﻿Taxonomic revision of the southern African species of the genus Cynoglossum L. (Boraginaceae)

﻿Abstract The aim of the study is to provide a revision of the genus Cynoglossum in southern Africa. The genus is taxonomically problematic within the family Boraginaceae, due to the morphological similarities it shares with other closely related genera in the family. Cynoglossum plants are low-growing biennial, perennial, or rarely annual herbs which are recognizable by their hairy stems and leaves, the latter are usually basal and long petiolate. Based on the latest checklist, a total of eight species of this genus are listed for the study region: C.alticola, C.amabile, C.austroafricanum, C.geometricum, C.hispidum, C.lanceolatum, C.obtusicalyx (endemic to South Africa), and C.spelaeum. The occurrence of C.amabile in the region, however, requires further investigation since the only existing specimen was collected within a protected area in the KwaZulu-Natal province. Two specimens collected in the Doornpoort area in Pretoria, Gauteng province, assigned to this species appear to have been misidentified. Diagnostic characters are described, correct nomenclature, synonyms, typification, distribution maps, as well as the key for identifying the studied species, are provided.


Introduction
The type genus Cynoglossum L. of the tribe Cynoglosseae belongs to the angiosperm family Boraginaceae (forget-me-not or borage family). Members of this genus have a worldwide distribution with many species occupying the temperate as well as were deposited in JRAU. The system by Edwards and Leistner (1971) was used for specimen citation under the section 'additional specimen examined'.
Data on vegetative morphology was obtained by analysing all the specimens provided per species. Inflorescence structures were studied from the freshly collected samples, herbarium samples, as well as from the original author's descriptions (in a case where all the specimens did not contain any inflorescence to study). Hand drawings representing both the vegetative and reproductive characters were made for all the species (except for C. amabile). The trichomes from the leaf samples and mature nutlets, from as many specimens as possible, were examined per species, except for C. amabile, where there was only one specimen available, and C. obtusicalyx, where three specimens were available. Original descriptions from JSTOR.org were also included in developing the diagnostic key. The specimens were studied using either the TESCAN VEGA3 scanning electron microscope (SEM) or the Phenom Desktop SEM. For TESCAN VEGA3 SEM analysis, samples were immersed in a mixture of 95% ethanol and isoamyl acetate (1:1) for 10 minutes and in pure isoamyl acetate for 15 minutes. After removing isoamyl acetate, the samples were placed on a holder for critical point drying for an hour. Then the dried samples were directly mounted on aluminium stubs and sputter-coated with a thin layer of gold before viewing under microscope. This was done to prevent charging of specimens due to accumulation of static electric field, and to increase the number of secondary electrons that can be detected from the surface of the specimen. For the Phenom Desktop SEM analysis, samples were only air-dried for an hour and were directly mounted on the aluminium stubs and viewed under microscope. Maps were plotted using the program CorelDraw Graphics Suite X7 (http://coreldraw.com).

Vegetative morphology
Members of this genus are either perennial, biennial, or rarely annual herbs which are recognizable by their hairy stems and leaves. Roots are thickened cream white taproots with small lateral roots. The stems are erect, hollow, simple at the base, and usually branched above. The basal leaves are deciduous, long petiolate, lanceolate-obtuse shaped, cross-venulate, with smooth margins, and are clustered at the lower parts of the stem forming a rosette, covered with simple trichomes on both the adaxial and abaxial surface. The stem leaves are alternate, sessile, or petiolate, lanceolate-obtuse shaped, with smooth margins. Trichomes on the leaves sometimes have a pustulate base typical of Boraginaceae members (C. austroafricanum, Cynoglossum coeruleum subsp. johnstonii var. mannii and C. lanceolatum). Vegetative morphology is of limited diagnostic value in distinguishing between southern African species, however a closer look at the trichomes has shown that they may be used to distinguish between similar species. For example, both C. alticola and C. obtusicalyx have a cluster of soft, woolly trichomes which differ in shape (cylindrical with a pointed tip in C. alticola, vs. flat surface and spherical with a blunt tip in C. obtusicalyx) and density (denser in C. alticola than in C. obtusicalyx) as observed in Figure 1.

Reproductive morphology
The inflorescence is a cyme which is often dichotomously branched with spreading panicles. Flowers are either pedicelled or subsessile, with five parted corollas; corolla white with a blue throat, blue, violet, or magenta (C. hispidum), or rarely white (C. spelaeum). The stamens are included and arise from the base of the tube, they have short filaments and elliptic to oblong shaped anthers. The style is short and relatively thick, with a capitate stigma. The fruit is a schizocarp of four nutlets attached apically to a narrowly conical gynobase. The nutlets are ovoid with a convex dorsal surface. At maturity, the nutlets produce glochidia, which are sharp hair-like spines or bristles tipped with barbs. The glochidia are either swollen at the base or not bulbous-based, they either cover the whole surface or are well spaced and vary in number. The structure and shape of glochidia display an important distinguishing character amongst the southern African species (Table 1), with each species portraying a unique character as can be seen in Figure 2.   Perennial herbs, 0.2-0.6 m in height. Basal leaves 76-270×8-18 mm, lanceolate, densely pubescent, persistent; margins entire. Stem leaves 35-120×5-10 mm, lanceolate, apex acute, base cuneate, margins entire, soft woolly hairs. Trichomes spread equally on both the adaxial and abaxial leaf surfaces, unicellular hair base, not bulbous on both leaf surfaces. Inflorescence racemose, clustered at the apex; pedicel 4-10 mm long, lengthening considerably in fruit. Calyx ca. 4 mm long, lobes elliptic-oblong, densely hairy on inner surface, apices obtuse. Corolla deep blue; lobe 4×3 mm diameter, oblong, round apex. Nutlets convex, 6-9×5-6 mm; glochidia short and thick at the base, densely packed on nutlet, tips multi-angular ( Figure 3).
Phenology. February to May. Conservation status. Least Concern (Raimondo et al. 2009). Diagnostic characters. Cynoglossum alticola can be distinguished by its thick, convex nutlets. Among the southern African species, it has a unique appearance due to the presence of woolly trichomes that cover the whole plant. Furthermore, it has larger nutlets (6-9×5-6 mm) than other species (less than 6×5 mm). According to Hilliard and Burtt (1986), this species is related to C. alpinum (Brand) B.L. Burtt from the highlands of Ethiopia, with which it shares nutlet shape and size, as well as the leaf texture and colour. The difference is observed on the fornices (small crests in the corolla tube of a plant), as in C. alticola they are broad and short while in C. alpinum they are long and narrow.

Cynoglossum amabile
Phenology. October to November. Conservation status. Not evaluated (Raimondo et al. 2009). Diagnostic characters. Amongst the southern African species, C. amabile can be confused with C. lanceolatum due to their small-sized nutlets (between 2-4×2.5-4) and flowers. However, the two species are easily distinguished by their flower colour (C. lanceolatum has white corolla with blue throat, whereas C. amabile has bluish purple corolla). This species was also reported by Stapf and Drummond (1906) and Kӧnig et al. (2015) to be like C. furcatum Wallich (from Nepal, China, Bhutan, Vietnam, Thailand, Philippines, and India), based on flower and fruit size. The difference can be observed in the inflorescences, whereby C. furcatum is a much larger plant with inflorescences up to 1 m tall, and C. amabile is up to 0.6 m tall.
Distribution and habitat. Cynoglossum amabile is widely distributed in southern China where it is usually grown for ornamental purposes and naturalised in many parts of the world (Xu et al. 2009). According to Germishuizen and Meyer (2003), this species is only found in KwaZulu-Natal Province (Figure 6 Taxonomic notes. Cynoglossum amabile has been described as a widespread species which grows in disturbed habitat and can be grown as an ornamental (Kӧnig et al. 2015). This species has only been collected once in South Africa by J. Stewart in 1977, since then there have been no later records or observations of this species in this region. Attempts to locate this species in the wild were futile. It is questionable whether this species occurs naturally in the southern African region since its single known locality is within a protected area in KwaZulu-Natal.  cuneate base, margins undulate, covered with stiff hairs. Trichomes unicellular, with thick round base on the adaxial surface, simple on the abaxial surface. Inflorescences dichotomously branched, loose cymes at the apex, pedicels 4-9 mm long, and lengthens considerably in fruit. Calyx ca. 2-3 mm long, lobes obtuse, pubescent on the outer surface, glabrous on the inner surface, apex acute. Corolla pale blue; lobes 2.75-4.25 mm in diameter, cruciform, apex obtuse. Nutlets ovoid, 3.0-3.5×2.5-3.0 mm; glochidia more spread towards the margins, thin, tip multiangular ( Figure 7).

Cynoglossum austroafricanum
Phenology. December to April. Conservation status. Least Concern (Raimondo et al. 2009). Diagnostic characters. Amongst the southern African species, Cynoglossum austroafricanum can be confused with either C. lanceolatum or C. coeruleum var. mannii. This species differs from the two by the colour of the corolla (white corolla with pale blue throat vs pale blue corolla throughout in C. austroafricanum). This latter observation was also noted by Hilliard and Burtt (1986). Taxonomic notes. According to Hilliard and Burtt (1986), this species was described by Dr. H. Weimarck who did not select a type specimen; therefore, in typifying it they retained the specific epithet. glochidia more marginal and on the median line (Figure 9).

Cynoglossum coeruleum
Phenology. December to April. Conservation status. Least Concern (Raimondo et al. 2009). Diagnostic characters. This variety can be easily confused with C. lanceolatum due to the dichotomous branching of the inflorescence but can be distinguished from it by the distribution and density of the glochidia in the nutlets. The glochidia on the nutlets of Cynoglossum coeruleum var. mannii are more marginal and on the median line, whereas they are equally distributed around the whole nutlet in C. lanceolatum.
Distribution and habitat. This variety is endemic to South Africa where it is known only from KwaZulu-Natal and Eastern Cape Provinces ( Figure 10). It is also reported from Malawi, Mozambique, Zambia, and Zimbabwe (Mill and Miller 1984). It is found in disturbed grassland and sandy areas. Taxonomic notes. (i) Cynoglossum geometricum was recorded in the southern African region (FSA) for the first time by Hilliard and Burtt (1986). Although the name seems to be accepted in the Flora of southern African region (FSA), for example, Germishuizen and Meyer (2003) and Burrows and Willis (2005), it is not the correct name for this taxon. The correct name is Cynoglossum coeruleum subsp. johnstonii var. mannii, as synonimized by Verdcourt (1991). It is worth noting that the latter name is erroneously listed as C. coeruleum var. mannii in websites such as Plants of the World Online (http://powo.sci- ence.kew.org/taxon/966730-1) and the World Flora Online (http://www.worldfloraonline.org/). Verdcourt (1991) relegated C. johnstonii to subspecies level under C. coeruleum and then transferred C. mannii to C. coeruleum as a variety of Cynoglossum coeruleum subsp. johnstonii. (ii) There are three sheets of Mann 2005 in Royal Botanic Gardens, Kew (K), the one with the barcode number K000418935 is chosen as a lectotype because it displays most of the important characters of the species which can be used to distinguish this species from the rest, such as the inflorescence character and the branching pattern.  isolectotype). [Note: The HAL specimen is chosen as a lectotype because the specimen displays the diagnostic characters of the species as described in the protologue]. Echinospermum enerve E.Mey. ex DC. Prodr. 10:154 (1846), nom. nud.
Phenology. October to March. Conservation status. Least Concern (Raimondo et al. 2009). Diagnostic characters. The species can be confused with C. lanceolatum with which it shares a similar branching pattern of the inflorescences and upright brittle hairs covering the whole plant. However, the two differ in the colour of the corolla (magenta-purplish vs. white with blue throat in C. lanceolatum) and pedicel length (2 cm long opposed to less than 2 cm long in C. lanceolatum).
Distribution and habitat. This species is widely distributed across all provinces of South Africa. It can also be found in eSwatini and Lesotho ( Figure 12  Taxonomic notes. Brand (1921) included C. hispidum as a synonym of C. lanceolatum. However, the two species have notable variations especially in the colour of the corolla (magenta-purplish vs. white with blue throat in C. lanceolatum) and pedicel length (2 cm long opposed to less than 2 cm long in C. lanceolatum).  Mill. in Notes Roy. Bot. Gard. Edinb.: 474 (1984). ♀♂Type: same as above.
Phenology. August to May. Conservation status. Least Concern (Raimondo et al. 2009). Diagnostic characters. Amongst the southern African species, C. lanceolatum is similar and possibly related to Cynoglossum coeruleum subsp. johnstonii var. mannii with which it shares the branching pattern of the inflorescence, flower colour, and nutlet size. The two species can be distinguished by the density of the glochidia on the nutlets and distribution. Cynoglossum lanceolatum nutlets are completely covered with glochidia, whereas in Cynoglossum coeruleum subsp. johnstonii var. mannii glochidia tend to be more marginal and acentric.
Distribution and habitat. Cynoglossum lanceolatum originates from Yemen (Kӧnig et al. 2015) but reported from Africa (Ge-Ling et al. 1995), Pakistan, India (Joshi 2016), the Mediterranean, and throughout Asia (Verdcourt 1991) and Madagascar (Kӧnig et al.2015). In South Africa it occurs widely in all provinces, it also occurs in eSwatini and Lesotho (Figure 14). It is a widespread species that grows in disturbed habitats throughout parts of Africa.  Diagnostic characters. The stems and leaves of Cynoglossum obtusicalyx are densely covered with soft, woolly trichomes which makes it similar to Cynoglossum alticola. However, the two species differ in flower colour and size (deep blue corolla, 4×3 mm vs. pale blue corolla, 5×7 mm in C. obtusicalyx). Cynoglossum obtusicalyx has pale blue flowers with corolla 5-7 mm long whereas C. austroafricanum has bright blue flowers with corolla 2.75-4.25 mm long. The two species also show variation in trichome texture and density, i.e., sparsely hairy with short, stiff hairs on both the stem and leaves in C. austroafricanum vs. densely arranged long, woolly hair on both the stem and leaves in C. obtusicalyx.
Phenology. December to March. Conservation status. Least Concern (Raimondo et al. 2009). Diagnostic characters. This species is characterised by leaves that are sparsely covered with hairs that have a softer feel, which distinguishes it sharply from all the other southern African species which have either brittle or woolly hairs. The abaxial leaf surface has a grey-greenish appearance which is also a unique character of this species.
Distribution and habitat. The species is distributed in South Africa (Eastern Cape, Free-State, and KwaZulu-Natal Provinces) and Lesotho (Figure 18). It grows in loose sandy soil at the edge of an overhang.  Taxonomic notes. Cynoglossum spelaeum is quite distinctive among the southern African species having uniquely shaped (i.e., spatulate to obtuse) and coloured (i.e., deep green adaxial surface, grey green abaxial surface) leaves, that almost seem leathery but contain few soft trichomes. It is also the only species among the southern African species that has completely white flowers. Although Hilger et al. (2015) indicated that the species does not belong to genus Cynoglossum, they did not elaborate the reasons for the exclusion. Nonetheless, the species fits the generic description of four glochidiate nutlets that are adapted for zoochory. In addition, preliminary molecular data (Madika 2020), showed a close relationship between C. coeruleum subsp. johnstonii var. mannii, C. lanceolatum, and C. spelaeum.