A Botanical Mystery Solved by Phylogenetic Analysis of Botanical Garden Collections: the Rediscovery of the Presumed-Extinct Dracaena Umbraculifera

A botanical mystery solved by phylogenetic analysis of botanical garden collections: the rediscovery of the presumed-extinct Dracaena umbraculifera

C HRISTINE E. EDWARDS,BURGUND B ASSÜNER,CHRIS B IRKINSHAW C HRISTIAN C AMARA,ADOLPHE L EHAVANA,PORTER P. LOWRY II, JAMES S. MILLER A NDREW W YATT and P ETER W YSE J ACKSON

Abstract Extinction is the complete loss of a species, but the discoveries and the importance of documenting and con- accuracy of that status depends on the overall information serving the flora of Madagascar. about the species. Dracaena umbraculifera was described Keywords Botanical garden, Dracaena reflexa, Dracaena in  from a cultivated plant attributed to Mauritius, but umbraculifera, extinction, living collections, Madagascar, repeated surveys failed to relocate it and it was categorized as Mauritius, phylogeny reconstruction Extinct on the IUCN Red List. However, several individuals labelled as D. umbraculifera grow in botanical gardens, suggesting that the species’ IUCN status may be inaccurate. The  goal of this study was to understand ( ) where D. umbracu- Introduction lifera originated, () which species are its close relatives, () whether it is extinct, and () the identity of the botanical xtinction is the complete and total loss of all individuals garden accessions and whether they have conservation Eof a species. In practice, extinction can be defined as ‘the value. We sequenced a cpDNA region of Dracaena from permanent absence of current and future records of a spe- Mauritius, botanical garden accessions labelled as D. um- cies’ (Ladle et al., ). Although this is a relatively straight- braculifera, and individuals confirmed to be D. umbraculi- forward concept, accurately determining and proving that a fera based on morphology, one of which is a living plant in a species is extinct is far from straightforward. Many species private garden. We included GenBank accessions of may be presumed to be extinct, but whether this is actually Dracaena from Madagascar and other locations and recon- the case may depend on factors such as the amount and ac- structed the phylogeny using Bayesian and parsimony ap- curacy of historical data on a species’ distribution; the size of proaches. Phylogenies indicated that D. umbraculifera is its range and the density of its populations; how its range has more closely related to Dracaena reflexa from Madagascar changed over time; the ease with which it can be observed in than to Mauritian Dracaena. As anecdotal information in- the field; and the number of knowledgeable scientists dicated that the living D. umbraculifera originated from searching for it, the effort they expend in searching for it, Madagascar, we conducted field expeditions there and lo- and their overall knowledge of the biota of a region (Ladle cated five wild populations; the species’ IUCN status should et al., ; Lee et al., ). The most convincing evidence therefore be Critically Endangered because ,  wild indi- for extinction involves species that were once widespread viduals remain. Although the identity of many botanical and well known but then experienced an observable decline garden samples remains unresolved, this study highlights in range and population size, and cannot now be relocated the importance of living collections for facilitating new despite a relatively complete knowledge of the biota of the region and extensive, repeated surveys by qualified biolo- gists. If there is robust evidence for the extinction of a species, IUCN Red List working groups may determine it to be Extinct or Extinct in the Wild. A considerable number of CHRISTINE E. EDWARDS (Corresponding author), BURGUND BASSÜNER,PORTER P. LOWRY II*, JAMES S. MILLER,ANDREW WYATT and PETER WYSE JACKSON plant species are known to be Extinct in the Wild but still Missouri Botanical Garden, P. O. Box 299, St. Louis, MO 63166, USA persist in cultivation, mainly in botanical gardens. E-mail [email protected] Dracaena umbraculifera (Asparagaceae sensu lato, Byng CHRIS BIRKINSHAW,CHRISTIAN CAMARA and ADOLPHE LEHAVANA, Missouri Botanical et al., ) is an example of a plant species that is currently Garden, Madagascar Research and Conservation Program, Antananarivo,  Madagascar categorized as Extinct on the IUCN Red List (Strahm, ). *Also at: Institut de systématique, évolution, et biodiversité, Unité mixte de To our knowledge it has never been observed by botanists in Recherche 7205 (Centre national de la Recherche scientifique/Muséum national the wild and as a consequence has remained a mystery for d’Histoire naturelle/École pratique des Hautes études, Université Pierre et Marie several centuries. Described in  by Nicolaus Joseph von Curie), Muséum national d’Histoire Naturelle, Sorbonne Universités, Paris, France Jacquin based on a plant cultivated in the greenhouses of the  Received  June . Revision requested  September . botanical garden at Schönbrunn in Vienna (see Plate a for Accepted  October . First published online  January . original illustration, Jacquin, ), this species has a

Oryx, 2018, 52(3), 427–436 © 2018 Fauna & Flora International doi:10.1017/S0030605317001570 Downloaded from https://www.cambridge.org/core. IP address: 170.106.33.19, on 29 Sep 2021 at 22:49:57, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0030605317001570 428 C. E. Edwards et al.

PLATE 1 (a) The original illustration of Dracaena umbraculifera from Jacquin (), (b) a paniculate inflorescence of Dracaena reflexa in Madagascar (photograph by P. Antilahimena, collection number ), (c) a diffuse paniculate infructescence of Dracaena floribunda in Mauritius (photograph by C. Edwards), (d) a young inflorescence of D. umbraculifera in Ile Sainte-Marie (photograph by Rova Malala Rakotoarivelo), (e) an inflorescence of D. umbraculifera in Ile Sainte-Marie in full flower (photograph by A. Lehavana), and (f) a vegetative individual of D. umbraculifera (photograph by P. Lowry).

distinctive morphology, with tightly clustered flowers in a present in the current collections of the Pamplemousses nearly umbellate inflorescence in the centre of the leaves Botanical Garden (now the Sir Seewoosagur Ramgoolam (Plate a), differing from the diffuse, paniculate inflores- Botanical Garden). cences that characterize all other Dracaena (Plate b). Dracaena umbraculifera was subsequently included in a Although Jacquin indicated that the cultivated plant on list of plants of Mauritius (Bojer, ) and described as which he based his description came from the island of growing in the thick forests in the centre of the island and Mauritius (Jacquin, ), its origin is unclear. It may have flowering very rarely. It was also listed as a Mauritian species been collected during a voyage made by Franz Boos and in the Flora of Mauritius and the Seychelles (Baker, ). In Georg Scholl, who sailed to South Africa and Mauritius dur- the more recent Flore des Mascareignes (Marais & Coode, ing – and sent back  cases of plants, assembled ), doubt was expressed about whether D. umbraculifera by M. Céré of the Pamplemousses Botanical Garden in was native to Mauritius, as the main evidence that it existed Mauritius. However, there is no surviving documentation of in Mauritius was Bojer’s description. It was suggested that the plants contained in this shipment nor is it known whether Bojer may have confused D. umbraculifera with Dracaena they were all Mauritian natives. Dracaena umbraculifera is not floribunda, which still occurs in central Mauritius, and

that if D. umbraculifera was indeed Mauritian it must be ex- from  botanical gardens in seven countries (Table ), as tinct. Following unsuccessful efforts over the previous cen- well as one cultivated accession of Dracaena reflexa from tury to locate living plants in the wild, the species was Missouri Botanical Garden. Individuals from botanical gar- categorized as Extinct in  (Strahm, ). dens identified as D. umbraculifera but that are not con- Despite the fact that no natural population of D. umbra- firmed by a flowering specimen are hereafter indicated in culifera has been documented in the wild in Mauritius, it quotes (i.e. ‘D. umbraculifera’). Although other gardens re- may not be extinct, as living plants labelled as D. umbracu- ported having individuals of D. umbraculifera, several flow- lifera have long been grown in botanical gardens around the ered recently and had diffuse-paniculate inflorescences, world. One such plant is growing at the Missouri Botanical confirming that they were incorrectly identified, and were Garden. This stimulated an interest in the true status of this not included in the study. Leaf material of a correctly iden- presumably extinct species and led us to initiate a study to tified, pressed specimen of D. umbraculifera was obtained try to elucidate its origins. The individual at Missouri from the University of Vienna herbarium (WU ; Botanical Garden was obtained in  from an unknown Table ). It was prepared in  from a plant cultivated in source and, if correctly identified, the IUCN status of the Hortus Botanicus Vindobonensis (now known as the D. umbraculifera should be updated to Extinct in the Botanical Garden of the University of Vienna), and has Wild (IUCN, ). Further research in the database of cul- the distinctive inflorescence of D. umbraculifera. Given tivated plants maintained by Botanic Gardens Conservation that Jacquin, who described D. umbraculifera, was the first International (BGCI, ) found  other gardens with living director of Hortus Botanicus Vindobonensis, we speculate individuals identified as D. umbraculifera. Communications that this specimen may have originated from the same with staff at these gardens indicated that most of these plants plant on which Jacquin based his description. have been in their collections for decades and their origins We also conducted fieldwork in Mauritius in January are unknown (other than in some cases in which they  to collect leaf tissue of all Dracaena species currently were obtained from other botanical gardens). As unequivo- known to be native to the island (Table ), including D. flori- cal morphological identification of these plants relies on the bunda, D. concinna and D. reflexa (including two of the presence of an inflorescence, yet they rarely, if ever, flower in three varieties of D. reflexa in Mauritius, var. reflexa and cultivation (the plant in Missouri Botanical Garden has not var. angustifolia). The collected tissue was preserved in silica been observed in flower for decades), the identity of these gel. During this trip, staff at Mauritius Herbarium informed specimens remains uncertain. It is important to determine us of an individual of D. umbraculifera growing in a private their identity because they may be the only remaining garden on the island. This plant had flowered recently and individuals of a species that is otherwise extinct. its identification was confirmed based on its distinctive, Our goal was to unravel the mystery of D. umbraculifera umbel-like inflorescence. The herbarium staff provided and, in particular, to understand where it originated, which leaf tissue in silica gel, which was used as a reference for species it is related to, whether it is indeed extinct in the comparison with the samples obtained from botanical gar- wild, whether the living plants in botanical gardens are cor- dens. Although the wild origin of the plant was not indicated rectly identified and whether they are of conservation value. on the herbarium specimen, a web search found an online We gathered samples from living individuals labelled as D. posting by the plant’s owner indicating that ‘it grows in umbraculifera from botanical gardens throughout the world, N-East of Madagascar in an island called Ste Marie, Illes travelled to Mauritius to collect specimens of the extant spe- Aux Nattes’ (Dave’s Garden, ). This information as cies of Dracaena, and investigated phylogenetic relationships well as the results from preliminary phylogenetic analyses using this material, as well as GenBank accessions from spe- prompted us to visit Ile Sainte-Marie and its small outlier cies from other islands of the Western Indian Ocean. As our Ile aux Nattes, off the east coast of Madagascar, in initial phylogenetic reconstruction based on DNA sequence October , where three samples of two native Dracaena data indicated that living plants identified as D. umbraculifera species were collected (Table ). were more closely related to species in Madagascar than to DNA was extracted from the leaf tissue samples using a those in Mauritius, we conducted fieldwork in Madagascar modified Cetyl trimethylammonium bromide (CTAB) ap- in an attempt to collect additional possible relatives and per- proach (Doyle & Doyle, ). As a recent molecular phylo- haps also locate the presumed-extinct D. umbraculifera. genetic study that investigated relationships in Malagasy D. reflexa (Buerki et al., ) employed a plastid marker span- Methods ning the trnL gene and trnL-F spacer, we selected this marker for our study to make use of previously generated DNA Sample collection and molecular methods sequence data. We conducted polymerase chain reactions (PCR) using the primers C and F (Taberlet et al., ) We obtained leaf samples preserved in silica gel from living and previously published amplification protocols (Shaw collections of  accessions identified as D. umbraculifera et al., ). PCR products were cleaned with ExoSAP

TABLE 1 Cultivated accessions of Dracaena umbraculifera and Dracaena reflexa used in phylogenetic analysis of Dracaena species.

GenBank Type of ID confirmed by accession Label Location Accession number collection inflorescence? Source number D. umbraculifera Vienna University WU0077694 (1895) Herbarium Yes Plant grown in MG020508 Herbarium, Vienna botanical gar- Austria den in Vienna (1895) D. umbraculifera Personal garden of Voucher deposited in Living Yes Unknown MG020494 I. Vencapah, Mont the Herbarium of the Choisy, Mauritius Missouri Botanical Garden: Vencapah 26071 ‘D. umbraculifera’ Wairere Nursery, Living No Chiltern Seeds MG020491 Auckland, New Zealand D. umbraculifera Dunedin Botanic Living No Chiltern Seeds MG020492 Garden, Dunedin, New Zealand ‘D. umbraculifera’ Toronto Zoo, Living No Unknown MG020497 Toronto, Canada ‘D. umbraculifera’ Botanic Garden of 6360PA Living No Unknown MG020493 Smith College Northampton, USA ‘D. umbraculifera’ Conservatoire 80-0330 Living No Jardin MG020505 Botanique National Botanique de Brest, France Genève ‘D. umbraculifera’ Moscow Main New & old greenhouses Living No Grown from MG020496 Botanic Garden, seed (1968) Moscow, Russia ‘D. umbraculifera’ Komarov Botanical Living No Pre 1917 MG020504 Institute, St. Petersburg, Russia ‘D. umbraculifera’ Missouri Botanical 1980-1301 Living No Unknown MG020495 Garden, St. Louis, (1904) USA ‘D. umbraculifera’ Jardin Botanique 1989.3.056, 1990.3.194 Living No Jardin MG020506 Nancy, France Botanique Genève ‘D. umbraculifera’ Muséum national 7460, 58406, 71595 Living No Unknown MG020507 d’Histoire naturelle, Paris, France D. reflexa Missouri Botanical 1989-4416 Living Joseph Hill Co MG020498 Garden, St. Louis, Tropical USA Foliage, Miami (1989)

(USB Corporation, Cleveland, USA) and sequenced in both ‘D. umbraculifera’ from a cultivated specimen at the Waimea directions using BigDye Chemistry (Applied Biosystems Arboretum and Botanical Garden, Haleiwa, USA (JQ, (ABI), Foster City, USA) on an ABI xl DNA Genetic Lu & Morden, ); three sequences of Dracaena from Asia Analyzer at the DNA analysis facility at Science Hill at and the Canary Islands, D. draco (KC), D. cambodiana Yale University, New Haven, USA. Forward and reverse se- (KC), and D. cochinchinensis (KC); multiple quences were assembled and edited in Geneious R (Kearse Asparagaceae s.l. outgroups, including Convallaria majalis et al., ). (EU), Maianthemum gongshanensis (EU), The new sequences generated in this study were aligned Disporopsis aspersa (EU), Polygonatum cyrtonema with the following  sequences from GenBank:  Dracaena (EU), and Cordyline cannifolia (KC); and one from Madagascar (EU-EU,Buerkietal.,); additional outgroup from another family of Asparagales,

TABLE 2 Wild accessions of Dracaena species used in phylogenetic analysis. All voucher specimens are deposited in the Herbarium of the Missouri Botanical Garden.

GenBank accession Species Location Voucher number D. floribunda On the west banks of the Papaya River, Henrietta, Mauritius Edwards 299 MG020499 D. reflexa var. reflexa Brise Fer Conservation Management Area, Mauritius Edwards 296 MG020500 D. reflexa var. reflexa Mondrain Reserve, Mauritius Edwards 300 MG020501 D. concinna St. Martin cemetery, Baie du Cap, Mauritius Edwards 301 MG020503 D. reflexa var. angustifolia Black River Gorges National Park, Mauritius Edwards 297 MG020502 D. umbraculifera Kalalao Forest, Ile Sainte-Marie, Madagascar Miller 10775 MG020509 D. umbraculifera Kalalao Forest, Ile Sainte-Marie, Madagascar Miller 10776 MG020511 D. xiphophylla Kalalao Forest, Ile Sainte-Marie, Madagascar Miller 10778 MG020510

Crinum oliganthum (AY; Amaryllidaceae), which was from the wild in Mauritius and Madagascar (Table ). The used to root the trees. Alignments were conducted using the aligned length of the trnL-F data matrix was  characters, default settings in Muscle (Edgar, )andwereadjusted of which  characters were constant,  were variable but manually in Geneious. parsimony-uninformative and  were parsimony-informative. Parsimony searches recovered , most-parsimonious trees with a length of  steps. Because the topologies of Data analysis the trees obtained from Bayesian and parsimony phylogeny reconstruction were almost identical, we present only the Phylogenetic analyses were carried out using parsimony and Bayesian tree, with Bayesian posterior probabilities (PP) Bayesian approaches. Parsimony analysis was implemented and parsimony bootstrap support (BS) indicated on branches in PAUP* .a (Swofford, ) using heuristic searches, (Fig. ). tree bisection–reconnection branch swapping and , Using Crinum as the outgroup, the remaining samples random addition replicates, saving  trees per replicate. were clustered into two well-supported clades (both with Bootstrap analyses (Felsenstein, ) with , replicates PP = .,BS=%), one comprising two cultivated sam- were employed to assess branch support, using a heuristic ples of ‘D. umbraculifera’ from New Zealand plus a sample search with tree bisection–reconnection branch swapping, of Cordyline, and the other containing all of the remaining with one random addition per replicate and saving no taxa. Within the latter clade the accession of ‘D. umbraculi- more than one tree per replicate. Bayesian phylogenetic ana- fera’ from the Botanic Garden of Smith College, lysis was conducted using MrBayes . (Ronquist et al., Northampton, USA, and the samples of M. gongshanensis, ). The optimal model of evolution for the data was se- C. majalis and D. aspersa + P. cyrtonema were weakly sup- lected using the Akaike Information Criterion in jmodeltest ported, successive sisters to a well-supported clade (PP = ., (Darriba et al., ). We ran two analyses, using four chains BS = %) containing all remaining Dracaena accessions each, three hot and one cold, the temperature set to the de- (Fig. ). This main Dracaena clade formed an unresolved fault of ., flat priors, and the GTR + G model of evolution polytomy comprising seven lineages: () a single GenBank as selected by jmodeltest. Analyses were run for  million accession of D. reflexa var. linearifolia,() a GenBank acces- generations, sampling a tree every , generations. To de- sion of ‘D. umbraculifera’ from the Waimea Arboretum and termine the burn-in value, we used Tracer v. . (Rambaut Botanical Garden, () a clade with PP = . and BS = % et al., ) to examine the stabilization of the run para- containing three GenBank accessions of Dracaena from meters and discarded trees saved before the stabilization Asia and the Canary Islands, () a clade with PP = . of the scores. Posterior probabilities were calculated using and BS = % that included one GenBank accession of the sumt command in MrBayes. Trees were visualized by Dracaena xiphophylla and two of D. reflexa var. angustifolia constructing a majority-rule consensus of trees in the pos- from northern Madagascar (i.e. clade C of Buerki et al., terior distribution using PAUP*. ), () a large clade with PP = . and BS = % containing  samples of D. reflexa from the far northern   Results Madagascar (i.e. clade A of Buerki et al., ), ( ) a clade with PP = . and BS = % containing all six Dracaena ac- The final data set contained  sequences:  GenBank accessions from Mauritius, and () a clade with PP = . and cessions and  sequences newly generated for this study, BS , % containing one single sample of D. xiphophylla  of which were obtained from cultivated individuals from Ile Sainte-Marie placed as the sister group to a clade (Table ) and eight of which were from plants collected with PP = . and BS , % containing an unresolved

FIG. 1 Bayesian phylogeny of wild and cultivated Indian Ocean Dracaena spp., with Bayesian posterior probabilities/parsimony support values indicated on the branches. Plants from Madagascar are in black font; plants from Mauritius are in turquoise; cultivated plants are in green, with the name of the garden; Dracaena from elsewhere (outgroups) are in yellow; and outgroups are in grey. D. umbraculifera individuals whose identification has not been confirmed by reproductive material are indicated by quotation marks.

FIG. 2 Locations of D. umbraculifera populations in Madagascar from which samples were collected for phylogenetic analysis.

polytomy of the two positively identified samples of D. um- umbraculifera and the overall phylogenetic placement of braculifera (from Mauritius and Vienna, respectively), two the species. The most likely reason that this species has re- samples from Ile Sainte-Marie, seven ‘D. umbraculifera’ ac- mained a mystery to botanists for centuries and has long cessions from botanical gardens (from Missouri, Moscow, been considered to be extinct is that it was attributed incor- Toronto, St. Petersburg, Paris, Brest and Nancy) and eight rectly to Mauritius. Records accompanying th century samples of D. reflexa from coastal north-eastern and north- material from the western Indian Ocean were often scanty central Madagascar (i.e. clade B of Buerki et al., ). at best, and it is likely that the original plant on which Jacquin based his description was obtained in a shipment of plants from the Pamplemousses Garden in Mauritius, Discussion leading to its misattribution to that island. That the species was never detected in Madagascar is not particularly sur- ’ The origin and closest relatives of Dracaena prising given the country s extensive botanical diversity umbraculifera and endemism, the high vegetative similarity and taxonomic uncertainty of Malagasy Dracaena, and the lack of compre- The phylogenetic analyses presented here indicate that all hensive, expert knowledge of the Dracaena of the region. species of Dracaena currently occurring in Mauritius form Although it is possible that D. umbraculifera once occurred a well-supported clade, whereas the two samples positively in the wild in Mauritius and went extinct, the phylogenetic identified as D. umbraculifera (i.e. the herbarium sample analysis indicates that D. umbraculifera probably originated from Vienna and the living plant in the private garden, in Madagascar, which would require a relatively recent col- with an umbel-like inflorescence) are more closely related onization event by D. umbraculifera from Madagascar to to a clade comprising multiple samples of D. reflexa from Mauritius, followed by its extinction in Mauritius, which Madagascar (i.e. clade B of Buerki et al., ), suggesting is a less parsimonious explanation. that D. umbraculifera may not be a Mauritian species. Previous phylogenetic analysis of D. reflexa in Madagascar recovered three clades that were generally distributed ac- Current status of Dracaena umbraculifera cording to geography, two that included samples from far northern Madagascar (i.e. clades A and C of Buerki et al., Surveys conducted during two field trips in  and  ) and one (i.e. clade B of Buerki et al., ) containing located a total of five wild populations of D. umbraculifera accessions from coastal north-eastern and north-central (Fig. ), all of which occur in low-elevation evergreen forest Madagascar, along with a few accessions from the southern on laterite and sandy soils. Three closely spaced populations portion of the country. Our analysis also showed that D. um- occur in partially degraded forest at Pointe à Larrée on braculifera formed a clade with individuals of D. reflexa be- mainland Madagascar (Fig. ), one inside the recently estab- longing to clade B (Buerki et al., ), which provided lished (in April ) protected area being managed jointly some indication of its likely geographical origin (i.e. coastal by the local community and Missouri Botanical Garden, north-eastern and north-central Madagascar). The inter- and two that are unprotected. A fourth population occurs pretation that D. umbraculifera may be of Malagasy origin in Ile aux Nattes (Fig. ), on private land in highly disturbed was consistent with the information provided by the owner secondary vegetation. The fifth population is located in of the positively identified plant in a private garden in largely intact, closed-canopy evergreen forest at Kalalao on Mauritius, which prior to this study was the only known liv- Ile Sainte-Marie (Fig. ). A few additional, apparently ing individual unambiguously attributable to the species. planted individuals occur on Sainte-Marie in private Fieldwork in Madagascar in  and  led to the discov- gardens. ery of several wild populations in Ile Sainte-Marie and Ile Although our findings demonstrate that D. umbraculi- aux Nattes, and further searches at Pointe à Larrée on the fera is clearly not extinct, it is nevertheless rare and faces mainland, directly across from Ile Sainte-Marie, found add- many threats. Despite being known from five populations itional wild populations (Fig. ). on Ile Sainte-Marie and mainland Madagascar, we estimate Although D. umbraculifera has always been considered that no more than  mature individuals of D. umbraculifera to be a Mauritian species, the geographical distribution of remain in the wild, all of which face threats from habitat the wild populations indicate that it is native to degradation, land clearing for agriculture and development, Madagascar. Phylogenies also suggest that it is more closely fires, and cyclones. The total number of individuals is below related to Malagasy species than Mauritian species, indicat- the threshold for Critically Endangered status under IUCN ing that D. umbraculifera most likely originated in Red List Criterion D (IUCN, ). The species has an  Madagascar. Because we included the herbarium sample Extent of Occurrence of  km and an Area of  from Vienna, which is probably derived from the type spe- Occupancy of  km , and is facing continued decline in cimen, we can be certain about the identification of D. the quality of its habitat and the number of mature

individuals. As with many threatened species in Madagascar, from New Zealand, which were placed among other efforts to conserve D. umbraculifera should focus on protect- Asparagaceae outgroups outside Dracaena in the phylogen- ing and restoring the forest fragments that harbour the wild etic tree. Our phylogenetic analyses did not, however, pro- populations. Establishing back-up collections of the species in vide adequate resolution to confirm the identification of the ex situ facilities should also be carried out to contribute to its remaining botanical garden accessions; their conservation long-term conservation. importance thus remains unresolved. Despite the fact that several botanical garden samples (from Missouri Botanical Garden, France, Russia and Toronto) were placed in the Taxonomic implications clade that contained individuals confirmed to be D. umbraculifera, this group also included accessions that belong to Our results indicate that D. reflexa, as currently circum- the D. reflexa clade B (Buerki et al., ), and we were un- scribed, is not monophyletic. Material from Mauritius as- able to determine to which group the botanical garden sam- signed to this species forms a clade with the two other ples belong. In the absence of flowering material, future Mauritian natives D. floribunda and D. concinna, indicating research using additional sampling and more informative that ‘D. reflexa’ in Mauritius is evolutionarily distinct from molecular markers (i.e. collected using next-generation populations in Madagascar and therefore should be referred DNA sequencing approaches) will be necessary to confirm to under a different name. Our results also show that D. re- the identity of these botanical garden samples as well as to flexa clade B of Buerki et al. () is more closely related to assess their genetic diversity and their value for conservation D. umbraculifera, which occurs in the same geographical or reintroduction. area, than to other populations from northern Madagascar This study highlights the value and importance of the liv- also assigned to D. reflexa, indicating that D. reflexa in ing collections maintained in botanical gardens, even those Madagascar is also not monophyletic. As currently circum- that are over a century old and lack information about their scribed, D. reflexa occurs throughout the islands of the origin. Genetic analysis of living collections can play an im- Western Indian Ocean region, including Madagascar, portant role in gaining a better understanding of poorly Mauritius, Rodrigues, Réunion and the Seychelles archipel- known species and making new discoveries of interest for ago, and also in Mozambique in continental Africa (Wright, biodiversity and conservation. In the past, plants of un- ; eMonocot, ). Given the geographical structuring known origin have often been regarded as being of little of lineages observed in Mauritius and Madagascar, it is pos- or no value for conservation purposes. However, this re- sible that populations currently assigned to ‘D. reflexa’ oc- search would never have been undertaken and D. umbracu- curring in these geographically isolated regions represent lifera would probably not have been rediscovered in distinct lineages and thus belong to more than one species. Madagascar if we had not used genetic analysis to elucidate It is also possible that some of these lineages occurring in the true identity of the living collections of ‘D. umbraculi- islands in the western Indian Ocean could be close relatives fera.’ Genetic analysis of living collections will unquestion- of D. umbraculifera. However, this study included only a ably bring new value to undocumented living collections in small portion of the diversity present in Madagascar and botanical gardens and facilitate their use for conservation Mauritius, did not include Dracaena from other locations purposes, such as for reintroductions and augmentations in the Indian Ocean region, and lacked the phylogenetic of depleted gene pools of wild populations of some resolution needed to elucidate phylogenetic relationships Critically Endangered species. fully. A future study employing more informative molecular Our findings also highlight the need for additional work markers (i.e. those generated using next-generation DNA in Madagascar and other islands of the western Indian sequencing approaches, such as RAD-seq or whole chloro- Ocean. Madagascar contains an estimated , vascular plast genomes) and more comprehensive taxon sampling of plant species, of which nearly % are thought to be endem- Dracaena in Madagascar, Eastern Africa and the islands of ic (Callmander et al., ; Madagascar Catalogue, ), and the Western Indian Ocean is needed to clarify species many are threatened as a result of extensive and ongoing de- boundaries, evolutionary relationships and patterns of di- forestation. Although Madagascar has been the focus of versity, and identify species in need of conservation. plant taxonomic work for over  centuries, comprehensive, expert knowledge of many taxa in the region is lacking, and Status of the botanical garden collections many endemic species are poorly known. As previously sta- ted, D. umbraculifera was overlooked by botanists in The phylogenetic analyses presented here have helped to re- Madagascar because of the extensive botanical diversity solve the identification of some of the sampled individuals and endemism there, the high vegetative similarity and identified as ‘D. umbraculifera’ in botanical gardens. Several taxonomic uncertainty of Malagasy Dracaena, and the are clearly misidentified, including those growing in the lack of comprehensive, expert knowledge of the Dracaena Botanical Garden at Smith College and the accessions of the region. An accurate understanding of the status of a

species is necessary to devise effective conservation strat- BGCI (BOTANIC GARDENS CONSERVATION INTERNATIONAL)() egies, and therefore we emphasize the urgent need to docu- PlantSearch. Https://www.bgci.org/plant_search.php [accessed   ment and conserve the biodiversity of this region, September ]. BOJER,W.() Hortus Mauritianus. Aimé Mamarot et Compagnie, particularly in poorly understood groups such as Dracaena. Mauritius. BUERKI, S., CALLMANDER, M.W., SCHÜPFER, F., RAVOKATRA, M., KÜPFER,P.&ALVAREZ,N.() Malagasy Dracaena Vand. ex Acknowledgements L. (Ruscaceae): an investigation of discrepancies between morphological features and spatial genetic structure at a small We are grateful to Imran Vencapah (Mauritius) for sharing evolutionary scale. Plant Systematics and Evolution, , –. his discovery of Dracaena umbraculifera in Madagascar; BYNG, J.W., CHASE, M.W., CHRISTENHUSZ, M.J.M., FAY, M.F., JUDD, W.S., MABBERLEY, D.J. et al. () An update of the Angiosperm and Michael Kiehn and Walter Till (Vienna University Phylogeny Group classification for the orders and families of Herbarium), Rob Nicholson (Smith College), Tom Myers flowering plants: APG IV. Botanical Journal of the Linnean Society, (Dunedin Botanic Garden), Jody Lusk (Wairere Nursery), , –. Elizabeth Miller (National Forestry Herbarium, New CALLMANDER, M.W., PHILLIPSON, P.B., SCHATZ, G.E., Zealand), Dmitry Geltman (Botanical Institute of ANDRIAMBOLOLONERA, S., RABARIMANARIVO, M., RAKOTONIRINA, N. et al. () The endemic and non-endemic St. Petersburg), Pete Phillipson (Missouri Botanical vascular flora of Madagascar updated. Plant Ecology and Evolution, Garden), Aleksander Demidov (Moscow Main Botanic , –. Garden), Claudia Baider (Mauritius Herbarium), Pei-Luen DARRIBA, D., TABOADA, G.L., DOALLO,R.&POSADA,D.() Lu (University of Hawaii at Manoa), Chad Husby (Fairchild jModelTest : more models, new heuristics and parallel computing.   Tropical Garden), Günter Gerlach (Munich Botanical Nature Methods, , . DAVE’S GARDEN () Dracaena or Yucca? Http://davesgarden.com/ Garden), Martin Callmander (Conservatoire et Jardin community/forums/t//#b [accessed  October ]. Botaniques de la Ville de Genève), Sven Buerki (Natural DOYLE, J.J. & DOYLE, J.L. () A rapid isolation procedure for small History Museum, London), Fanch Le Hir (Conservatoire quantities of fresh leaf tissue. Phytochemical Bulletin, , –. Botanique National de Brest), and staff at the Toronto EDGAR, R.C. () MUSCLE: a multiple sequence alignment method  – Zoo and the botanical gardens in Nancy for providing sam- with reduced time and space complexity. BMC Bioinformatics, , . ples. 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