. malifolia, with vascular bundles in open arch, B. circular in B. acerosa, with vascular bundles in open arch, C. concave-convex in B. membranifolia, with vascular bundles in open arch, D. concave-convex in B. parviflora, with vascular bundles in open arch, E. circular in B. martiniana, with vascular bundles in open arch with convolute extremities, F. plane convex in B. hatschbachii, with vascular bundles in open arch. Abbreviations: arrow = accessory bundles; P = phloem; X = xylem; Sc = sclerenchymatic sheath.

Sclerenchyma sheaths were recorded surrounding the vascular bundles in petioles of eight species [B. anisandra, B. campestris, B. hatschbachii (Fig. 2F), B. irwinii, B. latifolia, B. megaphylla, B. pulchra and B. schizoptera]. Disperse sclereids were observed in petioles of nine species (Fig. 2A), B. arborea, B. cipoensis, B. confusa, B. gardneriana, B. goiana, B. hirsuta, B. muricata, B. sellowiana, B. malifolia var. apressa, B. malifolia var. malifolia), and were absent in the remaining species (Fig. 2B-E).

Midribs presented a convex contour in both surfaces of leaf blades in most analyzed species (Fig. 3A-C, Tab. 2). Plane midribs were observed in the adaxial surface of leaf blades of eight species [B. anisandra (Fig. 3B), B. calcicola, B. gardneriana, B. irwinii, B. paraguariensis, B. prancei, and B. sellowiana]. Concave midribs were observed in the adaxial surface of leaf blades of five species [B. andersonii, B. angustifolia, B. arborea, B. martiniana var. martiniana (Fig. 3C) and B. muricata]. The epidermis is single-layered in most species (Fig. 3D), except in the abaxial surface of leaf blades in B. martiniana (Fig. 3E). This species presented two layers of cells and different layers of non-chlorophyll cells in some regions below the epidermis. Most species also showed unistratified adaxial epidermis (Fig. 3D, Tab. 2), and 16 species presented a bistratification (Fig. 3E-F, Tab. 2) in some regions of the adaxial epidermis. Also, several layers of non-chlorophyll cells were observed in the varieties of B. martiniana (Fig. 3E).

Figure 3
Anatomical characterization of leaf blades of Banisteriopsis: A. midrib transversal section of B. hatschbachii, B. midrib transversal section of B. anisandra, C. midrib transversal section of B. martiniana var. martiniana, D. mesophyll transversal section of B. arborea, E. mesophyll transversal section of B. martiniana var. martiniana, F. mesophyll transversal section of B. acerosa. Abbreviations: P = phloem; X = xylem; Sc = sclerenchymatous fibers; EU = unistratified epiderm; EM = multistratified epiderm; EB = bistratified epiderm; PP = palisade parenchyma; SP - spongy parenchyma.

The mesophyll is isobilateral (Fig. 3D, F) in most species (Tab. 2), except in B. adenopoda, B. anisandra, B. argyrophylla, B. calcicola, B. gardneriana, B. parviflora, B. sellowiana, B. martiniana varieties, B. megaphylla, B. multifoliolata and B. nummifera in which the mesophyll is dorsiventral (Fig. 3E). All species showed collateral vascular bundles (Fig. 3A-C).

Paracytic stomata were observed in all species, with large subsidiary cells covering guard cells (Fig. 4A). Stomatal crests are absent (Fig. 4A), except in the varieties of B. martiniana (Fig. 4B), with subsidiary cells not covering guard cells. Malpighiaceous hairs were observed in most species (Fig. 4C), but it was not possible to distinguish their morphotypes (T, Y or V-shaped), due to these hairs being deciduous in most species, probably due to the re-hydratation method. In Banisteriopsis acerosa and B. caapi, we observed scars from deciduous hairs. On the other hand, B. hatschbachii, B. latifolia, B. parviflora, B. sellowiana, B. stellaris and in the varieties of B. martiniana, all leaves were always glabrous without hair scars (Fig. 4E-F). The extension of the parenchymal (Fig. 4C) or sclerenchymatic sheath was observed in most species, except in Banisteriopsis anisandra, B. argyrophylla, B. byssacea, B. caapi, B. gardneriana, B. harleyi, B. hatschbachii, B. laevifolia, both varieties of B. martiniana, B. membranifolia, B. nummifera, B. paraguariensis, B. parviflora, B. pulchra, B. quadriglandula, B. scutellata, B. sellowiana, B. stellaris and B. vernoniifolia. The presence of prismatic crystals (Fig. 4D) was observed in most of the analyzed species.

Figure 4
Mesophyll transversal sections and diaphanization of species of Banisteriopsis: A. detail of the stomata with subsidiary cells projected over guard cells in B. byssacea, B. details of the stomata with stomatal crests in B. martiniana, C. sheath extension in B. andersonii, D. prismatic crystals in B. muricata, E. the presence of quintenary veins in B. adenopoda, F. the absence of quintenary veins in B. sellowiana.

All species showed pinnate vein pattern, absence of basal veins, with secondary veins ramified in loops, not reaching the margin of the leaf blade. Angle variation among midrib and secondary veins was uniform, with the secondary veins being decurrent and the last marginal vein looped. Quaternary veins were recorded in most species (Fig. 4E-F), except in B. acerosa, B. angustifolia, B. anisandra, B. arborea, B. argyrophylla, B. byssacea, B. calcicola, B. goiana, B. harleyi, B. muricata, B. oxyclada, B. paraguariensis, B. quadriglandula and B. vernoniifolia. Quintenary veins were recorded only in B. adenopoda (Fig. 4E) and B caapi. Areoles were observed in B. argyrophylla and B. byssacea, with veinlets ending freely in most species, except in B. argyrophylla, B. byssacea and B. goiana, in which it was absent.

A total of three morphological patterns for leaf glands were observed in almost all species, except in B. quadriglandula, in which glands were absent. Rounded, concave and stalked glands (Fig. 5A) were recorded in B. adenopoda, B.arborea, B. argyrophylla, B. basifixa, B. calcicola and B. scutellata. On the other hand, B. acerosa, B. andersonii, B. angustifolia, B. anisandra, B. byssacea, B. caapi, B. cipoensis, B. gardneriana, B. goiana, B. hatschbachii, B. hirsuta, B. irwinii, B. laevifolia, B. latifolia, B. malifolia, B. megaphylla, B. multifoliolata, B. nummifera, B. parviglandula, B. prancei, B. quadriglandula, B. scutellata and B. vernoniifolia all glands were rounded and sessile, without concavity (Fig. 5B). In the remaining species, glands were rounded concave and sessile (Fig. 5C) .

Figure 5
Shape of leaf glands in Banisteriopsis. A. B. scutellata ( Rounded gland with concavity and stalked, B. B. goiana ( Rounded gland without concavity and sessile, and C. B. campestris ( Rounded gland with concavity and sessile. Co = concavity, arrow = stalk.

An identification key based on morphoanatomical characters for all studied species of Banisteriopsis is presented below:

1. Mesophyll dorsiventral (except for B. laevifolia), petals yellow in anthesis…...................... 2

1’. Mesophyll isobilateral (except for B. argyrophylla), petals pink to white in anthesis (Fig. 1M)..............................................................................................................................................10

2. Unistratified epidermis ...................................................................................B. parviglandula

2’. Bistratified epidermis at some portions …….........................................................................3

3. Guard cells with crests ……................................................................................B. martiniana

3’. Guard cells with projections of subsidiary cells......................................................................4

4. Leaf glands rounded and stalked ……………………….......................................... B. basifixa

4’. Leaf glands rounded and sessile .............................................................................................5

5. Leaf glands concave ...............................................................................................................6

5’. Leaf glands flat ......................................................................................................................7

6. Petioles transversally concave-convex................................................................. B. parviflora

6’. Petioles transversally plane-convex................................................................... B. sellowiana

7. Mesophyll isobilateral............................................................................................B. laevifolia

7’. Mesophyll dorsiventral .........................................................................................................8

8. Vascular system of petioles in open arch with convoluted extremities ..............B. gardneriana

8’. Vascular system of petioles in open arch without convoluted extremities………………....9

9. Midrib adaxially plane…………..……................................................................ B. anisandra

9’. Midrib adaxially convex..................................................................................... B. nummifera

10. Flowers with white petals in anthesis...................................................................................11

10’. Flowers with pink petals in anthesis...................................................................................25

11. Epidermis bistratified in some regions……........................................................................12

11’. Epidermis uniestratified….................................................................................................16

12. Leaf glands rounded and stalked………………..................................................B. scutellata

12’. Leaf glands rounded and sessile…………..........................................................................13

13. Glands with concavity…….................................................................................................14

13’. Glands without concavity…..................................................................................B. acerosa

14. Accessory bundles present…...............................................................................................15

14’. Accessory bundles absent……..............................................................................B. stellaris

15. Presence of a sclereid sheath around vascular bundles in the petiole…………B. schizoptera

15’. Absence of a sclereid sheath around vascular bundles in the petiole.......................B. harleyi

16. Petiole contour concave-convex………..............................................................................17

16’. Petiole contour plane-convex……….................................................................................18

17. Presence of sheath extension in the mesophyll...................................................B. adenopoda

17’. Absence of sheath extension in the mesophyll...........................................B. paraguariensis

18. Mesophyll dorsiventral.................................................................................. B. argyrophylla

18’. Mesophyll isobilateral........................................................................................................19

19. Midrib contour adaxially plane…………….………............................................... B. irwinii

19’. Midrib contour adaxially concave...……………………...................................................20

20. Leaf glands rounded with concavity…………………......................B. confusa, B. variabilis

20’. Leaf glands rounded without concavity…………………...…...........................................21

21. Sheath extension present………..........................................................................................22

21’. Sheath extension absent………..........................................................................................23

22. Sclereid sheath extension present around petioles’ vascular bundles..................... B. latifolia

22’. Sclereid sheath extension absent around petioles’ vascular bundles....................... B. hirsuta

23. Mesophyll sclereids present…….……………...............................................B. vernoniifolia

23’. Mesophyll sclereids absent………………….....................................................................24

24. Sclereid sheath extension present around petioles’ vascular bundles……….B. hatschbachii

24’. Sclereid sheath extension absent around petioles’ vascular bundles....................B. byssacea

25. Epidemis unistratified……………......................................................................................27

25’. Epidermis bistratified in some regions………....................................................................26

26. Midrib adaxially concave……….…………....................................................B. angustifolia

26’. Midrib adaxially convex………………..............................................................B. cipoensis

27. Leaf glands present……….................................................................................................28

27’. Leaf glands absent………….....................................................................B. quadriglandula

28. Leaf glands rounded and stalked………………………......................................................29

28’. Leaf glands rounded and sessile…………………..............................................................30

29. Mesophyll dorsiventral.........................................................................................B. calcicola

29’. Mesophyll isobilateral...........................................................................................B. arborea

30. Glands with concavity…….................................................................................................31

30'.Glands without concavity…................................................................................................35

31. Petiole’s contour plane-convex……....................................................................................32

31’. Petiole’s contour concave-convex......................................................................................33

32. Sheath extension present………........................................................................B. campestris

32’. Sheath extension absent……….............................................................................B. pulchra

33. Midrib adaxially concave…………….….............................................................B. muricata

33’. Midrib adaxially convex.....................................................................................................34

34. Mesophyll sheath extension present………..........................................................B. oxyclada

34’. Mesophyll sheath extension absent……….................................................B. membranifolia

35. Petiole transversally concave-convex…………..................................................................36

35. Petiole transversally plane-convex…………......................................................................38

36. Midrib adaxially plane………………...................................................................B. prancei

36’. Midrib adaxially convex.....................................................................................................37

37. Mesophyll dorsiventral.....................................................................................B. megaphylla

37’. Mesophyll isobilateral.............................................................................................B. goiana

38. Midrib adaxially concave……….………..........................................................B. andersonii

38’. Midrib vein adaxially convex.............................................................................................39

39. Sheath extension present………..........................................................................................40

39’. Sheath extension absent……….................................................................................B. caapi

40. Mesophyll dorsiventral…..............................................................................B. multifoliolata

40’. Mesophyll isobilateral ..........................B. malifolia var. malifolia, B. malifolia var. apressa

Leaf anatomy of outgroups species

The outgroup species show yellow petals, unistratified epidermis, presence of accessory bundles in petioles (Fig. 6A-B), midrib adaxially convex (Fig. 6C), and course of secondary veins brochidodromous. Rounded and sessile leaf glands were present in D. lutea (with concavity), D. valvata, and P. tomentosa (without concavity), and absent in S. cavernulosum. The mesophyll is isobilateral (Fig. 6D) only in P. tomentosa and dorsiventral (Fig. 6E) in the remaining species. Sheath extension (Fig. 6F) was present only in P. tomentosa. S. cavernulosum showed a concave-convex petiole contour (Fig. 6A), while the remaining species were plane-concave (Fig. 6B). Vascular bundles of petioles in D. valvata and S. cavernulosum are the open arch type with convoluted extremities (Fig. 6A), while in D. lutea and P. tomentosa it is only open arch type (Fig. 6B). Areoles were found only in D. lutea and D. valvata.

Figure 6
Anatomical characters of petioles and leaf blades of outgroups: A. petiole in transversal section of S. cavernulosum, B. petiole in transversal section of D. lutea, C. midrib in transversal section of P. tomentosa, D. mesophyll in transversal sections of P. tomentosa, E. mesophyll in transversal sections of S. cavernulosum, F. sheath extension in P. tomentosa. Abbreviations: P = phloem; PP = palisade parenchyma; Sc = sclerenchymatous fibers; SE = sheath extension; SP - spongy parenchyma; X = xylem.

Multivariate analysis

The similarity analysis with UPGMA (Fig. 7) arranged all Banisteriopsis species in four groups, besides the outgroups. Banisteriopsis was grouped based on the concave shape of its leaf glands, and primary vein adaxially convex. The first group comprises B. nummifera, B laevifolia, B. sellowiana, B. martiniana, and B. gardneriana, which share the presence of yellow petals and quaternary veins. The second group comprises B. adenopoda, B. argyrophylla and B. calcicola, which share the presence of a dorsiventral mesophyll, unistratified epidermis on both surfaces, and rounded and stalked glands with a concavity. The third group comprises B. paraguariensis, B. prancei, B. muricata, B. harleyi, B. confusa, B. latifolia, B. caapi and B. pulchra, which do not share exclusive characters but show the presence of accessory vascular bundles, isobilateral mesophyll and vascular system of petioles in open arch. And a fourth group comprising B. acerosa and B. angustifolia, which share the presence of sheath extensions and rounded, and sessile leaf glands.

Figure 7
Cluster analysis (UPGMA) of species of Banisteriopsis. Abbreviations: Bace B. acerosa, Bade B. adenopoda, Bcon B. confusa, Blat B. latifolia, Bpul B. pulcra, Bhar B. harleyi, Bpara B. paraguariensis, Bmur B. muricata, Bang B. angustifolia, Bpra B. prancei, Bcaa B. caapi, Blae B. laevifolia, Bnum B. nummifera, Bgar B. gardneriana, Bsel B. sellowiana, Bmart B. martiniana, Barg B. argyrophylla, Bcal B. calcicola, Dlutea Diplopterys lutea, Dvalv D. valvate, Ptomen Peixotoa tomentose, Scaver Stigmaphyllon cavernulosum.

Discussion

Leaf anatomy

The anatomical and macromorphological characters used on the identification key allowed the distinction of 40 from the 43 sampled species. The most relevant characters used to differentiate the species: petal color, type of leaf glands, presence/absence of bistratified epidermis, shape of petioles, shape of the midrib, position of vascular bundles on petioles, presence/absence of sheath extension, presence/absence of sclereid sheath around petiole's bundles, and mesophyll type.

A total of three patterns of petiole contour were observed in the 42 analyzed species of Banisteriopsis. This difference was considered relevant to distinguish the species, corroborating the results of Araújo et al. (2010Araújo JS, Azevedo AA, Silva LC, Meira RMSA. 2010. Leaf anatomy as an additional taxonomy tool for 16 species of Malpighiaceae found in the Cerrado area (Brazil). Plant Systematics and Evolution 286: 117-131.) that the petiole contour is a resourceful character to help identifying species of Malpighiaceae. Petiole anatomical characters are regarded as useful to distinguish taxa due to its stability in response to different environmental variables (Metcalfe & Chalk 1979Metcalfe CR, Chalk L. 1979. Anatomy of the dicotyledons. Vol. 1. Systematic anatomy of the leaf and stem . 2nd. edn. Oxford, Oxford Claredon Press .). This character has successfully been applied to comparative anatomical studies in several plant families (Rio et al. 2005Rio MC, Kinoshita LS, Castro MM. 2005. Anatomia foliar como subsídio para a taxonomia de espécies de Forsteronia G. Mey. (Apocynaceae) dos cerrados paulistas. Revista Brasileira de Botânica 28: 713-726.; Costa et al. 2010Costa FF, Lima HRP, Cunha M, Santos IS. 2010. Leaf anatomy and histochemistry of Macropeplus dentatus (Perkins) I. Santos and Peixoto and Macropeplus ligustrinus (Tul.) Perkins (Monimiaceae). Acta Botanica Brasilica 24: 852-861.; Moraes et al. 2011Moraes TMS, Rabelo GR, Alexandrino CR, Silva Neto SJ, Cunha M. 2011. Comparative leaf anatomy and micromorphology of Psychotria species (Rubiaceae) from the Atlantic Rainforest. Acta Botanica Brasilica 25: 178-190.; Ogundare & Saheed 2012Ogundare CS, Saheed SA. 2012. Foliar epidermal characters and petiole anatomy of four species of Citrus L. (Rutaceae) from south-western Nigeria. Bangladesh Journal Plant Taxon 19: 25-31.; Almeida Jr. et al. 2013Almeida-Jr EB, Araújo JS, Santos-Filho FS, Zickel CS. 2013. Leaf morphology and anatomy of Manilkara Adans. (Sapotaceae) from northeastern Brazil. Plant Systematics and Evolution 299: 1-9.; Coutinho et al. 2013Coutinho IAC, Francino DMT, Meira RMSA. 2013. Leaf anatomical studies of Chamaecrista subsect Baseophyllum (Leguminosae, Caesalpinioideae): new evidence for the up-ranking of the varieties to the species level. Plant Systematics and Evolution 299: 1709-1720.). Previously, only two types of petiole contour were reported in the literature for Malpighiaceae, concave-convex, and plane-convex (Araújo et al. 2010Araújo JS, Azevedo AA, Silva LC, Meira RMSA. 2010. Leaf anatomy as an additional taxonomy tool for 16 species of Malpighiaceae found in the Cerrado area (Brazil). Plant Systematics and Evolution 286: 117-131.). The results obtained in this study corroborate those data and add a third new type of petiole contour to the family, the circular one.

The petiole vascular bundles showed two different types of conformation in the studied species. Our results showed that the position of vascular bundles on petioles is useful to the group and distinguish species of Banisteriopsis, corroborating the data of Metcalfe & Chalk (1950Metcalfe CR, Chalk L. 1950. Anatomy of the dicotyledons. Vol. 2. Oxford, Oxford Claredon Press.) and Araújo et al. (2010Araújo JS, Azevedo AA, Silva LC, Meira RMSA. 2010. Leaf anatomy as an additional taxonomy tool for 16 species of Malpighiaceae found in the Cerrado area (Brazil). Plant Systematics and Evolution 286: 117-131.) for Malpighiaceae. Characters related to vascularization patterns of petioles have been widely used to improve the taxonomy of different families of flowering plants, such as Melastomataceae (Reis et al. 2004Reis C, Proença SL, Sajo MG. 2004. Vascularização foliar e anatomia do pecíolo de Melastomataceae do cerrado do Estado de São Paulo, Brasil. Acta Botanica Brasilica 18: 987-999.), Myrtaceae (Oliveira et al. 2011Oliveira MIU, Funch LS, Santos FAR, LR Landrum. 2011. Aplicação de caracteres morfoanatômicos foliares na taxonomia de Campomanesia Ruiz and Pavón (Myrtaceae). Acta Botanica Brasilica 25: 455-465.), Rubiaceae (Moraes et al. 2011Moraes TMS, Rabelo GR, Alexandrino CR, Silva Neto SJ, Cunha M. 2011. Comparative leaf anatomy and micromorphology of Psychotria species (Rubiaceae) from the Atlantic Rainforest. Acta Botanica Brasilica 25: 178-190.), Sapotaceae (Almeida Jr. et al. 2013Almeida-Jr EB, Araújo JS, Santos-Filho FS, Zickel CS. 2013. Leaf morphology and anatomy of Manilkara Adans. (Sapotaceae) from northeastern Brazil. Plant Systematics and Evolution 299: 1-9.) and Leguminosae (Francino 2006Francino DMT. 2006. Anatomia foliar de espécies de Chamaecrista Moench. (Leguminosae/Caesalpinioideae) ocorrentes em campo rupestre.MsC Thesis, Universidade Federal de Viçosa, Viçosa.; Coutinho et al. 2013Coutinho IAC, Francino DMT, Meira RMSA. 2013. Leaf anatomical studies of Chamaecrista subsect Baseophyllum (Leguminosae, Caesalpinioideae): new evidence for the up-ranking of the varieties to the species level. Plant Systematics and Evolution 299: 1709-1720.). Accessory bundles occurred in 40 of 42 species of Banisteriopsis, varying in the number of bundles, sometimes in specimes from the same species. Even though the occurrence of accessory bundles is an important character to help distinguishing species within Malpighiaceae (Araújo et al. 2010Araújo JS, Azevedo AA, Silva LC, Meira RMSA. 2010. Leaf anatomy as an additional taxonomy tool for 16 species of Malpighiaceae found in the Cerrado area (Brazil). Plant Systematics and Evolution 286: 117-131.) and different families of flowering plants (Bieras & Sajo 2004Bieras AC, Sajo MG. 2004. Anatomia foliar de Erythroxylum P. Browne (Erythroxylaceae) do Cerrado do estado de São Paulo, Brasil. Acta Botanica Brasilica 18: 601-612.; Moraes et al. 2011Moraes TMS, Rabelo GR, Alexandrino CR, Silva Neto SJ, Cunha M. 2011. Comparative leaf anatomy and micromorphology of Psychotria species (Rubiaceae) from the Atlantic Rainforest. Acta Botanica Brasilica 25: 178-190.; Coutinho et al. 2013Coutinho IAC, Francino DMT, Meira RMSA. 2013. Leaf anatomical studies of Chamaecrista subsect Baseophyllum (Leguminosae, Caesalpinioideae): new evidence for the up-ranking of the varieties to the species level. Plant Systematics and Evolution 299: 1709-1720.; Almeida Jr. et al. 2013Almeida-Jr EB, Araújo JS, Santos-Filho FS, Zickel CS. 2013. Leaf morphology and anatomy of Manilkara Adans. (Sapotaceae) from northeastern Brazil. Plant Systematics and Evolution 299: 1-9.), it was inconstant and not taxonomically significant (homoplastic) in our study. Only six from the 42 analyzed species did not present hairs, and due to the lack of studies on leaf ontogeny for Banisteriopsis, it is not possible to assert if those species are truly glabrous or just glabrescent at age.

The bistratified epidermis was found in 15 from the 42 analyzed species, indicating that this character is typical in the genus. The bistratified epidermis was mentioned by Metcalfe & Chalk (1950Metcalfe CR, Chalk L. 1950. Anatomy of the dicotyledons. Vol. 2. Oxford, Oxford Claredon Press.) for species of Acridocarpus, Banisteriopsis, Blepharandra, Hiraea, and Tetrapterys. Additionally, Araújo et al. (2010Araújo JS, Azevedo AA, Silva LC, Meira RMSA. 2010. Leaf anatomy as an additional taxonomy tool for 16 species of Malpighiaceae found in the Cerrado area (Brazil). Plant Systematics and Evolution 286: 117-131.) mentioned this character for Banisteriopsis, Byrsonima, and Heteropterys. Although this character has limited applicability in a phylogenetic context, it has already been used to distinguish species of Manilkara Adans. (Sapotaceae) (Almeida Jr. et al. 2013Almeida-Jr EB, Araújo JS, Santos-Filho FS, Zickel CS. 2013. Leaf morphology and anatomy of Manilkara Adans. (Sapotaceae) from northeastern Brazil. Plant Systematics and Evolution 299: 1-9.). Stomata with subsidiary cells larger than guard cells and absence of stomatic crests occurred in 41 from 42 species. Those characters were previously useful to distinguish species in Banisteriopsis (Araújo et al. 2010Araújo JS, Azevedo AA, Silva LC, Meira RMSA. 2010. Leaf anatomy as an additional taxonomy tool for 16 species of Malpighiaceae found in the Cerrado area (Brazil). Plant Systematics and Evolution 286: 117-131.) and Byrsonima (Beiguelman 1962Beiguelman B. 1962. Contribuição para o estudo anatômico das plantas do cerrado: II. Anatomia da folha e do caule de Byrsonima coccolobifolia Kunth. Revista de Biologia 3: 111-123.), the latter also showing stomata crests, but further studies in the remaining genera of Malpighiaceae are needed in order to test its taxonomic relevance at the generic level.

Isobilateral mesophyll was predominant in relation to dorsiventral one, being found in 30 from 42 studied species. The occurrence of those types of mesophyll was previously reported for Banisteriopsis and other genera of Malpighiaceae such as Acridocarpus, Banisteriopsis, Blepharandra, Byrsonima, Heteropterys, Hiraea, and Tetrapterys (Metcalfe & Chalk 1950Metcalfe CR, Chalk L. 1950. Anatomy of the dicotyledons. Vol. 2. Oxford, Oxford Claredon Press.; Pereira 1953Pereira E. 1953. Contribuição ao conhecimento da família Malpighiaceae. Separata dos Arquivos do Serviço Florestal, Ministério da Agricultura 7: 12-66.; Araújo et al. 2010Araújo JS, Azevedo AA, Silva LC, Meira RMSA. 2010. Leaf anatomy as an additional taxonomy tool for 16 species of Malpighiaceae found in the Cerrado area (Brazil). Plant Systematics and Evolution 286: 117-131.). However, this is the first time that it is pointed out as a taxonomically relevant character at the generic level for Banisteriopsis. The type of mesophyll has been used in different anatomical studies applied to taxonomy, and allied to additional characters, was useful in distinguishing species of Apocynaceae (Rio et al. 2005Rio MC, Kinoshita LS, Castro MM. 2005. Anatomia foliar como subsídio para a taxonomia de espécies de Forsteronia G. Mey. (Apocynaceae) dos cerrados paulistas. Revista Brasileira de Botânica 28: 713-726.), Celastraceae (Gomes et al. 2005Gomes SMA, Silva EAM, Lombardi JA, Azevedo AA, Vale FHA. 2005. Anatomia foliar como subsídio à taxonomia da subfamília Hippocrateoideae (Celastraceae). Acta Botanica Brasilica 19: 945-961.), Leguminosae (Coutinho et al. 2013Coutinho IAC, Francino DMT, Meira RMSA. 2013. Leaf anatomical studies of Chamaecrista subsect Baseophyllum (Leguminosae, Caesalpinioideae): new evidence for the up-ranking of the varieties to the species level. Plant Systematics and Evolution 299: 1709-1720.), Myrtaceae (Gomes et al. 2009Gomes SM, Somavilla NSDN, Gomes-Bezerra KM, Miranda SC, De-Carvalho PS, Graciano-Ribeiro D. 2009. Anatomia foliar de espécies de Myrtaceae: contribuições à taxonomia e filogenia. Acta Botanica Brasilica 23: 223-238.), and Piperaceae (Gogosz et al. 2012Gogosz AM, Boeger MRT, Negrelle RRB, Bergo C. 2012. Anatomia foliar comparativa de nove espécies do gênero Piper (Piperaceae). Rodriguésia 63: 405-417.).

The variation in the shape of the midrib observed in the studied species was useful to elaborate an identification key, corroborating its taxonomic relevance for Malpighiaceae (Araújo et al. 2010Araújo JS, Azevedo AA, Silva LC, Meira RMSA. 2010. Leaf anatomy as an additional taxonomy tool for 16 species of Malpighiaceae found in the Cerrado area (Brazil). Plant Systematics and Evolution 286: 117-131.). In the study of Bieras & Sajo (2004)Bieras AC, Sajo MG. 2004. Anatomia foliar de Erythroxylum P. Browne (Erythroxylaceae) do Cerrado do estado de São Paulo, Brasil. Acta Botanica Brasilica 18: 601-612., this character was also useful to distinguish species of Erythroxylaceae. Data on the venation patterns from the 42 species of Banisteriopsis studied showed limited variation, with a conserved venation pattern that might be taxonomic significant at the generic level. Future studies on character evolution in the Stigmaphylloid clade might shed some light on this matter.

Araújo & Meira (2016Araújo JS, Meira RMSA. 2016. Comparative anatomy of calyx and foliar glands of Banisteriopsis C. B. Rob. (Malpighiaceae). Acta Botanica Brasilica 30: 112-123.) have already studied the anatomy and macromorphology of leaf glands in Banisteriopsis. These authors observed a variation pattern in relation to the shape of theses structures, but have not observed any significant difference in the anatomy of these glands. The same patterns were observed in this study, with the macromorphological differences being important in differentiating species in Banisteriopsis.

Multivariate analysis

Banisteriopsis was characterized in the UPGMA analysis and distinguished from Diplopterys, Peixotoa and Stigmaphyllon by leaf glands concave, primary vein adaxially convex and veinlets ending freely. Since Davis & Anderson (2010Davis CC, Anderson WR. 2010. A complete generic phylogeny of Malpighiaceae inferred from nucleotide sequence data and morphology. American Journal of Botany 97: 2031-2048.) recovered Banisteriopsis as monophyletic in the last molecular phylogeny for the family, those anatomical characters might be recovered as morphological synapomorphies for the genus in future character-mapping studies. It seems that only the type of mesophyll characterized the two main groups recovered by the UPGMA analysis, as described in the first step of the abovementioned identification key. These two major groups roughly represent the two subgenera currently recognized in Banisteriopsis (subg. Banisteriopsis and Hemiramma) (Gates 1982Gates B. 1982. Banisteriopsis, Diplopterys (Malpighiaceae). Flora Neotropica 30: 1-237.; Anderson & Davis 2006Anderson WR, Davis CC. 2006. Expansion of Diplopterys at the expense of Banisteriopsis (Malpighiaceae). Harvard Papers in Botany 11:1-16.), and were recovered as monophyletic by Davis & Anderson (2010)Davis CC, Anderson WR. 2010. A complete generic phylogeny of Malpighiaceae inferred from nucleotide sequence data and morphology. American Journal of Botany 97: 2031-2048.. Thus, future studies of character mapping might recover these anatomical characters as synapomorphies for the subgenera of Banisteriopsis.

Our results also corroborate some of the informal groups of Banisteriopsis proposed by Gates (1982Gates B. 1982. Banisteriopsis, Diplopterys (Malpighiaceae). Flora Neotropica 30: 1-237.) in the last taxonomic revision for this genus. Those informal groups are partially recovered as monophyletic by Davis & Anderson (2010Davis CC, Anderson WR. 2010. A complete generic phylogeny of Malpighiaceae inferred from nucleotide sequence data and morphology. American Journal of Botany 97: 2031-2048.), with four major lineages in the genus. Group 1 from Davis & Anderson (2010)Davis CC, Anderson WR. 2010. A complete generic phylogeny of Malpighiaceae inferred from nucleotide sequence data and morphology. American Journal of Botany 97: 2031-2048. comprises B. prancei, B. martiniana, B. sellowiana, B. nummifera, and B. gardneriana. It coincides with the group formed in the similarity analysis, except by the presence of B. laevifolia. This group is almost entirely represented by species from the informal group B. martiniana and B. nummifera from Gates (1982)Gates B. 1982. Banisteriopsis, Diplopterys (Malpighiaceae). Flora Neotropica 30: 1-237.. Group II from Davis & Anderson (2010)Davis CC, Anderson WR. 2010. A complete generic phylogeny of Malpighiaceae inferred from nucleotide sequence data and morphology. American Journal of Botany 97: 2031-2048. comprises B. latifolia, B. confusa, B. adenopoda, B. pulchra, and B. caapi, which partially corresponds to group III of the similarity analysis. This group contains species from four different informal groups proposed by Gates (1982)Gates B. 1982. Banisteriopsis, Diplopterys (Malpighiaceae). Flora Neotropica 30: 1-237. and was only grouped by sharing homoplastic characters. On the other hand, group II obtained in the similarity analysis comprises B. calcicola, B. argyrophylla, and B. adenopoda. Those species are assigned to three different informal groups by Gates (1982)Gates B. 1982. Banisteriopsis, Diplopterys (Malpighiaceae). Flora Neotropica 30: 1-237. and three clades by Davis & Anderson (2010)Davis CC, Anderson WR. 2010. A complete generic phylogeny of Malpighiaceae inferred from nucleotide sequence data and morphology. American Journal of Botany 97: 2031-2048.. The same pattern was found in group IV from the similarity analysis, which comprises B. angustifolia and B. acerosa, both assigned to two different informal groups by Gates (1982)Gates B. 1982. Banisteriopsis, Diplopterys (Malpighiaceae). Flora Neotropica 30: 1-237., and two clades by Davis & Anderson (2010)Davis CC, Anderson WR. 2010. A complete generic phylogeny of Malpighiaceae inferred from nucleotide sequence data and morphology. American Journal of Botany 97: 2031-2048..

Conclusions

The present study included 75 % of all currently accepted species of Banisteriopsis and 90 % of all species occurring in Brazil. Our results evidenced that characters related to the shape of leaf glands, the shape of the midrib in the adaxial surface of leaves and veinlets ending freely might be important in characterizing genera in Malpighiaceae. Additionally, we corroborated part of the informal groups currently recognized in Banisteriopsis, which are already confirmed as monophyletic, opening the possibility to recognize them as infrageneric ranks in future studies of character evolution in Banisteriopsis.

Acknowledgements

We thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Finance Code 001) for funding; the curators and staff from HUEFS, INPA, MBM, RB and SP herbaria for their assistance; CNPq (SECTES and grant #422747/2016-5), FAPEMIG and Floresta Escola for funding our field and herbarium studies. JSA thanks Universidade Estadual do Piauí and FAPEPI for granting her a Ph.D scholarship; RFA thanks CAPES for his postdoctoral scholarship at UFMG and RMSAM thanks CNPq for her produtividade grant (DEB #307762/2016-5).

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Publication Dates

  • Publication in this collection
    20 Mar 2020
  • Date of issue
    Jan-Mar 2020

History

  • Received
    08 Aug 2019
  • Accepted
    12 Dec 2019
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