Tarennella, a new Pavetteae (Rubiaceae) genus from eastern Madagascar

1Meise Botanic Garden, Nieuwelaan 38, BE-1860 Meise, Belgium 2Kew Madagascar Conservation Centre, Lot II J 131 Ambodivoanjo, Ivandry, Antananarivo, Madagascar 3Parc Botanique et Zoologique de Tsimbazaza, Antananarivo-101, Madagascar 4Swedish Museum of Natural History, Department of Botany, Box 50007, SE-104 05 Stockholm, Sweden 5Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AE, UK *Corresponding author: petra.deblock@meisebotanicgarden.be RESEARCH ARTICLE


INTRODUCTION
With ca 700 species and 20 genera currently recognized, the Pavetteae (sensu De ) are one of the larger tribes in the Rubiaceae. The tribe has a paleotropical distribution with a similar number of species occurring in Asia/Oceania and in Africa/Madagascar. However, while species numbers are more or less equally divided, this is not the case for the genera. Fourteen Pavetteae genera are endemic to Africa/Madagascar, three to Asia/Oceania, and two genera have a paleotropical distribution (Pavetta L. and Tarenna Gaertn.).
Members of the tribe Pavetteae are characterized by fewto many-flowered terminal inflorescences (with the exception of Coptosperma supra-axillare (Hemsl.) Degreef), 4-or 5-merous flowers with a hypocrateriform (salver-shaped) corolla tube (but campanulate in Kindia gangan Cheek) and corolla lobes contorted to the left in bud, bilocular ovaries with a single to numerous ovules per locule, 3-or 4-colporate tectate pollen grains, drupaceous fruits, seeds with an adaxial hilar cavity and ruminate or entire endosperm, and exotestal cells that are parenchymatic or have thickenings along the outer tangential wall and the upper part of the radial walls to accommodate a species previously thought to belong to Coptosperma, but differing from representatives of this genus by having keeled triangular stipules with welldeveloped awns and three ovules pendulous from a small placenta attached to the upper half of the septum .
The Malagasy representatives of the Pavetteae show low sequence divergence in molecular phylogenetic analyses Kainulainen et al. 2017), which is interpreted as the result of recent rapid radiation. Kainulainen et al. (2017) found that the Pavetteae of the Western Indian Ocean Region are strongly supported as a monophyletic group and estimated the crown age at 8.5 Ma. The Malagasy Pavetteae are, however, morphologically very diverse. Madagascar is a centre of diversification for the tribe and different lineages have developed in dry and humid woody vegetation types . It would therefore be imprudent to unite all Malagasy representatives of the Pavetteae in a single genus (under the oldest name Coptosperma), which would then comprise the morphological variation of the entire tribe . Further integrative studies in taxonomy, morphology, and phylogenetics are needed to clarify the complex evolutionary history of the Malagasy Pavetteae. This paper aims to contribute to this goal by taxonomically treating one of the well-supported but unnamed terminal clades in the phylogenetic tree of the Malagasy Pavetteae.
We describe and illustrate five new species. All have the morphological characters of members of the tribe Pavetteae. However, they are unique within the tribe by the strongly pubescent style and stigma. Another distinguishing character is the superficial hilar cavity. In most Pavetteae genera having seeds with entire endosperm, the adaxial hilar cavity is deep and surrounded by a thickened annulus, whereas it is superficial and not surrounded by an annulus in the genera having seeds with ruminate endosperm (e.g. Coptosperma, Rutidea DC., Schizenterospermum). The only known exceptions to date are the genera Homollea and Paracephaelis, the laterally flattened seeds of which have a linear or elongated superficial hilum and entire endosperm. In the five new species studied here, the hilar cavity has a triangular or ovate shape but is superficial and not surrounded by a thickened annulus. A molecular analysis shows that the new species form a monophyletic group, which is not nested within any of the currently recognized Pavetteae genera. To accommodate them, the new genus Tarennella, endemic to eastern Madagascar and comprising five species, is described.

Phylogenetic analysis
Taxon sampling -The taxon sampling used in this study (supplementary file 1) was identical to that in  except for the addition of specimens of three undescribed species. The analysis comprised all Malagasy Pavetteae genera as well as the East African genus Tennantia and Asian representatives of the genus Tarenna, all belonging to the Afro-Madagascan clade of De . Two continental African Pavetteae species, Leptactina mannii Hook.f. and Tarenna precidantenna N.Hallé, not belonging to the Afro-Madagascan clade (De Block et al. 2018), were chosen as outgroup taxa. DNA isolation, amplification, and sequencing -Methods for DNA isolation, amplification, and sequencing of the chloroplast markers rps16, trnT-F, petD, and accD-psa1, the nuclear marker ITS, and the MADS-box gene marker PI (the floral homeotic gene PISTILLATA) followed De . Sequence alignment and phylogenetic analyses -Loci that were impossible to amplify (mainly PI and ITS) were considered as missing data in the sequence data matrix (supplementary file 1). Potentially informative indels were coded as separate characters, following the "simple indel coding" method of Simmons & Ochoterena (2000). Newly generated sequences were submitted to GenBank (supplementary file 1). Sequences were automatically aligned with MAFFT (Katoh et al. 2002) under the E-INS-I algorithm with a scoring matrix of 100PAM/k=2 and a gap open penalty of 1. The automatically aligned data matrix was subsequently fine-tuned by hand with Geneious v.11.1.
Putative topological conflicts between different datasets were inferred by visually inspecting the resolution and support values of the different topologies (hard vs soft incongruence; Johnson & Soltis 1998). A threshold with a maximum likelihood bootstrap (ML-BS) value ≥ 80% was applied to indicate a strongly supported incongruence between different data sets. The best-fit nucleotide substitution model for each plastid and nuclear dataset was determined using jModelTest v.2.1.4 (Posada 2008) under the Akaike information criterion (AIC). For ITS, petD, and PI, the GTR+I+G model was found as best fit, whereas the GTR+G model was shown to be the best substitution model for rps16 and trnT-F, and the HKY+I model for accD-psa1. Maximum likelihood analyses were computed using RAxML v.7.2.8 (Stamatakis et al. 2008) under the GTRGAMMA model. Non-parametric bootstrapping was carried out with 1000 bootstrap replicates.

Taxonomic study
Descriptions were based on dried and alcohol-preserved samples from BR, K, MO, P, and TAN. Abbreviations of herbaria follow Holmgren et al. (1990), and Thiers (continuously updated). Terminology followed Robbrecht (1988) but leaf shape was described according to the terminology of simple symmetrical plane shapes (Anonymous 1962). Methods followed normal practice of herbarium taxonomy (De Vogel 1987). For vegetative characters, colours and sizes were given for dried plant parts; for flower and fruit characters, colours were given for living material except when specified differently. Sizes of flowers and fruits covered the range of dried and alcohol-preserved material.
Specimens were cited per region, alphabetically by collector, with localities as given on the specimen labels. All specimens cited were seen unless indicated differently. Methods for pollen acetolysis followed De Block & Robbrecht (1998). Methods for light microscopy and microtomy followed Vrijdaghs et al. (2015). SEM images were obtained with a JEOL JSM-7100F scanning electron microscope. The distribution maps were drawn using QGIS Desktop v.3.4.11 (QGIS Development Team 2020).
Provisional conservation status was assessed by applying the IUCN Red List Category criteria (IUCN 2017) based on metrics produced via GeoCAT (Geospatial Conservation Assessment tool; Bachman et al. 2011). Google Earth Pro v.7.3.3.7699 (https://www.google.com/earth/) was used to infer the state of the natural vegetation in the localities and locations of the representatives of Tarennella species (Brummitt et al. 2015).

Phylogenetic analysis
For this study, we generated 23 sequences for representatives of the new species, which were analysed with the 299 sequences generated in previous studies (Bremer & Eriksson 2009;De Block et al. 2015, representing a total of 60 accessions and 50 species (supplementary file 1). Sequence variation within the individual datasets is summarised in table 1. The majority-rule consensus topologies from the separate ML analyses of the rps16, trnT-F, petD, accD-psa1, PI, and ITS data revealed similar topologies but did not provide much resolution. No hard incongruences were observed between the different datasets and they were combined for further analyses. The combined ML phylogenetic tree of the six markers is shown in fig. 1.
The monophyly of the ingroup (clade "A" in fig. 1), which corresponds to clade IV of De , later named the Afro-Madagascan clade (De Block et al. 2018 monospecific East African genus Tennantia was resolved as sister to a poorly supported clade (ML-BS = 55) formed by the rest of the ingroup taxa (clade "B"). Clade "B" contained two main clades, clade "C" (ML-BS = 80) and clade "D" (ML-BS = 100). Clade "C" comprised all sampled Asian/ Oceanic Tarenna species included in the study. Clade "D" contained continental African, Malagasy, and Indian Ocean species and is further named the Afro-Madagascan-Indian Ocean clade. Within clade "D", the continental African Coptosperma graveolens and C. peteri were successive sisters to a moderately supported large lineage (clade "F",   without a long needle-like awn, large placentas with several impressed ovules, and large angular, hemispherical, or hemi-ovoid seeds, and differing from them by having compact inflorescences with axes completely reduced and flowers sessile (vs inflorescences lax, inflorescence axes well-developed, and pedicels 5-50 mm long), mostly longitudinally ribbed fruits (vs fruits smooth), and seeds with a small superficial hilum not surrounded by a thickened annulus (vs hilum large and deep, surrounded by a thickened annulus); differing from Malagasy representatives of both genera by having a densely pubescent style with hairs that continue on the abaxial surfaces of the fused stigmatic lobes (vs style glabrous or pubescence ending somewhat below the papillate zone). Description -Small shrubs or treelets up to 2.5 m tall, sometimes litter-collecting, or rarely trees up to 5 m tall; vegetative parts glabrous or sparsely to densely covered with minute, erect hairs (only visible using hand lens or stereomicroscope); colleter exudate sometimes visible on young inflorescences (most conspicuous in T. sanguinea where it is orange-reddish and may occur as a droplet on the youngest stipule pair); bark often flaking. Leaves petiolate, with petioles canaliculate above; blades subcoriaceous; domatia absent; tips acuminate; midrib prominently raised on lower surface, impressed on upper surface (in T. coronata only at the base of the leaf); number of secondary nerves 10-18; tertiary and higher order nerves mostly inconspicuous on both surfaces. Stipules caducous, 2.5-10 mm long, sheath triangular to ovate with tip acute or shortly acuminate to 1.5 mm long or sheath truncate. Inflorescences terminal, becoming pseudo-axillary at a later stage, sessile, compact, 0.5-1.5 × 0.5-1 cm (corollas not included), with 15-60 flowers; inflorescence axes completely reduced; bracts and bracteoles present. Flowers sessile (but sometimes pedicels 1-2 mm long in fruit), hermaphroditic, (4-)5-merous. Calyx with short tube 0.5-1.5 mm long in flowering stage, in some species elongating up to 6 mm long in fruiting stage; lobes narrowly to broadly triangular, 0.5-9 mm long. Corolla white, turning yellow with age, in T. sanguinea the outer surface of corolla tube and lobes may appear orange because of orange colleter exudate; tube narrowly cylindrical, widening at the throat, ≤ 5 mm long, upper half moderately covered with erect hairs inside with pubescence continuing in the throat and on the corolla lobes; lobes oblong, somewhat fleshy, contorted to the left in bud and spreading at anthesis, as long as or somewhat shorter than corolla tube. Stamens sessile or subsessile, inserted in the sinuses of the corolla lobes at the level of the throat, basalmost part included in the corolla tube at anthesis; anthers 3-4.5 mm long, basimedifixed, with sagittate base and conspicuous sterile apical appendix, 0.3-0.5 mm long; filaments 0-0.3 mm long. Disc annular, fleshy, glabrous. Ovary cup-shaped, bilocular; placentation axile, placentas large, attached to the middle of the septum, with (2-)3 impressed ovules. Style and stigma white, exserted from the corolla tube at anthesis; style (except for the very base) and abaxial surfaces of stigmatic lobes densely covered with long, erect or ascending hairs, stigmatic lobes fused over their entire length. Fruits drupaceous, crowned by persistent and sometimes accrescent calyx, spherical or ovoid, 0.9-1.3 × 0.8-1.2 cm (not including persistent calyx), with 10 longitudinal ribs (but ribs absent in T. coronata and fruit of T. puberula unknown), orange or reddish when ripe (but noted as reddish purple in T. coronata); endocarp crustaceous. Seeds 2-6 per fruit, angular, hemi-ovoid or hemispherical, 4-7 × 3-6 mm, brown and glossy; hilum superficial, small, often triangular in shape, not surrounded by a thickened annulus; exotesta cells with continuous platelike thickenings along the outer tangential wall and the upper part of the radial walls, with a narrow ring-shaped intrusion from the cell lumen, without elongation of the exotesta cells around the hilum; endotesta consisting of partially crushed parenchymatic cell layers with or without prismatic calcium oxalate crystals; endosperm horny, entire. Pollen grains 3-zonocolporate, exine perforate to microreticulate, supratectal elements present in the form of microgemmae.
A genus with five species, endemic to littoral and lowland to mid-elevation humid evergreen forest in eastern Madagascar; 0-900 m a.s.l. Etymology -In the herbaria, material of the new genus was either unnamed or annotated as 'cf. Tarenna'. Within the Malagasy representatives of the tribe Pavetteae, Tarennella most resembles the Malagasy Tarenna species, especially those with the anthers and style/stigma partly included in the corolla tube at anthesis, hence the name of the new genus. inflorescences with small, white, 5-merous flowers and longitudinally ribbed fruits; differing from C. mitochondrioides by the shape and size of the stipules (sheath ovate, 4-10 mm long with acute to shortly acuminate tip vs sheath ovate-oblong to obovate, 6-18 mm long with rounded tip in C. mitochondrioides), a densely pubescent style and stigma (vs glabrous or pubescence restricted to lower half of style), orange-red fruits with 2-6 seeds (vs brown fruits with a single seed) and entire endosperm (vs ruminate endosperm). Description -Small shrub or treelet, 1-2.5 m tall, sometimes litter-collecting, or, rarely, small tree up to 5 m tall; shoots and branches usually glabrous, but rarely densely covered with minute (only visible using hand lens or stereomicroscope), erect hairs (Rabevohitra et al. 3907); young shoots drying bisulcate and blackish brown, smooth; branches brown (but reddish-brown in Razafimandimbison et al. 1239, fig. 3A), flaking. Leaves 15-30 × 4-9.5 cm, narrowly obovate or, more rarely, narrowly elliptic; blades drying brown or greenish brown and not discolorous, glabrous above, sparsely to moderately covered but midrib and sometimes secondary nerves densely covered by minute erect hairs below; base cordate or rounded; apex acuminate, acumen 15-35 mm long; 13-18 secondary nerves on each side of the midrib. Petioles robust, up to 5 mm long, glabrous or sparsely to moderately covered with minute, erect hairs. Stipules 4-10 mm long; sheath triangular to ovate, keeled, glabrous outside or, more rarely, moderately covered with Key to the species of Tarennella  minute, erect hairs especially in the region of the keel and the tip; tip acute or shortly acuminate, up to 1 mm long.

Tarennella cordatifolia
Inflorescences with bracts and bracteoles broadly triangular, 3-4 × ca 3 mm and 1.5-2.5 × ca 2 mm respectively, glabrous outside, with a ring of large colleters interspaced with hairs at the base and sparsely to moderately covered with appressed hairs near the tip inside, margins ciliate, tips acute to acuminate. Calyx with tube 1-1.5 mm long in flowering stage, up to 6 mm long in fruiting stage, glabrous outside, glabrous and without colleters inside; lobes broadly triangular, 1.5-2 mm long, sparsely to moderately covered with appressed hairs especially centrally and in the upper half outside, without colleters but moderately covered with appressed hairs in the upper half inside, margins ciliate, tips acute to obtuse. Corolla with tube 4-4.5 mm long, glabrous outside, upper half moderately covered with erect hairs inside; lobes 4-4.5 mm long, glabrous outside, moderately covered with erect hairs at the base inside, margins ciliate, tips blunt. Anthers 4-4.5 mm long. Ovary 1.5-2 mm long, glabrous. Style and stigma ca 9 mm long, exserted from the corolla tube for ca 4.5 mm at anthesis. Fruits ovoid, 1-1.3 × 0.9-1.2 cm (persistent calyx not included), longitudinally ribbed, orange or red when ripe; calyx tube accrescent, up to 6 mm long in fruiting stage. Seeds 4-6 per fruit, angular, 5-7 × 3.5-6 mm. Etymology -The species epithet is based on the shape of the leaf bases. Habitat -Eastern littoral or lowland humid evergreen forest; 5-600 m a.s.l. Distribution -Tarennella cordatifolia is only known from the Maroantsetra-Masoala Peninsula region in eastern Madagascar and is restricted to the northern part of the Analanjirofo Region ( fig. 9A). Phenology -Flowering: October-November; fruiting: April-June. Critical notes -1. Of the seven specimens known, six are described as small shrubs or treelets up to 2.5 m tall. Only  Tarennella cordatifolia is estimated to be 207 km 2 , but more than half of this covers marine area (Antongil Bay) and should not be taken into account. Therefore, T. cordatifolia is considered to have an EOO of less than 100 km 2 , which complies with criterion B1 for the Critically Endangered category. Using 2 × 2 km 2 grid cells, its area of occupancy (AOO) is 20 km 2 , which complies with criterion B2 for the Endangered category. The species is known from eight specimens, all collected relatively recently (from 1987 onwards). These herbarium specimens represent six unique occurrences, four subpopulations, and two locations, since the three subpopulations within Masoala National Park are counted as a single location. This complies with subcriterion 'a' of criterion B2 for the Endangered category. The fourth location (Davis et al. 4538) is relatively close to Makira Natural Park and it is possible that the species is present in this protected area with humid lowland forests. The main threat to T. cordatifolia is a decline of its habitat, which is an issue both inside and outside the protected areas. Masoala National Park is part of the World Heritage Centre named 'Rainforests of the Atsinanana'. While Masoala National Park is nominally protected, human encroachment in the form of illegal logging, wildfires, clearing for tavy (shortterm cultivation of crops), subsistence hunting, and artisanal mining continues inside the national park (Nicoll & Langrand 1989;Kremen 2003;UNESCO 2020). The disturbance is especially strong close to the city of Ambanizana (Google Earth, satellite imagery of 23 Nov. 2019), so a reduction of the habitat of the species is inferred. Also, Makira Natural Park is under threat of slash-and-burn agriculture, illegal logging, and mining from communities living around the protected area (WCS Madagascar 2020). These human activities are likely to increase with growing human populations, so a further reduction of the habitat of the species is projected for the future. Because of the small EOO and AOO (< 100 km 2 and 20 km 2 , respectively), the low number of locations (two) and the inferred present and projected future decline in the extent and quality of the habitat, Tarennella cordatifolia is assessed as Endangered EN B1ab(iii)+2ab(iii  Critical notes -1. The infructescences have three or four mature fruits. The number of flowers per inflorescence is probably higher. -2. The species is only known from the type material. Flowers have not been collected.

IUCN Red List assessment (provisional) -Critically
Endangered (Possibly Extinct): CR B2ab(iii). The extent of occurrence (EOO) of Tarennella coronata cannot be calculated since the species is only known from a single specimen. Using 2 × 2 km 2 grid cells, its area of occupancy (AOO) is estimated to be 4 km 2 , which complies with criterion B2 for the Critically Endangered category. The species is known from a single location, which complies with subcriterion 'a' of criterion B2 for the Critically Endangered category. It was collected on Andriantantely Mountain in 1922 and has not been recorded since. Andriantantely Mountain is situated ca 28 km WNW of Ampasimanolotra (Brickaville). It is part of the lowland humid eastern forest and is known for its high species diversity and high level of endemism (Emberton et al. 2010), but it is not protected. While some parts of Andriantantely Mountain are still relatively undisturbed, the native forest is being encroached by human activity on all sides (Google Earth, satellite imagery of 6 Sep. 2019), so a reduction in the habitat of Tarennella coronata is inferred. This encroachment will only increase with growing populations, so a further decline in the habitat of the species is projected for the future. The main threat to the species is a decline of its habitat as a result of slash-and-burn agriculture, logging, wildfires, etc. All along the eastern coast of Madagascar, the lowland forests outside protected areas are threatened with destruction because of human activity and this is also the case for Andriantantely Mountain. Emberton et al. (2010) mention that the remaining native forests on Andriantantely Mountain occur from 250 m to 700 m elevation, whereas the lower elevations are deforested. We have no information on the elevation at which T. coronata occurs. It is therefore not known whether suitable habitat for the species is still available at this location. Since the species was not collected after 1922, there is a possibility that it is extinct in the only known location. A targeted survey is needed to confirm or deny the presence of the species at the type locality and in the surrounding areas.
Because of the small AOO (4 km 2 ), the single location and the inferred present and projected future decline in the extent and quality of the habitat, Tarennella coronata is assessed as Critically Endangered (Possibly Extinct) CR B2ab(iii).  area of occupancy (AOO) of T. homolleana is estimated to be 64 km 2 , which complies with criterion B2 for the Endangered category. The species is known from 19 specimens, two of which were not taken into account for this assessment since no locality data is available. The earliest specimen was collected in 1892. Eleven specimens were collected after the year 1995. The seventeen herbarium specimens used for this assessment represent 17 unique occurrences, 11 subpopulations, and 10 locations (because the different localities within the Masoala National Park form a single location), which is the upper limit for the Vulnerable category under subcriterion 'a' of criterion B2. Some locations are in protected areas, notably Betampona Integrated Reserve, Makira Natural Park, Mangerivola Special Reserve, and Masoala National Park. Tarennella homolleana occurs in lowland to mid-elevation humid forests, which form a narrow belt along the eastern coast of Madagascar. This vegetation type has suffered from deforestation for centuries and is now heavily degraded and fragmented (Green & Sussman 1999;Dufils 2003; WWF 2020). The forests have been reduced to isolated patches separated from each other by agricultural areas or have degenerated to secondary savoka (degraded humid forest) as a result of slash-and-burn agriculture (tavy). This is the case in several of the known locations of Tarennella homolleana, e.g. in the region of Soanierana-Ivongo (Google Earth, satellite imagery of 27 Mar. 2019), so a reduction in the extent of the habitat of the species is inferred. The presence of the species in protected areas does not guarantee its continued existence. Masoala National Park is nominally protected but forest clearing for short-term cultivation of crops, illegal logging of rosewood, subsistence hunting, and artisanal mining continues inside the park (Nicoll & Langrand 1989;Kremen 2003;Zhu 2017;UNESCO 2020). Makira Natural Park is under threat of slash-and-burn agriculture, illegal logging, and mining from communities living around the protected area (WCS Madagascar 2020). Also, the Mangerivola Special Reserve is under high human pressure as the result of slash-andburn agriculture and wildfires (BirdLife International 2020). Because forest conversion is ongoing in several of the known locations of the species, we infer a reduction in the extent and quality of the habitat. These human activities will only increase with a growing human population, so a further reduction of the habitat of the species is projected for the future. The main threat to T. homolleana is clearing of its habitat both inside and outside protected areas for agricultural purposes. Secondary threats include burning from wildfires, logging for timber and charcoal, artisanal mining, overexploitation of decorative forest species, cyclone damage, and invasion of exotic species such as guava (Lowry et al. 1997;Britt et al. 2003;Kremen 2003;Rakotondratsimba et al. 2008;Wingen 2011;Randrianarijaona 2017). Because of the (relatively) low EOO (17,350 km 2 ), AOO (64 km 2 ) and number of locations (ten) and because of the current inferred and future projected reduction in the quality and extent of its habitat, Tarennella homolleana is listed as Vulnerable VU B1ab(iii) + 2ab(iii). of Tarennella puberula cannot be calculated since the species is only known from a single specimen. Using 2 × 2 km 2 grid cells, its area of occupancy (AOO) is estimated to be 4 km 2 , which complies with criterion B2 for the Critically Endangered category. The species is known from a single location, which complies with sub-criterion 'a' of criterion B2 for the Critically Endangered category. A single specimen was collected in 1994 in Antalavia littoral forest. Antalavia is the only littoral forest (ca 8 × 1.5 km) on the western side of the Masoala Peninsula. Large parts of the forest, notably around the coastal towns of Antalavia and Marofototra, have already been cleared for slash-and-burn agriculture (Razafimahatratra et al. 1999; Google Earth, satellite imagery of 22 Jan. 2018), so a reduction in the extent and quality of the habitat of Tarennella puberula is inferred. These human activities will only increase with growing human populations, so a further reduction of the habitat is projected for the future. Antalavia littoral forest is part of the Masoala National Park, which is part of the World Heritage Centre named 'Rainforests of the Atsinanana'. Human activities impacting the integrity of the national park are clearing of the vegetation for slash-and-burn agriculture, logging for timber and charcoal, wildfires, subsistence hunting, and artisanal mining (Nicoll & Langrand 1989;Kremen 2003;UNESCO 2020). A targeted survey of the Antalavia littoral forest and surrounding areas is needed to get a better understanding of the distribution area of T. puberula. Based on the low AOO (4 km 2 ), the single location and the current inferred and future projected reduction in the quality and extent of its habitat, T. puberula is listed as Critically Endangered. blades drying greenish or greyish brown above and brown below, glabrous on both surfaces but midrib and secondary nerves sometimes densely covered with minute, erect hairs below; base attenuate to cuneate; apex broadly acuminate, acumen 7-16 mm long; 9-16 secondary nerves on each side of the midrib. Petioles slender, 6-16 mm long, glabrous or sparsely to moderately covered with minute, erect hairs. Stipules 2.5-4 mm long; sheath irregularly truncate, more rarely triangular with obtuse tip, sometimes mildly keeled, usually densely covered with minute, erect hairs outside, often minutely ciliate. Inflorescences with bracts and bracteoles broadly ovate, 1.5-3 × 2.5-4 and 1-1.5 × 2-3 mm respectively, glabrous or moderately to densely covered with minute, erect hairs outside, glabrous but with many colleters all over the surface inside, margins ciliate, tips rounded. Calyx moderately to densely covered with minute, erect hairs outside, glabrous but with many colleters all over the surface inside; tube 0.5-1 mm long, truncate or with short lobes; lobes broadly triangular, ≤ 0.5 mm long, slightly keeled, margins ciliate, tips acute to obtuse. Corolla orange outside because of coverage with orange colleter exudate; tube 2.5-3 mm long, glabrous outside, upper half densely covered with ascending hairs inside; lobes 3-4 mm long, glabrous outside, densely covered with erect hairs at least in the lower half and often higher up in a central line inside, tips blunt. Anthers ca 3 mm long. Ovary 1-1.5 mm long, densely covered with minute, erect hairs. Style and stigma 6.5-8 mm long, exserted from the corolla tube for 3. .5 mm at anthesis. Fruits spherical, 0.9-1.1 × 0.8-1.1 cm (short persistent calyx included), longitudinally ribbed, orange or red when ripe, sparsely to densely covered with minute, erect hairs all over. Seeds 2-4 per fruit, angular or hemi-ovoid, 5-6 × 4-5 mm. Etymology -The species is named for the reddish colleter exudate, visible as droplets on the youngest stipule pair and often completely covering young inflorescences ( fig. 8C-F).

IUCN Red List assessment (provisional) -Endangered: EN B1ab(iii)+2ab(iii). The extent of occurrence (EOO) of
Tarennella sanguinea is estimated to be 1,032 km 2 , which complies with criterion B1 for the Endangered category. Using 2 × 2 km 2 grid cells, its area of occupancy (AOO) is estimated to be 28 km 2 , which complies with criterion B2 for the Endangered category. The species is known from 16, mostly recent, specimens, one of which has no locality data and cannot be taken into account for this assessment. The 15 remaining specimens constitute 11 unique occurrences, 6 populations, and four locations, which complies with subcriterion 'a' of criterion B2 for the Endangered category. The species occurs in three protected areas: Analalava Protected Area (10 specimens), Zahamena National Park (2 specimens) and Betampona Integrated Reserve (1 specimen). Each protected area constitutes a single location. Three other localities occur close to the western border of Zahamena National Park and are considered as a single location. Tarennella sanguinea occurs in lowland to midelevation humid forests (25-650 m a.s.l.), which are under severe threat from anthropogenic actions. The species is currently known only from a small area between 17°30′S and 18°00′S and between 48°55′E and 49°30′E. The main threat to the species is decline of its habitat both inside and outside the protected areas as a result of slash-and-burn agriculture, logging for timber and charcoal, subsistence hunting, wildfires, and cyclone damage (Nicoll & Langrand 1989;Project ZICOMA 2001;Rakotoarinivo et al. 2010). Almost all eastern lowland and mid-elevation humid forests outside the protected areas have been destroyed, with only small remnant patches remaining in between agricultural land (Google Earth, satellite imagery of 12 May 2019). Therefore, a decline in the extent and quality of the habitat of T. sanguinea is inferred. In the future, with growing populations, the pressure on the remaining forests will only increase, so a further reduction in extent and quality of the habitat is projected. Because of the small EOO (1,032 km 2 ), AOO (28 km 2 ), the low number of locations (four), and the current inferred and future projected reduction in quality and extent of its habitat, Tarennella sanguinea is assessed as Endangered EN B1ab(iii)+2ab(iii).

Phylogenetic analysis
Our analysis confirms the monophyly of the Afro-Madagascan clade Kainulainen et al. 2017) and shows the division of this clade into two main subclades, the first one comprising the Asian/Oceanic species of Tarenna (clade "C") and the second one the Afro-Madagascan-Indian Ocean clade (clade "D"). Our results differ from an earlier analysis in that the monospecific East African genus Tennantia is sister to the rest of the Afro-Madagascan clade (De Block et al. 2018;Kainulainen et al. 2017) rather than to the Asian/Oceanic Tarenna clade , yet this relationship is only poorly supported. Our analyses confirm the polyphyly of the genus Tarenna as already shown by De Block et al. (2015 and Kainulainen et al. (2017). However, as only 17 out of ca 200 species are represented in our phylogenetic analysis, we will not address this issue here. The backbone of the Afro-Madagascan-Indian Ocean clade "D" is unresolved, which was also the case in De  and Kainulainen et al. (2017). This probably reflects a quick radiation of the group following its arrival in Madagascar and precludes hypotheses about the relationships between the Malagasy genera. However, while the backbone of the Afro-Madagascan-Indian Ocean clade is unresolved, there is high support for many of the distal nodes in the clade, which mostly correspond to groups of species recognized at generic level. All currently recognized endemic Malagasy Pavetteae genera are confirmed as monophyletic, as was already shown by De Block et al. (2015. This is the case with high support for the Malagasy endemics Exallosperma, Helictosperma, Homollea, Pseudocoptosperma, Robbrechtia, Tulearia, and the more widely distributed Paracephaelis, occurring in eastern Africa, Madagascar, and the Comoros. The monophyly of the Malagasy Tarenna species is only moderately supported. Clade "I", comprising three of the five new species described here, is also well-supported as monophyletic ( . A representative of Tarennella cordatifolia (i.e. Razafimandimbison et al. 1239), albeit identified as "Pavetteae sp.", was included in an earlier analysis by Kainulainen et al. (2017). Similar to our results, Kainulainen et al. (2017) showed that the earliest divergent lineages in the Afro-Madagascan-Indian Ocean clade are continental African Coptosperma species. In contrast, they have T. cordatifolia (as "Pavetteae sp.") as the most earlydivergent Malagasy lineage, which is not the case in our tree. However, in both Kainulainen et al. (2017) and in the present analysis, the new species studied here do not group with other Pavetteae genera but form a distinct monophyletic clade. Because of this, and because of the morphological differences between the new species and all other Malagasy Pavetteae, we formally recognise this clade as the new genus Tarennella.
The analysis also confirms the paraphyly of the largest member of the Pavetteae in Madagascar, the genus Coptosperma Kainulainen et al. 2017). The position of Coptosperma graveolens and C. peteri as earliest divergent lineages in the Afro- Madagascan-Indian Ocean clade, also shown by De Block et al. (2015, is confirmed with good support values. This would indicate that at least C. graveolens can be considered as a separate genus. Another example of the paraphyly of Coptosperma is the position of the small genus Schizenterospermum in the Coptosperma clade "G". Furthermore,  recently described the monospecific genus Pseudocoptosperma for a species that was previously considered to belong to Coptosperma. Schizenterospermum, Pseudocoptosperma, and Coptosperma are all characterized by fruits with a single (partly) ruminate seed but there is morphological variation in characters, such as merosity, placentation, shape of stipules, pollen ornamentation, degree of rumination, etc. (De Block et al. 2002). It should be noted that in the two clades "G" and "H", which comprise Malagasy Coptosperma species, the earliest divergent lineages are not, or not strictly, Malagasy: C. borbonicum is restricted to Réunion and Mauritius, C. littorale to eastern and southern Africa, and C. supra-axillare occurs in Madagascar, eastern and southern Africa, and Aldabra (Seychelles). However, the type species, C. nigrescens, which is present in Madagascar, eastern and southern Africa, and the Comoros, seems to have a Malagasy origin. Until a largescale phylogenetic and taxonomic study of Coptosperma can be undertaken, we refrain from making taxonomic changes for the following reasons: 1) The majority of the Malagasy Coptosperma species remain undescribed and their morphological characters unknown, 2) Only 13 out of ca 50 species are included in our phylogenetic analysis, and 3) Certain Coptosperma species (e.g. C. graveolens) switch position in different analyses Kainulainen et al. 2017) indicating that the markers currently used do not provide sufficient support to completely clarify the delimitation of this species complex.

Tarennella
Distribution and habitat -Tarennella occurs in the understorey of eastern littoral and lowland to mid-elevation humid forest. The five species have narrow distribution ranges and are poorly collected, with up to 20 specimens for T. homolleana and T. sanguinea, less than ten specimens for  (•) and T. coronata (♦). B. T. homolleana (•). C. T. puberula (♦) and T. sanguinea (•).
T. cordatifolia, and a single specimen for T. coronata and T. puberula. As a result, the descriptions for the last two species are incomplete. We hope that the description of Tarennella will inspire botanists to search for its species, especially for T. coronata which may be extinct. Further collecting of all Tarennella species would give us a better understanding of their distribution, their abundance, and the threats they face. Observations on pollination and seed dispersal would also be useful. Tarennella as a member of the tribe Pavetteae -Tarennella shows all key characters of the tribe Pavetteae, notably terminal inflorescences, 5-merous flowers with a hypocrateriform corolla tube and corolla lobes contorted to the left in bud, permanently fused stigmatic lobes, bilocular ovaries with several ovules per locule impressed in a large placenta, small drupaceous fruits comprising several angular seeds, exotestal cells with plate-like thickenings along the outer tangential wall and the upper part of the radial walls, and 3-colporate tectate pollen grains (Robbrecht 1984;Bridson & Robbrecht 1985;De Block 1997). The genus is unique within the tribe by the seeds with a round or ovate, superficial adaxial hilar cavity not surrounded by a thickened annulus, and by the densely pubescent style and stigma (abaxial surfaces of the stigmatic lobes).
The five species are relatively homogeneous in their morphology but can be distinguished using various characters such as the shape and size of the calyx lobes, the shape of the leaf base, the presence or absence of pubescence, etc. Some of the less obvious characters are discussed below. Litter-collecting -Some Tarennella species exhibit occasional litter-collecting with roots present in the axils of the leaves, notably T. cordatifolia (Lowry et al. 4474 and Davis et al. 4538),and T. homolleana (Davis et al. 4526). In Rubiaceae, litter-collecting occurs typically in low woody plants, either monocaul (single-stemmed) or poorly branched, occurring in the forest understorey. In these plants, the leaf bases are often rounded or cordate and the leaves are sessile or have short petioles. Litter-collecting is known in some species of Pavetta (Pavetteae; Hawthorne 2013) and in some other Rubiaceae species such as Coffea magnistipula Stoffelen & Robbr. (Coffeeae;Stoffelen et al. 1997 In bracts and bracteoles of T. cordatifolia and T. coronata, colleters are present in a basal line and the inner surface is densely pubescent in the first and partially pubescent (base and tip) in the second species. In T. sanguinea and T. homolleana, the adaxial surface of bracts and bracteoles is glabrous and colleters are homogeneously distributed all over. It was impossible to observe the adaxial surface of bracts and bracteoles in T. puberula.
Colleters are absent from the adaxial surface of the calyx, except in T. homolleana and T. puberula, where one or a few colleters are present in the sinuses of the calyx lobes, and in T. sanguinea, where many colleters occur all over the inner surface of calyx tube and calyx lobes. In this species, the colleters produce copious, orange-red colleter exudate that often covers the entire young inflorescence ( fig. 8D-F). Also, a droplet of colleter exudate may be observed emerging from the youngest stipule pair ( fig. 8C). Colleter exudate covering part or all of the young inflorescences can also be observed in other Tarennella species, but is less common. The colour of the colleter exudate in these species is unknown. It should be noted that T. sanguinea, the species with the most copious colleter exudate, is the only species in the genus with numerous colleters in the calyx.
The presence of colleters in stipules, bracts, and bracteoles is typical for Pavetteae and, in fact, also for Rubiaceae. Robbrechtia, another Malagasy endemic Pavetteae genus, also produces copious colleter exudate that covers parts of the inflorescences (De Block 2003). The orange-red colour of the colleter exudate in T. sanguinea is rare within the Pavetteae, but it is also found in the monospecific genus Kindia Cheek from Western Africa (Cheek et al. 2018) and in some as yet undescribed Coptosperma species from Madagascar (Petra De Block pers. obs.). Due to the fact that the stipules in Tarennella are caducous and that little material is available, it was impossible to check the presence of colleters inside the stipules. Style and stigma -Style and stigma are exserted at anthesis and have a total length of 6-9.5 mm. The stigmatic lobes are permanently fused and only their very tips are free. Papillae are present on the adaxial surfaces of these free tips and along the line of fusion between the stigmatic lobes, a situation which is typical for the Pavetteae. The styles of Tarennella (unknown as yet in T. coronata) are distinct within the tribe because of the dense covering with long erect or ascending hairs present from ca 1 mm from the base to the very tip of the stigma (on the abaxial surfaces of the fused stigmatic lobes). Pubescent styles are common in representatives of the Afro-Madagascan clade of  with only Homollea and Tennantia providing an exception at generic level (Verdcourt 1988;. But the pubescent styles of all other Malagasy Pavetteae genera differ from those in Tarennella in that the pubescence is restricted to part of the style, i.e. the lower or upper half, and that it ends somewhat below the papillate zone. As such, the dense pubescence over almost the entire length of style and stigma in Tarennella is a unique character within the Malagasy Pavetteae. Pubescence on the abaxial surfaces of the fused stigmatic lobes is also found in the paleotropical genus Pavetta, which is absent from Madagascar. Fruits -The fruits of Tarennella are drupaceous, spherical or ovoid in shape. In the Pavetteae, the fruit colour is usually black (e.g. Pavetta, Tarenna). In Tarennella, however, fruit colour is orange or red in T. sanguinea ( fig. 8G), T. cordatifolia, and T. homolleana. In T. coronata, the label of the only specimen states that the fruit colour is reddish purple. Orange fruits occur in continental Africa in the genera Leptactina Hook.f., Rutidea, and sporadically in Tarenna (e.g. T. pallidula Hiern, T. vignei Hutch. & Dalziel). In Madagascar, Coptosperma nigrescens has fruits that first become orange and then turn black at full maturity : figure 1J).
The fruits of Tarennella cordatifolia, T. homolleana, and T. sanguinea are longitudinally ribbed but ribs are absent in fruits of T. coronata and fruits are unknown for T. puberula. Longitudinally ribbed fruits are rare within the Pavetteae but can be found in some species of Coptosperma (e.g. C. mitochondrioides Mouly & De Block;Mouly & De Block 2008). In T. homolleana and T. cordatifolia, the calyx tube is accrescent in the fruiting stage, reaching a length of up to 3 and up to 6 mm, respectively. The endocarp is crustaceous without a special opening mechanism. Seeds and seed coat -The fruits contain 2-6 seeds, which are angular, hemi-ovoid, or hemispherical in shape (depending on the seed number). The seed coat consists of a single-layered, large-celled and thickened exotesta, and a multi-layered endotesta ( fig. 10C-E). The endotesta cells are parenchymatic and they become partially crushed during the development of the seed, so that the endotesta, or at least its  basal cell layers, is seen as an amorphous thin layer in the mature seed ( fig. 10C, D). Only in the region of the hilum, the endotesta cell layers remain intact ( fig. 10E). In some species, the endotesta contains calcium oxalate crystals (e.g. T. homolleana) but these are absent in other species (e.g. T. coronata, T. sanguinea). The observed calcium oxalate crystals are prismatic in shape, which is the common type in the tribe Pavetteae. The exotesta cells have straight walls ( fig. 10A) and thickenings along the outer tangential and the upper part of the radial walls ( fig. 10C-E). The thickenings are plate-like and continuous but have a narrow ring-shaped intrusion from the cell lumen; in a longitudinal section this ring-like intrusion is visible as two narrow canals running to the outer edges of the cells (fig. 10C). This is the common type of thickening in Pavetteae possessing seeds with entire endosperm. In most continental African Pavetteae genera with this exotesta type, the seeds have a large, deep, and round adaxial hilar cavity that is surrounded by a thickened annulus, caused by the strong elongation of the exotesta cells (e.g. Cladoceras Bremek., Robbrecht & Bridson 1984: figure 2C, D;Leptactina, Robbrecht 1984: figure 3E; Pavetta, Robbrecht 1984: figure 5D-F). In contrast, in Tarennella, the adaxial hilar cavity is small, triangular or ovate in shape, and superficial. No annulus is present and the exotesta cells around the hilar cavity do not show elongation ( fig. 10E). This is also the case in other Pavetteae genera from the Afro-Madagascan clade of De , notably in Homollea and Paracephaelis (Bridson & Robbrecht 1985: figure 8C-E, as Tarenna trichantha) which have a linear hilum and laterally flattened seeds and in the East African Tennantia (Bridson & Robbrecht 1985: figure 1D, E) with a small round hilum and angular seeds. Small superficial hilar cavities are usually present in taxa with ruminate seeds (e.g. Coptosperma), which, however, usually have exotesta cells without thickenings. Pollen -Pollen of Tarennella ( fig. 11) is characterized by a perforate to microreticulate sexine dotted with supratectal elements in the form of microgemmae (pollen type 3 of De Block & Robbrecht 1998). This pollen type is also present in the genera Homollea and Paracephaelis and in certain Malagasy species of the genera Coptosperma and Tarenna (C. nigrescens; T. grevei; Petra De Block, pers. obs.). Supratectal elements are also present in Pavetta subgenus Pavetta, which is absent from Madagascar.

SUPPLEMENTARY FILE
Supplementary file 1 -List of taxa used in the phylogenetic analyses with voucher information (geographic origin, collection, herbarium) and GenBank accession numbers for the plastid and nuclear markers rps16, trnT-F, ITS, petD, accD-psa1, and PI. https://doi.org/10.5091/plecevo.2021.1756.2367 Fernandez are acknowledged for undertaking the line drawings. Ms Iris Van der Beeten is acknowledged for doing pollen acetolysis, for taking the pollen photographs and for colouring microtomy slides. Mr Sven Bellanger and Ms Liliane Tytens kindly prepared fig. 1. The use of field photographs was gracefully allowed by Dr Olivier Lachenaud, Dr Kent Kainulainen, and Ms Catherine Caney-Dunod. Mr Wim Baert is thanked for help with the molecular work. Fieldwork was conducted in collaboration with Kew Madagascar Conservation Centre (KMCC). We thank Mr Stuart Cable and Dr Hélène Ralimanana for giving us access to the KMCC facilities, Ms Tianjanahary Randriamboavonjy and the staff at the KMCC facilities for their help and hospitality. Ms Jacqueline Razanatsoa (TAN) is gratefully acknowledged for help during the fieldwork. We thank PBZT (Parc Botanique et Zoologique de Tsimbazaza) for help with the application of a research and collection permit and DSAP (Direction du Système des Aires Protégées) for granting this permit. Financial support for fieldwork was provided by the Research Foundation -Flanders (FWO).