Revision of the Afro-Madagascan genus Costularia (Schoeneae, Cyperaceae): infrageneric relationships and species delimitation

A recent molecular phylogenetic study revealed four distinct evolutionary lineages in the genus Costularia s.l. (Schoeneae, Cyperaceae, Poales). Two lineages are part of the Oreobolus clade of tribe Schoeneae: the first being a much-reduced genus Costularia s.s., and the second a lineage endemic to New Caledonia for which a new genus Chamaedendron was erected. The other two lineages were shown to be part of the Tricostularia clade of tribe Schoeneae. Based on morphological and molecular data, the genus Costularia is here redelimited to represent a monophyletic entity including 15 species, which is restricted in distribution to southeastern Africa (Malawi, Mozambique, South Africa, Swaziland, Zimbabwe), Madagascar, the Mascarenes (La Réunion, Mauritius), and the Seychelles (Mahé). Molecular phylogenetic data based on two nuclear markers (ETS, ITS) and a chloroplast marker (trnL-F) resolve the studied taxa as monophyletic where multiple accessions could be included (except for Costularia laxa and Costularia purpurea, which are now considered conspecific), and indicate that the genus dispersed once to Africa, twice to the Mascarenes, and once to the Seychelles. Two endemic species from Madagascar are here described and illustrated as new to science, as is one additional species endemic to La Réunion. Two taxa previously accepted as varieties of Costularia pantopoda are here recognised at species level (Costularia baronii and Costularia robusta). We provide a taxonomic revision including an identification key, species descriptions and illustrations, distribution maps and assessments of conservation status for all species.


Nomenclature and taxonomy
A nomenclatural study including the taxonomic history of the genus and its species, critical for the correct coining of the new names and the proper use of prior ones, was performed. The electronic version of this article in portable document format will represent a published work according to the International Code of Nomenclature for algae, fungi and plants (ICN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. In addition, new names contained in this work which have been issued with identifiers by IPNI will eventually be made available to the Global Names Index. The IPNI LSIDs can be resolved and the associated information viewed through any standard web browser by appending the LSID contained in this publication to the prefix 'http://ipni.org/'. The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central and CLOCKSS.

Molecular study
All known Costularia s.s. species, except Costularia microcarpa (Cherm.) Kük. which is only known from its type and Costularia brevifolia Cherm. which is rare in collections, were sampled (representing c. 80% of the diversity of the genus, that is, nine out of 11 species and two out of three heterotypic varieties recognised by Govaerts et al. (2018) using multiple accessions per taxon where possible. Additionally, samples were included from several taxa potentially representing new species. The outgroup taxa, selected based on Larridon et al. (2018a), consist of nine species representing the other four genera of the Oreobolus clade of tribe Schoeneae. A total of 36 samples (15 newly sequenced) from 24 different taxa were used for this study. The samples with species names, voucher information, origin and GenBank accession numbers for the sequences, are given in Table S1. The DNA extraction protocol, markers (ETS, ITS and trnL-F), and material and methods for PCR amplification and sequencing and for obtaining alignments follow Larridon et al. (2018a). Sequences were assembled and edited in Geneious R8 (http://www.geneious.com, Kearse et al., 2012), aligned using MAFFT 7 (Katoh, Asimenos & Toh, 2009;Katoh & Standley, 2013) with 'maxiterate' and 'tree rebuilding number' set to 100 (long run), afterwards, alignments were checked manually in PhyDE 0.9971 . The alignments used to produce the phylogenies are available as a Data S1.
We first inferred the gene trees for each of the three regions separately to identify potential incongruence. As there were no instances of conflict at well-supported nodes (Figs. S1-S6), the matrices of the three regions were concatenated for the downstream analyses. PartitionFinder 2.1.1 (Lanfear et al., 2012) was used to determine an appropriate data-partitioning scheme from potential partitions that were defined a priori (in this case, each marker was treated as a separate partition), as well as the best-fitting model of molecular evolution for each partition, using the Bayesian Information Criterion. PartitionFinder confirmed the a priori data-partitioning scheme, and the GTR+I+C (invgamma) model of sequence evolution was determined to be the best-fitting model for the two nrDNA markers, while the GTR+C (gamma) model of sequence evolution was determined to be the best-fitting model for the trnL-F partition in the concatenated data set.
Maximum likelihood (ML) analyses of the optimally partitioned data were performed using RAxML 8.2.10 (Stamatakis, 2014). The search for an optimal ML tree was combined with a rapid bootstrap analysis of 1,000 replicates. Additionally, partitioned analyses were conducted using Bayesian Inference (BI) in MrBayes 3.2.6 (Ronquist et al., 2012). Rate heterogeneity, base frequencies, and substitution rates across partitions were unlinked. The analysis was allowed to run for 100 million generations across four independent runs with four chains each, sampling every 10,000 generations. Convergence, associated likelihood values, effective sample size values and burn-in values of the different runs were verified with Tracer 1.5 (Rambaut & Drummond, 2007). The first 25% of the trees from all runs were excluded as burn-in before making a majority-rule consensus of the 30,000 posterior distribution trees using the 'sumt' function. All phylogenetic analyses were run using the CIPRES portal (http://www.phylo.org/; Miller, Pfeiffer & Schwartz, 2011), and were executed for both full and reduced sampling alignments. Trees were drawn using TreeGraph2 (Stöver & Müller, 2010).

Morphological study
Herbarium specimens of BR, G, GENT, K, L, MAU, P, REU, TAN and UPOS (Thiers, 2018) were studied morphologically using a Leica (Leica Microsystems, Wetzlar, Germany) binocular microscope. Measurements where made with a ruler (e.g. leaf and culm length), or using a binocular microscope with graticule (e.g. spikelet and glume length). When measuring width, this was done near the middle of the organ (e.g. middle of the culm). The term peducles represents the main inflorescence branches measured from base of primary inflorescence bract to second order bract. Where possible, links to imaged type specimens are provided (Catalogue des herbiers de Genève, 2018;HerbCat, 2018;Muséum national d'Histoire naturelle, 2018).

Species distributions and conservation assessments
Information on locality data was obtained from the studied herbarium records (see Taxonomic Treatment and Data S2). Georeferenced localities were used to map the distribution of the Costularia species studied in SimpleMappr (Shorthouse, 2010). The extent of occurrence (EOO) and area or occupancy (AOO) of the species were calculated in GeoCAT (Bachman et al., 2011), where the AOO was based on a user defined cell width of two km in line with IUCN Red List criteria (IUCN, 2012). Conservation assessments were prepared according to the guidelines to the IUCN Red List categories and criteria (IUCN, 2012;IUCN Standards and Petitions Subcommittee, 2014).

Morphological study, species distributions and conservation assessments
Morphological results, species distributions and conservation assessments are elaborated in the Taxomic Treatment. The additional herbarium specimens studied per taxon are listed in Data S2.

DISCUSSION
Four clades are here retrieved in the Oreobolus clade of tribe Schoeneae ( Fig. 1): Costularia, Chamaedendron, Capeobolus + Cyathocoma and Oreobolus, in line with recent studies Semmouri et al., 2018). Viljoen et al. (2013) reconstructed the ancestral areas for tribe Schoeneae but did not obtain a clear result for the ancestral area of the Oreobolus clade. Both Capeobolus and Cyathocoma are found in the Cape Floristic Region, while Chamaedendron is endemic to New Caledonia, and Oreobolus has a wider distribution in the souther hemisphere (Malesia to Australasia, Hawaiian Islands, Costa Rica to Falkland Islands; Govaerts et al., 2018).
Of the four main clades in Costularia, only clade B is restricted to Madagascar, while the others include Madagascar endemics and species found on the Indian Ocean islands and/or mainland Africa (Fig. 1). In clade A, the Madagascan endemic species Costularia leucocarpa and Costularia andringitrensis are sister to Costularia natalensis from southeastern Africa. Costularia itremoensis from South Central Madagascar is sister to these three species (Fig. 1). Clade B consists of the Costularia pantopoda species complex with Costularia baronii and Costularia robusta forming well supported monophyletic lineages separate from the typical Costularia pantopoda (Fig. 1). Costularia robusta was first described as Costularia baronii var. robusta Cherm. This concurs with our results in which Costularia baronii and Costularia robusta are sister species (Fig. 1). A last taxon part of this clade, sister to the others, is currently identified as Costularia cf. pantopoda. This taxon needs further study since it is only known from a single collection with little metadata information. Its morphology appears intermediate between Costularia pantopoda and Costularia itremoensis. Potentially related to clade B is Costularia microcarpa, a species first described by Chermezon (1937) under Costularia baronii (as Costularia baronii C.B.Clarke var. microcarpa Cherm.), and later raised to species level by Kükenthal (1939). Clade C contains two well supported subclades, one of which includes specimens identified as Costularia laxa and Costularia purpurea. These taxa were not always recovered as monophyletic (Figs. S1-S6). Morphological study confirmed that the delimitation between these taxa is unclear, resulting in the decision to combine the two species under a single species name: Costularia purpurea (see Taxonomic Treatment). The second well supported subclade of Clade C consists of individuals of Costularia melicoides (Fig. 1). Costularia melicoides is endemic to the Mascarenes where it is found on both the islands of La Réunion and Mauritius. Clade D includes four species: Costularia xipholepis, a recently rediscovered species endemic to the Seychelles , a single accession of Costularia melleri from Central Madagascar, and a subclade including Costularia cadetii and Costularia humbertii. The newly discovered Costularia cadetii and the species Costularia humbertii stand out due to their smaller stature and shorter leaves. Both are restricted to high-elevation zones, but what is remarkable is that while Costularia humbertii is found in the northeast of Madagascar (Marojejy National Park), Costularia cadetii is endemic to La Réunion where it is limited to peaks of the island's volcanoes. This sister relationship points at a long-distance dispersal event likely from the mountain tops of northeastern Madagascar to those of La Réunion. A species potentially related to Costularia humbertii is Costularia brevifolia with which it shares characters such as a robust caudex, short stature, and short broad leaves, although it is biogeographically (southeastern Madagascar) and ecologically (low-mid elevation) isolated from it (Fig. 2).
Of the 15 species of Costularia recognised here, three-quarters are threatened with extinction because of their restricted distribution ranges and human impact (see Taxonomic Treatment). In Madagascar, habitat destruction and deterioration are the major threats. Additional threats may relate to climate change as some species exclusively occur at (very) high elevation (e.g. Costularia cadetii, Costularia humbertii, Costularia robusta), or to invasive species (e.g. in the Mascarenes). Two species were assessed as critically endangered (CR), six as endangered (EN) and three as Vulnerable (VU) according to IUCN Red List categories and criteria (IUCN, 2012;IUCN Standards and Petitions Subcommittee, 2014). Two endemic but widely distributed species from Madagascar (Costularia leucocarpa, Costularia purpurea) were assessed as least concern (LC), as was Costularia natalensis, the only species occurring in mainland Africa. A final species (Costularia microcarpa) could not be assessed at this time due to lack of information and is considered data deficient (DD). Further research and fieldwork are needed to study the species of Costularia, their populations and the threats they face.  Goetghebeur (1986)).

TAXONOMIC TREATMENT
Perennial herbs, small to tall, tufted or more rarely shortly rhizomatous, caudex sometimes present. Culms scapose or with few nodes. Leaves usually both basal and caudal; basal leaves with poorly defined sheaths; cauline leaves enveloping up to ½ internode length; margins scabrid, spirodistichous, eligulate, blade sometimes deciduous. Inflorescence terminal, (contracted) paniculate with few to numerous spikelets; primary bracts ± leaf-like, sheathing. Spikelets with several distichous, deciduous glumes, of increasing length, the upper (1-)2 glumes each subtending a flower, enclosed by the wings of the next glume. Flowers, lower one (functionally) male (rarely bisexual or absent), upper one bisexual or functionally female (rarely functionally male). Perianth bristles 6, fimbriate to ciliate, mostly longer than the nutlet and deciduous with it. Stamens 3. Style trifid, style base often distinct (at anthesis), thickened, persistent, often scabrid. Nutlet ovoid or oblong, rounded trigonous, often 3-ribbed, ± stipitate, beaked, surface smooth or rugulose. Diagnosis: Costularia andringitrensis differs from all other Costularia species from Madagascar by its small stature with the flowering culm scarcely exceeding the leaves. In this aspect it mostly resembles Costularia cadetii from La Réunion from which it can easily be distinguished by the latter maturing two nutlets per spikelet.

Distribution
The species is only known from south-central Madagascar, where it was found in the Andringitra National Park, Haute Matsiatra region, Fianarantsoa province ( Fig. 2).

Ecology
This species is found in near rocks in grassland to ericoid shrubland vegetation at 2,000-2,500 m in elevation.

Phenology
Immature inflorescence observed in November, while the specimen collected in April had already shed its ripe nutlets.

Etymology
The species is named for the Andringitra National Park in Madagascar.

Conservation status
Costularia andringitrensis is a small perennial herb endemic to Madagascar, where it is only known from two specimens and occurs in a restricted area in the Andringitra National Park. It is only known from a single location and a minimum AOO of eight km 2 . However, there are other potential areas of occurrence for the species that have not yet been explored. The species is threatened by cattle grazing and by fires started for pastoral reasons which can easily get out of control and enter the National Park (I. Larridon, 2010, personal observation;F. Rakotonasolo, 2017, personal observation). Therefore, it is assessed as Critically Endangered: CR B2ab(ii,iii).

Notes
As is commonly seen in tropical Cyperaceae species occurring at high elevation (I. Larridon, 2010, personal observation), Costularia andringitrensis is characterised by very dark spikelets. In the molecular phylogenetic hypothesis ( Fig. 1), it is retrieved as sister to Costularia leucocarpa.
Although species of tribe Schoeneae are adapted to natural fire, if fire frequency is increased, especially by herders, this can threaten their regeneration (A.M. Muasya, 2010, personal observation). However, complete absence of fire can also be a threat as most species occur in habitats where open/forest are alternative states. Forests are kept out by the fire, whose absence could lead to forest encroachment. Most species of tribe Schoeneae are shade intolerant and thus would die if shaded. Type (lectotype designated here). Madagascar, Central Madagascar, R. Baron 3316 (lectotype: K000244885!, isolectotype: MNHN-P-P00459989!).

Ecology
It has been found growing in rocky areas (e.g. rock crevices along a stream bank), ericoid shrubland at elevations of 1,300 to almost 2,200 m.

Phenology
Flowering specimens were collected from March to May, fruiting plants in October, while plants collected in December and January had either shed their nutlets or bore very young inflorescences.

Conservation status
Costularia baronii is distributed in the Antananarivo, Fianarantsoa and Toliara provinces of Madagascar, and occurs in at least four protected areas, that is, Andringitra, Ankaratra Massif, Andohahela, Ibity Massif and Pic d'Ivohibe. Threats to this taxon need further investigation but in the Andrigitra National Park, its habitat and area of occupancy are impacted negatively by cattle grazing and by fires started for pastoral reasons which can easily get out of control and enter the National Park (I. Larridon, 2010, personal observation;F. Rakotonasolo, 2017, personal observation). Based on 10 georeferenced herbarium specimens, the species occurs in at least seven locations and has an estimated AOO of 36 km 2 and an EOO of 16,292 km 2 . Using IUCN criteria, it can be assessed as VU B1ab(ii,iii)+2ab(ii,iii).

Distribution
Endemic to southeastern Madagascar and only known from the Atsimo Atsinana and Anosy regions in the Fianarantsoa and Toliara provinces (Fig. 2).

Ecology
It has been found growing on humid rocks in peatlands, on laterite and granite in tropical forest, and in faults of gneiss rock escarpments, at elevations of (200-)600-900 m. Phenology Flowering specimens were collected from in March. Young inflorescences can be observed on the specimens collected in February, while old inflorescences remain on the plants until October-November.

Conservation status
Costularia brevifolia is a robust perennial herb, limited in distribution to the forested mountain ranges of south-eastern Madagascar at mid-elevation. It is known from only four locations. The estimated EOO is 2,463 km 2 and the area of occupancy is 20 km 2 . According to the limited metadata available this species likely occurs in the protected areas of Midongy du Sud and Andohahela. Fire (natural or man-made) and disturbance or elimination as a result of deforestation for agricultural extension are the major threats wich affect this species. Hence, it is assessed as EN B1ab(i,ii,iii,iv)+B2ab(i,ii,iii,iv).

Notes
One of  Diagnosis: This species is closely related to Costularia humbertii from northern Madagascar, from which it differs in its smaller habit, absence of a caudex, the basal leaves equaling or overtopping the flowering culm, and having two bisexual flowers. It can be distinguished from the only other species of Costularia on La Réunion by its much smaller habit and having two bisexual flowers.

Distribution
Costularia cadetii is a small perennial herb, endemic to La Réunion and found only in the Parc National de La Réunion at elevations of 1,700-2,400 m (Fig. 10).

Ecology
Found growing in rocky areas, montane grasslands and ericoid vegetation close to volcanic crater edges at high elevation. Phenology Flowering specimens were collected in January, fruiting specimens in February and March. The specimens collected by Cadet in May had shed their glumes and nutlets, while the plants collected in November and December were vegetative or immature.

Etymology
The first record of this species (Cadet 454) was collected by in 1965, and on its label the following note is written 'Costularia sp. Further material needed!'. Thérésian Cadet ) was a botanist from La Réunion specialised in the vegetation from the Mascarene Islands. He taught plant biology at the University of La Réunion and was one of the main authors of the Flore des Mascareignes. This species is named in his honour.

Conservation status
Costularia cadetii is a small perennial herb, endemic to La Réunion. It is known only from three locations within the Parc National de La Réunion. The area, extent and quality of habitat of this species is threatened by fire, volcanic activity and climate change.
Based on the seven known herbarium collections, the minimum estimated area of occupancy is 20 km 2 and the minimum estimated EOO is 250 km 2 . It is hence categorised as EN B1ab(iii)+B2ab(iii).

Distribution
Endemic to the Antsiranana province of Madagascar where it is restricted to the high-elevation zone of the Marojejy National Park (Fig. 2). Ecology Found growing in swamps in high elevation ericoid vegetation, and on gneiss and quartzite rocks of the mountain ridge, at elevations of 1,400-2,200 m.

Phenology
Flowering specimens were collected from March to early April. Young inflorescences can be observed on the specimens collected in November-December.

Conservation status
Costularia humbertii is endemic to the Antsiranana province of Madagascar and is limited in distribution to the high-elevation zone of the Marojejy National Park. The minimal area of occupancy was calculated as 24 km 2 , the estimated EOO is 17 km 2 and the species is only known from one location. Fire (natural and man-made) and disturbance of its habitat as a result of logging, firewood collection and charcoal are the major threats which may affect this species. Costularia humbertii is only known from seven herbarium collections and has not been collected since 1989. Research is needed to investigate its current status at the single known location. Here, we assess the species as CR B1ab(i,ii,iii).

Notes
One of two short leaved Costularia species in Madagascar, the other being Costularia brevifolia. In the molecular phylogenetic hypothesis (Fig. 1), Costularia humbertii appears to be closely related with a small high-elevation species from La Réunion (Costularia cadetii). Diagnosis: This species resembles most closely Costularia pantopoda var. pantopoda from which it can be distinguished by having longer peduncles (longest 5.5-11 vs. 4-5 cm) and more emptyglumes (6-11 vs. 3-6).

Distribution
Costularia itremoensis is endemic to Madagascar and is found in the highlands of South Central Madagascar, in the Fianarantsoa province (Fig. 5).

Ecology
The habitat in which this species is found consists of bare rocks and/or grassland in the Itremo massif (L. Rabarivola, 2014, personal observation). In Isalo, its habitat is dominated by wooded grassland-bushland mosaic and/or plateau grassland-wooded grassland mosaic (Moat & Smith, 2007) between 800 and 1,700 m in elevation.

Phenology
Flowering/fruiting specimens were collected from July to September, plants collected from January to April were immature.

Conservation status
Costularia itremoensis is endemic to Madagascar and is found in the highlands of South Central Madagascar, in the Fianarantsoa province. Based on the limited metadata available it likely occurs in the Itremo new protected area and Isalo National Park. The estimated EOO was calculated as 7,169 km 2 and the minimal area of occupancy is 20 km 2 . This species is only known from three locations and is threatened by grazing and uncontrolled fire from pastures fire. Its habitat is also threatened by deforestation from logging, firewood collection and mining. Therefore, this species is assessed as Endangered: EN B2ab(i,ii,iii).

Ecology
The species occurs at mid to (very) high elevation, and has been collected along mountain ridges, from thickets on rock formations, and in open forest.

Phenology
Flowering specimens were collected in December-January, while fruiting specimens were collected in February and March.

Conservation status
Costularia leucocarpa is endemic to Madagascar and found in Antsiranana, Antananarivo, Toamasina, Fianarantsoa and Toliara provinces, where it has been collected along mountain ridges, from thickets on rock formations. The species occurs in Ranomafana National Park, Tsaratanana Reserve Naturelle Intégrale and Manongarivo Special Reserve. The species has a large distribution range (AOO = 124 km 2 ) and its estimated EOO is 7,636 km 2 , which is much larger than the threshold for a threatened category. Despite its habitat being under various anthropogenic pressures, Costularia leucocarpa is here assessed as LC because (1) no specific threats to its survival have been observed, (2) it is widely distributed in Madagascar, and (3) occurs in several protected areas. Notes Since Costularia recurva shares syntypes with the older name Costularia leucocarpa, both can be lectotypified to the same specimen (Baron 399 K000244883) rendering Costularia recurva superfluous. Previously, the number and position of flowers has been unclear. Chermezon (1937) (in general for the genus) and Kükenthal (1939) (for Costularia leucocarpa) described the male flower to be born by the third glume from the top of the spikelet, the bisexual flower to be born by the second glume from the top, and the topmost glume to be empty and reduced. At first glance, this appears correct, but when comparing Costularia leucocarpa spikelets with those of the other Costularia species where the topmost glumes are fertile and the lower glumes are sterile, and taking in consideration the common metatopic displacement (epicaulescence) of the glumes and flowers on the rachilla in spikelets with distichous glumes of species of Cyperaceae subfamily Cyperoideae (Vrijdaghs et al., 2010(Vrijdaghs et al., , 2011, we believe that Costularia leucocarpa represents the common pattern observed in the rest of the genus. Perennial herb with short rhizome with stiff fibres. Culm 35-100 cm Â 1.5-4 mm, striate, minutely puncticulate. Basal leaves crowded, distichous; leaf sheaths four to six cm long, indistinct, straw-coloured to purple, multiveined; leaf blades c. 27-60 cm Â 2.5-5 mm, flat, indistinctely keeled, tapered at the tip, edges minutely serrulate. Cauline leaves 3-4, very distant; leaf sheaths, long, green-purplish, mouth oblique. Inflorescence an elongate panicle, 30-85 cm long, with c. 9-11 partial inflorescences, distantly spaced; inflorescence bracts longer than the partial inflorescence they subtend, sheaths purplish. Peduncles unequal, up to c. 12 cm. Pedicels of the spikelets erect, flattened, margins slightly scabrid, 4-15 mm long. Spikelets oblong-lanceolate, 5.5-7.5 Â 1.5-2 mm, somewhat flattened. Glumes 5-8, distichous, lanceolate-ovate, acuminate, reddish-black, with colourless-whitish margins; lower glumes empty, scabrid on the midvein, mucronatae-aristulate; two upper glumes fertile, barely mucronate; rhachilla short and erect. Flowers 2, lower bisexual, upper male. Perianth bristles 6, longer than the nutlet, pale to rusty-coloured, antrorsely densely ciliate-scabrid. Stamens 3; anthers linear yellow; connective short, bent, purple. Style trifid, base elongate-conical, triquetrous, pale, margins hispidulous, persistent. Nutlet swollen-trigonous, longitudinaly trisulcate, pale, smooth, 2-2.3 mm long, base long cuneate; beak narrow, 2-2.5 mm long.

Distribution
Endemic to the Mascarene Islands of La Réunion and Mauritius (Fig. 10).

Ecology
Costularia melicoides prefers mid to higher elevation on the island of La Réunion: (500-) 900-1,700 (-2,000) m where it occurs in ericoid thickets (avounes), moist tropical forest, forest with Acacia heterophylla (tamarinaie), and humid tickets with Pandanus (C. Fontaine, 2018, personal communication). However, in Mauritius, it is found on boulders or in clumps in seasonally-flooded upland marshes near Petrin in the Black River Gorges National Park at elevations of c. 600-700 m, in upland marshes and thickets in Perrier Nature Reserve at c. 550 m in elevation, and in the district Flacq it was found at an elevation of 280 m.

Phenology
Flowering specimens were collected in February (La Réunion) and June (Mauritius), fruiting specimens were collected in April and May and from October to January (La Réunion).

Conservation status
Costularia melicoides occurs in the four regions of La Réunion, and it has been recorded from two regions of Mauritius (Flacq and Plaines Wilhems). Likely, the location at Flacq does not exist anymore (C. Baider, 2018, personal communication). The species prefers mid to higher elevations on La Réunion, while it it is found at lower elevations on Mauritius. It grows in ericoid thickets, forests, on boulders or in clumps in seasonallyflooded upland marshes. Its area of occupancy was estimated as 64 km 2 and its EOO as 6,805 km 2 , and it occurs at four locations (the Parc National de La Réunion, and in the Black River Gorges National Park, the Perrier Nature Reserve and the protected areas of the Bambou Mountains on Mauritius). The habitat of the species in Le Réunion is threatened by invasive alien species, disturbance due to human activities, and climate change. In Mauritius, similar threats to the habitat of the species exist, in particular due to invasive alien species and the patchiness of the remaining native vegetation. Therefore, Costularia melicoides is here assessed as Endangered EN B2ab(i,ii,iii,iv).

Notes
In Costularia melicoides, the lower fertile flower is bisexual and the upper fertile flower is male (or sterile), in contrast with the mainland African and Madagascan Costularia species (with lower fertile flower male or sterile, and upper fertile flower bisexual), and in contrast with Costularia cadetii from La Réunion with two bisexual flowers.

Ecology
Marshes, humid areas in forest, an elevation of 1,000-1,500 m.

Phenology
Specimens with very young inflorescences were found in October, November and April, flowering specimens were collected in November and January, fruiting specimens in December. Specimens collected in March and April had already lost their ripe nutlets.

Conservation status
The conservation status of Costularia melleri was previously assessed by Faranirina (2017) as EN B2ab(i,ii,iii,iv,v) based on an estimated AOO of 45 km 2 (within the limits for EN status under the criterion B2) and five known locations. Only one subpopulation occurs in a protected area (Ranomafana National Park; Larridon et al. 2010-0249), the other subpopulations are known from unprotected areas subject to agriculture activity (Faranirina, 2017). Faranirina (2017) projected that the ongoing loss of its habitat will induce a strong continuous decline in the number of subpopulations and mature individuals in the next ten years as well as a continuing decline in its EOO and AOO.

Distribution
Endemic to the Ihorombe region of Fianarantsoa province in Madagascar (Fig. 5).

Ecology
Found growing on shaded, humid sandstone in Isalo National Park at an elevation of c. 1,000 m.

Phenology
Only known specimen was collected in October as flowering.

Conservation status
Costularia microcarpa is endemic to Madagascar. It is only found in Ihorombe region of Fianarantsoa province at elevation 1,000 m in Isalo National Park. There is insufficient information available to assess the conservation status of this species since it is only known from its type specimen. Therefore, it is categorised as DD. Research is needed to investigate whether the population of this species at the only known location in Isalo National Park is still present.

Notes
This is one of only two Costularia species that could not be sampled for this study, as it is only known form the type specimen. Although unsure at this time, this species is likely part of the Costularia pantopoda species complex. Chermezon (1937) originally published this as a variety under Costularia pantopoda subsp. baronii (as Costularia baronii var. microcarpa) though Kükenthal (1939) later recognised this taxon at species level. Type (lectotype designated here). South Africa, KwaZulu-Natal, (without stated locality but probably Noodsberg (Burtt, 1988;Browning & Gordon-Gray, 1996)), J. Buchanan 152 (lectotype: K000244893!).

Distribution
Costularia natalensis is restricted in its distribution to southeastern Africa (Fig. 16). In particular, the species is present at higher elevation (1,070-2,130 m) along the chain of individually isolated highlands roughly paralleling part of the coastline, for example, in South Africa the Wolkberg, Sabie and Graskop areas of the Mpumalanga Drakensberg (Browning & Gordon-Gray, 1996). Mount Mulanje in Malawi is the northernmost known locality, and the southernmost distribution of the species reaches the area of Pietermaritzburg in Kwazulu-Natal (South Africa).

Ecology
According to observations by Browning & Gordon-Gray (1996), populations are mostly very localised, often small, and in KwaZulu-Natal, frequently limited to a few scattered, solitary plants which grow on steep, rocky slopes, associated with coarse grasses in the zone between forest and grassland. A slightly more extensive population grows along banks of small streams and among boulders, where nutrients particularly phosphates are in short supply and other vegetation is scare (Restionaceae and short grasses), in the Chimanimani National Park (Zimbabwe; Browning & Gordon-Gray, 1996). In Mozambique, several (small) subpopulations are also found on quartzite sandstone in the Chimanimani Mts, and on rocks in the submontane grasslands of Mt Gorongosa and Serra Choa. Plants of this species have been collected from Mount Mulanje in Malawi (which is composed of seynite, quartz-seyinite and granite rock materials), in particular from the eastern zone of the Biosphere Reserve (Lichenya and Chambe). It is restricted to higher elevations.

Phenology
Flowering/fruiting specimens were collected from November to May. Conservation status Costularia natalensis is restricted in its distribution to southeastern Africa (Malawi, Mozambique, Zimbabwe, Swaziland, South Africa). It is found at higher elevations in rocky areas in grassland and shrubland. Threats affecting part of the range of the species include fire, fuelwood collection, illegal logging of natural forests and plantation forestry, invasive species and potential mining. Although the population of this species is believed to be decreasing (Browning & Gordon-Gray, 1996), it currently does not fall within the criteria for any of the threat categories, and is therefore assessed as LC. However, further research is needed to investigate threats and population size.

Notes
In our molecular phylogenetic results (Fig. 1), Costularia natalensis is found in a clade with three other Costularia species, two of which are here described as new to science, that is, Costularia andringitrensis and Costularia itremoensis. Although Burtt (1988) indicated some morphological variety between plants of different localities, Browning & Gordon-Gray (1996), who studied specimens from the entire distribution range of Costularia natalensis, found no clear discontinuities that may provide a basis for subdivision of the species.

Conservation status
Costularia pantopoda is restricted in its distribution to south-central Madagascar, and occurs in at least one protected area, that is, the Andringitra National Park. Threats to this taxon need further investigation but in the Andrigitra National Park, where most collections have been made, its habitat and area of occupancy are impacted negatively by cattle grazing and by fires started for pastoral reasons which can easily get out of control and enter the National Park (I. Larridon, 2010, personal observation;F. Rakotonasolo, 2017, personal observation). In other areas, fire (natural and man-made) and disturbance of its habitat as a result of logging, firewood collection and charcoal may also affect this species. Based on 10 georeferenced herbarium specimens, the taxon occurs in at least six locations and has an estimated AOO of 40 km 2 and an EOO of 9,478 km 2 . Using IUCN criteria, this variety can be assessed as VU B1ab(ii,iii)+2ab(ii,iii).

Distribution
Costularia pantopoda var. pantopoda occurs in the Fianarantsoa province and in the south of the Antananarivo province of Madagascar (Fig. 5).

Phenology
Flowering specimens were collected from December to April, fruiting specimens from September to November.

Distribution
Known from a single collection made near Antsirabe in the Antananarivo province of Madagascar (Fig. 5).

Ecology
The only known collection was found growing in a marsh at c. 1,600 m in elevation.

Phenology
The taxon was collected in flower in January. Kükenthal (1939) described this new variety based on a single specimen (Perrier de la Bâthie 2729). Though likely present in P, the lectotype could not be traced. This variety most closely resembles Costularia pantopoda var. pantopoda.

Ecology
The species is found infrequent on granitic formations in ericaceous shrubland, grassland and open forests at mid to high elevations (500-1,850 m).

Phenology
Flowering/fruiting specimens were collected from November to May. Young inflorescences can be observed on the specimens collected in September-October, while old inflorescences remain on the plants until September.

Conservation status
The species occurs in a range of protected areas including: Analamazaotra (Périnet), Andohahela, Didy National Park (NP), Kalambatritra, Manjakatompo Ankaratra, Manongarivo Reserve, Marojejy NP, Masoala NP, and Ranomafana NP. Based on its known and projected distribution, it is likely also present in among others Midongy du Sud NP. Since no specific threats are known to the species, and because it has a wide distribution in Madagascar (AOO = 132 km 2 , EOO = 218,948 km 2 ) and occurs in a range of protected areas, Costularia purpurea is here assessed as LC.

Notes
The specimen Hildebrandt 3752a was listed as a syntype of Costularia recurva (accepted name Costularia leucocarpa) but conforms to circumscription of Costularia purpurea. A lot of confusion existed between Costularia leucocarpa and Costularia purpurea, with many Costularia purpurea specimens at the G, K and P herbaria identified as Costularia recurva. However, these species are quite different in morphology, with Costularia purpurea characterised by longer, flatter, narrower, darker spikelets generally bearing more glumes compared to Costularia leucocarpa.
Most herbarium specimens listed as Costularia laxa by Chermezon (1937) and Kükenthal (1939) are very immature and difficult to identify. Although the clade with two accessions originally identified as Costularia laxa and three accessions identified as Costularia purpurea is well supported in the molecular phylogenetic hypothesis, the taxa themselves are not (Fig. 1). Chermezon (1937) and Kükenthal (1939) distinguished Costularia laxa from Costularia purpurea based on it laxer inflorescence, fewer empty glumes (3-4 vs. 5-9) and perianth bristles much overtopping the nutlets. However, this distinction does not hold as variation in inflorescence branching and number of spikelets per inflorescence is gradual, even in the specimens listed by Chermezon (1937) as Costularia laxa spikelets often have more than four empty glumes, and in Perrier de la Bâthie 2639 (MNHN-P-P00459983), selected as hololectotype of Costularia laxa, the size of the nutlets varies from 1.6 to 2 mm plus a beak of 0.7-1.3 which is not that much shorter than the perianth bristles and similar to many specimens conforming to the description of Costularia purpurea.
This species is sister to Costularia melicoides of the Mascarenes. Costularia melicoides is unusual in perfecting a nutlet in lower of the two fertile glumes, while most Costularia species perfect a nutlet in the upper fertile glume. In Costularia purpurea, the number and sex of the flowers is variable with many of the collected specimens also perfecting a nutlet in the lower fertile glume. These sister species also share a similar build and size.
Costularia laxa var. macrantha Cherm. (1925: 21) is here excluded from Costularia laxa as we consider it to be synonymous with Costularia robusta (see more discussion under that taxon).
14. Costularia robusta (Cherm.) Larridon, comb. et stat. nov Very robust and tall perennial herb with a strongly developed and long (c. one m) caudex. Culms 0.8-2 m Â c. 6 mm, robust. Basal leaves with very wide leaf sheaths (15-20 mm), persistent at the base of the culm above the caudex. Inflorescence an elongate, narrow panicle with very numerous, crowded spikelets; inflorescence brances erect, not more than five cm long. Pedicels of the spikelets short, not patent. Spikelets six to seven mm long. Glumes 12-14, up to seven mm long, purplish black.

Distribution
Costularia robusta is only known from the Manongarivo, Marojejy and Tsaratanana protected areas and their environs in the Antsiranana province of Madagascar (Fig. 5).

Phenology
Inflorescences are initiated in April and flower/fruit between October and January. Old inflorescences are still visible on the plants in April when the new inflorescence are formed.

Conservation
Costularia robusta is restricted in its distribution to the Antsiranana province of Madagascar, and occurs in at least three protected areas, that is, Manongarivo, Marojejy and Tsaratanana. Threats to this taxon need further investigation but fire (natural and man-made) and disturbance of its habitat as a result of logging, firewood collection and charcoal may affect this species. Based on 11 georeferenced herbarium specimens, this species occurs in at least six locations and has an estimated AOO of 28 km 2 and an EOO of 2,947 km 2 . Using IUCN criteria, Costularia robusta can be assessed as VU B1ab(ii,iii)+2ab(ii,iii). Kükenthal (1939) also listed the specimen Humbert 3344 when creating the combination under Costularia pantopoda. We here exclude this specimen collected at Pic d'Ivohibe Reserve in the Fianarantsoa region from Costularia robusta and place it in Costularia baronii. Kükenthal (1939) listed specimen Perrier de la Bâthie 16398 under Costularia pantopoda var. baronii. Although this specimen could not be found in the P herbarium, it is very likely to be Costularia robusta as it is from the same locality as the other positively identified specimens of Costularia robusta by the same collector at the same time.

Notes
Costularia laxa var. macrantha looks very similar to Costularia robusta but has paler glumes. Since its type specimen was collected at a somewhat lower elevation than the specimens identified as Costularia robusta, this is unsurprising since glume colour in tropical Cyperaceae often darkens with elevation in the same species (I. Larridon, 2010, personal observation). Kükenthal (1939) discusses the presence of a caudex in Costularia robusta. Although this is not clearly visible in the specimens he cited, the type specimen of Costularia laxa var. macrantha clearly has a well developed and long caudex. We here consider Costularia laxa var. macrantha to be a synonym of Costularia robusta. Kükenthal (1939) did realise a potential relationship between Costularia laxa var. other by unsuitable habitat (i.e. wet forests) are within the Morne Seychellois National Park and appear healthy, with limited risks from invasive species. The AOO for the Congo Rouge population is 10 m 2 , Copolia 6,000 m 2 , and Pérard 20,000 m 2 . The two newly discovered sub-populations, at Mont Sébert and at Glacis Sarcelles are not well known, but the Mont Sébert one is about the same size as the one of Congo Rouge (small), while the Glacis Sarcelles population is comparable to the Copolia one (B. Senterre, 2018, personal communication) resulting in an estimated AOO totalling approx. 0.032 km 2 . Its EOO was estimated at approximately 5.9 km 2 (Bachman et al., 2011). Both AOO and EOO fall within the limits of CR status under criterion B. According to Henriette et al. (2015) and based on IUCN (2012) criterion B, with an EOO < 5,000 km 2 , AOO < 500 km 2 , number of locations 5, and a projected decline of the quality of the habitat as a result of climate change, this species can be classified as Endangered EN B1ab(iii)+2ab(iii).
Notes Henriette et al. (2015) noted that among the known species of Costularia s.s. only one presents some similarity with Costularia xipholepis, that is, Costularia baronii from Madagascar, as both species have long leaves and hypogynous bristles much longer than the nutlet with relatively few empty glumes. However, our molecular phylogenetic results point at a sister relationship with Costularia melleri. The sister pair Costularia melleri (Madagascar) and Costularia xipholepis (Seychelles) in turn are sister to a clade encompassing the species Costularia humbertii (Madagascar) and Costularia cadetii (La Réunion).

CONCLUSIONS
The genus Costularia is redelimited to represent a monophyletic entity including 15 species. Although the species diversity is largely found in Madagascar, our results indicate that the genus dispersed once to Africa (Malawi, Mozambique, South Africa, Swaziland, Zimbabwe), twice to the Mascarenes (La Réunion, Mauritius), and once to the Seychelles (Mahé). Three-quarters of the species are threatened with extinction, because of restricted distribution ranges and human impact. A full taxonomic treatment is provided, including an identification key to all species, formal descriptions of two new species from Madagascar (Costularia andringitrensis and Costularia itremoensis) and one new species from La Réunion (Costularia cadetii), and two taxa previously recognised as varieties of Costularia pantopoda are here recognised at species level (Costularia baronii and Costularia robusta).
Martin Xanthos performed the experiments, authored or reviewed drafts of the paper, approved the final draft. A. Muthama Muasya performed the experiments, contributed reagents/materials/ analysis tools, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft.

Field Study Permissions
The following information was supplied relating to field study approvals (i.e. approving body and any reference numbers): Permits to collect and export these specimens were issued by the Madagascar authorities: a collecting permit for Cyperaceae in Madagascar (N 082/10/MEF/SG/ DGF/DCB.SAP/SLRSE-Isabel Larridon) was provided by ANGAP Madagascar National Parks authority.

DNA Deposition
The following information was supplied regarding the deposition of DNA sequences: The newly generated sequence data have been deposited to GenBank (accession numbers MH512812 to MH512851), and are available as part of Data S2 (sequence alignment used for the analyses).

Data Availability
The following information was supplied regarding data availability: DNA sequence data has been submitted to GenBank (https://www.ncbi.nlm.nih.gov/ genbank/). GenBank accession numbers are included in Table S1 (voucher information) and in Data S1 (sequence alignment used for the analyses).
Herbarium specimens studied are accessible in public herbaria (contact info via: http://sweetgum.nybg.org/science/ih/). The additional herbarium specimens studied per taxon are listed in Data S2.

Supplemental Information
Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.6528#supplemental-information.