Satyrium Liltvedianum: a Newly Discovered Orchid Species from the Kogelberg Mountains of the Cape Floristic Region (South Africa)

South African Journal of Botany 111 (2017) 126–133

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South African Journal of Botany

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Satyrium liltvedianum: A newly discovered orchid species from the Kogelberg Mountains of the Cape Floristic Region (South Africa)

T. Van der Niet

School of Life Sciences, P. Bag X01, University of KwaZulu-Natal, 3209 Scottsville, South Africa article info abstract

Article history: Individuals of plant populations with traits which are inconsistent with any existing species description may rep- Received 21 July 2016 resent intraspecific variants, products of hybridisation, or a novel species. To distinguish among these possibilities Received in revised form 26 December 2016 for a population of unusual Satyrium individuals from the Kogelberg Mountains in the Cape Floristic Region (CFR), Accepted 9 March 2017 morphological traits and floral scent were documented, and phylogenetic analyses implemented. Plants from the Available online 23 March 2017 Kogelberg population were characterised by long-spurred white flowers and a bifid rostellum. Floral scent was fl β Edited by GV Goodman-Cron dominated by the common oral monoterpene volatile -linalool. Although these traits characterise several southern African members of the genus, DNA sequences from the nuclear and plastid genomes of an accession Keywords: from the Kogelberg population were highly distinct from other Satyrium species. The Kogelberg accession occu- β-Linalool pied an isolated phylogenetic position within the ‘Satyrium clade’ and was not sister to any other species with Moth pollination similar traits. There was weak support for membership of a clade of species with which plants from the Kogelberg Orchidaceae population share the possession of lateral sepals that project at a perpendicular angle to the median sepal, and Palaeoendemic cover the side of the labellum, and which also produce β-linalool as dominant scent compound. Given the con- Floral scent gruence of phylogenetic relationships inferred from plastid and nuclear DNA sequences respectively, a hybrid status of the Kogelberg population was rejected. Based on these results, the new species, Satyrium liltvedianum, which is uniquely characterised by the size, shape and orientation of sepals and lateral petals, is described in this study. Other Satyrium species with similar floral traits are pollinated by crepuscular moths, which therefore can also be inferred for the new species. A dichotomous key to the white-flowered, long-spurred Satyrium species of South Africa is provided. The restricted distribution range, a typical phenomenon for many CFR plant species, in combination with the isolated phylogenetic position, suggests that S. liltvedianum represents a palaeoendemic species. © 2017 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction Region (e.g. Van der Niet and Johnson, 2009; Verboom et al., 2009; Schnitzler et al., 2011). Therefore, if an orchid population which is The Orchidaceae is one of the largest plant families worldwide, and characterised by traits that do not fit those of any previously described is well-represented in the Cape Floristic Region of South Africa species is encountered, then two alternative scenarios must be ruled (Goldblatt and Manning, 2002). The number of recognised orchid spe- out before describing a new species: (1) variation might represent cies is highly contentious, and taxonomic opinions may differ over ten- previously undocumented intraspecific (e.g. clinal) variation, and fold with regard to the number of actual species within a particular (2) hybridisation might have given rise to individuals with unusual group (e.g. Bateman et al., 2011; Vereecken et al., 2011). Of the currently (intermediate) characters. A phylogenetic analysis can reveal a 71,391 available orchid species names, 56.0% are synonyms (http:// population's evolutionary origin, and a comparative analysis of trait var- www.theplantlist.org/; 7 July 2016), suggesting that species naming iation can be used to establish whether traits are uniquely derived. may often have been done too liberally. This is not only problematic Therefore, together these analyses can be used to decide whether recog- for applications such as conservation biology, but also affects the accura- nition of a new species is indeed warranted (e.g. Van der Niet et al., cy of studies which use species as operational taxonomic units, such as 2011). the many phylogenetic studies that have been performed to understand Satyrium is an orchid genus with centres of diversity in the Fynbos the evolutionary origin of the exceptionally species-rich Cape Floristic Biome of the Cape Floristic Region (CFR) of South Africa, grasslands on mountains along the rift valley in southern and eastern Africa, and with a few species occurring in Madagascar, West Africa, and South E-mail address: [email protected]. East Asia (Van der Niet et al., 2005). The non-resupinate flowers of

Satyrium are characterised by the presence of two spurs which are usually nectar-secreting. Variation in floral traits, such as colour, scent, shape and spur length is extensive and associated with different, specialised pollination systems (Johnson, 1997a; Van der Niet et al., 2010; Johnson et al., 2011; Van der Niet et al., 2015). Morphological trait variation has been dealt with taxonomically in several ways. First, morphological variation is represented in over 90 currently recognised species (Kurzweil and Linder, 1999), of which more than 30 occur in South Africa. Secondly, intraspecific variation appears common and has led to the recognition of several varieties and subspecies (Hall, 1982), often associated with different pollination systems (e.g. Johnson, 1997b). Finally, several taxa have been deemed hybrids in previous taxonomic treatments and were therefore not recognised as species (Hall, 1982). Indeed, hybrid zones have been identified in Satyrium (Ellis and Johnson, 1999; Liltved and Johnson, 2012), and sig- natures of past hybridisation events were detected from incongruent to- pologies resulting from separate phylogenetic analyses of plastid and nuclear DNA sequences (Van der Niet and Linder, 2008). Together, this suggests that hybridisation might be a fairly common phenomenon in Satyrium. AgroupofSatyrium species with scented pale-coloured, long- spurred flowers, characterised by moth-pollination and which are par- Fig. 1. Satyrium liltvedianum in situ on 10 November 2009 at the type locality in the ticularly well-represented in the CFR, has provided taxonomical chal- Kogelberg Mountains after the veld fire, showing maroon coloration of the stem, lenges (Van der Niet et al., 2011, 2015). Comparative morphological sheathing leaves and abaxial side of the bracts. Photograph by Herbert Stärker. studies in this group have resulted in the recent recognition of two new species (Van der Niet et al., 2009, 2011). Phylogenetic analyses have revealed that the moth-pollinated species do not form a monophy- letic clade, and are in several cases closely related to species with differ- “Satyrium sp. nov. ined. ‘Kogelberg’” by Liltved and Johnson (2012)). ent floral morphologies and pollination systems, including bee- and The population of c. 50 plants (herein referred to as ‘Kogelberg popula- bird-pollination (Johnson, 1996; Johnson, 1997a; Van der Niet et al., tion’ for plant measurements, or ‘Kogelberg accession’, for the phyloge- 2011, 2015). Characters that are used to distinguish superficially similar netic analyses) occupied a relatively small area of several 100 m2 in species include variation in the dimensions and orientation of the peri- extent. anth, the rostellum, and composition of floral scent (Van der Niet et al., In November 2009 three inflorescences were collected, scent- 2011, 2015). These characters are often relatively conserved within, but sampled (see below), and lodged in the Compton Herbarium as pressed not among the various clades to which moth-pollinated Satyrium spe- specimens. Several individual flowers from three individuals were pre- cies belong (Van der Niet et al., 2005). served in 70% ethanol and leaf material was placed in silica gel for later Although the CFR is botanically well-studied, new species are still molecular phylogenetic analyses. In November 2010, further plant mea- regularly discovered and described (e.g. Linder and Hitchcock, 2006; surements were made in the field. Van der Niet et al., 2009; Steiner, 2011; Manning et al., 2016). Early 2009, a veld fire occurred in the Kogelberg Mountains, one of the most species-rich areas of the CFR (Goldblatt and Manning, 2000). Fires stim- 2.2. Plant trait analyses ulate flowering of orchids and other geophytic plant species in the CFR (Bytebier et al., 2011; Kraaij and Van Wilgen, 2014). During the austral 2.2.1. Morphology spring of 2009, a population of c. 50 Satyrium plants with long-spurred, To compare morphological characters among similar Satyrium spe- scented, white flowers was discovered in the Kogelberg Mountains cies, the same traits that were used in Van der Niet et al. (2011) were (Fig. 1). These plants superficially resembled several other species of characterised. These included: orientation of basal leaves; projection Satyrium, but also differed in several aspects. The aim of this study of bracts; projection of lateral sepals; size and degree of fusion and re- was therefore to assess whether the plants from the Kogelberg popula- flexion of sepals and petals; orientation of the labellum apex; presence tion in fact represented an entirely distinct new species. Morphological of a dorsal labellum ridge; orientation of the spurs; total spur-length traits and floral scent were specifically documented, and the phyloge- from its apex to where the two spurs fuse; and position and shape of netic relationship of individuals from the Kogelberg population to the viscidia. These diagnostic traits were recorded and measured in Satyrium species reconstructed, to compare traits with other species in the field in eleven individual plants, and from flowers preserved in a phylogenetic context. To assess whether the population could poten- 70% ethanol. Comparisons were made to the Satyrium species covered tially be the product of current and ancient hybridisation, phylogenetic in Van der Niet et al. (2011): S. acuminatum, S. candidum, S. patterns derived from plastid and nuclear DNA sequences respectively, eurycalcaratum, S. humile, S. situsanguinum,andS. stenopetalum,as were compared (cf. Gravendeel et al., 2004; Van der Niet and Linder, these are the most morphologically similar species in the CFR. 2008). Additional morphological traits recorded in the field and from spirit- preserved flowers included leaf-, stem-, and inflorescence characters, 2. Materials and methods and floral coloration. Terminology used to describe these morphological traits follows Beentje (2010). 2.1. Study site Herbarium specimens of Satyrium species compared in Van der Niet et al. (2011) were examined to assess whether individuals resembling On 10 November 2009 a striking and unusual Satyrium species that those from the Kogelberg population, either previously collected at, or could not readily be identified was discovered near Grabouw (Fig. 1), in the vicinity of the Kogelberg Mountains, or from other CFR localities, in the Steenbras Catchment Area of the Kogelberg Mountains, which is were present among historical collections in the Bolus and Compton part of the Kogelberg Biosphere Reserve (previously referred to as herbaria in Cape Town, South Africa. 128 T. Van der Niet / South African Journal of Botany 111 (2017) 126–133

2.2.2. Floral scent characterisation clade to which the Kogelberg population was unambiguously assigned To characterise and quantify floral scent, Gas Chromatography in separate analyses of plastid and nuclear DNA sequences (see results), coupled with Mass Spectrometry (GC–MS), was used. Floral scent was which comprises S. rupestre, S. ligulatum, S. emarcidum, S. carneum,and sampled in the evening for 30 min in the laboratory in Cape Town S. acuminatum, S. muticum, S. outeniquense, S. coriifolium, and both from the headspace of three individuals separately. To distinguish floral S. stenopetalum subspecies, was included. Satyrium bicorne was used as scent compounds from possible contaminants at the sampling locality, an outgroup for this test. The HomPart command in PAUP version 4.0b the ambient air was sampled simultaneously as a control for the same was used to run the ILD test. Datasets were randomised 1000 times, time period as that of sampling the orchid inflorescences. Sampling and the branch and bound search routine for the most parsimonious and analysis protocols followed those of Van der Niet et al. (2015).For tree was implemented. each compound identified, its presence in the scent of the moth- If morphological variation represents intraspecific variation, DNA pollinated Satyrium species covered in Van der Niet et al. (2015) was sequences are expected to be identical or similar to those of an al- assessed for comparison. ready described species (cf. Lahaye et al., 2008). To identify species To quantify absolute rates of scent emitted per inflorescence, per with the most similar DNA sequences to the Kogelberg accession, a minute, the peak area of plant compounds in chromatograms of orchid pairwise calculation of uncorrected (p) distance in Mesquite version samples was compared to the peak area produced by a known quantity 3.04 (Maddison and Maddison, 2015), was performed in this study. of β-linalool, the dominant scent compound in individuals of the To assess whether the degree of sequence similarity to the most sim- Kogelberg population. ilar species is high or low, the same calculation for each Satyrium species included in the matrix that is member of the ‘Satyrium 2.2.3. Non-morphological plant features clade’ (with duplicate accessions removed) was performed, and the The ecology of the Kogelberg population, including habitat charac- distance to the most similar species was compared among all teristics and flowering time, was recorded in the field and was based Satyrium species to the value calculated for the Kogelberg accession. on patterns observed in the two consecutive years, 2009 and 2010. 3. Results 2.3. Phylogenetic analyses 3.1. Morphology To test whether plants from the Kogelberg population are related to Satyrium species with similar traits, a phylogenetic analysis using DNA Plants from the Kogelberg population are characterised by a unique sequences as characters was performed. DNA regions that were used combination of morphological traits that is not present in any previous- in previous phylogenetic analyses of Satyrium were sequenced, includ- ly described Satyrium species (Supplementary Table S2). Although sev- ing the nuclear ribosomal Internal Transcribed Spacer (ITS), and the eral traits are shared with other white-flowered, long-spurred species, plastid trnL–trnF intron and intergenic spacer (trnLF), the trnS–trnG in particular S. candidum, the shape, size, and orientation of the sepals intergenic spacer (trnSG) and a portion of the trnK intron and the and lateral petals are unique (Fig. 2). matK gene (trnK and matK) (Van der Niet and Linder, 2008). All molec- No other Satyrium specimens with the same combination of charac- ular protocols and primer identities for PCR amplification and sequenc- ters were detected among herbarium collections. ing have followed those of Van der Niet and Linder (2008), but for sequencing trnK and matK the primers used in this study were 19F, 3.2. Floral scent characterisation 580F, 1382F, and R1 (Van der Niet and Linder, 2008). Forward and re- verse sequences of all regions were assembled into contigs in The floral scent of plants from the Kogelberg population is Sequencher version 4.1.4. Genbank accession numbers of the DNA se- characterised by 32 compounds, most of which are acyclic monoter- quences of the Kogelberg accession are given in Supplementary penes, emitted at 0.22 ± 0.12 μginflorescence−1 min−1 (Table 1). The Table S1. Consensus sequences were added to the matrices and aligned six unknown compounds only made up 0.05% of the entire blend. Floral by eye. The ITS DNA sequence matrix used by Van der Niet et al. (2011) scent is dominated by β-linalool, which represents 94.95% ± 1.07 of the was supplemented with the sequence of the Kogelberg accession. The entire blend (Table 1). Of the 24 identified compounds, four were not plastid DNA sequence matrix in Van der Niet and Linder (2008) was found in a range of moth-pollinated Satyrium species (Table 1). Most pruned to include only members of the ‘Satyrium clade’ (cf. Van der compounds were found in common with those present in S. ligulatum Niet et al., 2005)andS. cristatum Sond. var. cristatum (BB2297) as (16) and S. acuminatum (15), including β-linalool, whereas only three rooting taxon, and supplemented with sequences of the Kogelberg compounds were found to exhibit commonality with the scent bouquet accession. of S. pallens. To infer phylogenetic relationships, Bayesian inference was implemented following the methods described by Van der Niet and Linder 3.3. Phylogenetic analyses (2008), and used the same models of sequence evolution. Given several cases of well-supported topological incongruence resulting from phylo- The accession from the Kogelberg population is strongly supported genetic analyses of plastid and ITS data sets respectively (Van der Niet (PP in both ITS and plastid analysis = 1.0) as part of a clade that contains and Linder, 2008), separate phylogenetic analyses using the plastid two subclades: the ‘S. acuminatum subclade’ which includes and nuclear character matrices, were performed. Analyses were carried S. acuminatum, S. carneum, S. emarcidum, S. ligulatum, S. rupestre, and out using the default settings in Mrbayes 3.2.6 with the Monte Carlo S. muticum, and the ‘S. coriifolium subclade’ which includes Markov Chain set to run for five million generations, sampling trees S. coriifolium, S. outeniquense, and both subspecies of S. stenopetalum and parameters every five thousand generations, and discarding the (Fig. 3). In the majority rule consensus tree of the Bayesian analysis of first 20% as burn-in. Convergence of parameter estimates was con- plastid sequences, the position of the Kogelberg accession is unresolved firmed in Tracer v 1.4 (effective sample sizes of all parameters were with respect to the two subclades, whereas there is weak support shown to be well above 200). (PP = 0.50) for a sister relationship between the Kogelberg accession To test whether phylogenetic patterns resulting from plastid and nu- and the ‘S. acuminatum subclade’ in the majority rule consensus tree clear datasets were congruent, the parsimony-based Incongruence of the Bayesian analysis of ITS sequences (Fig. 3). Length Difference (ILD) test (Farris et al., 1994) implemented in The ILD test detected no significant topological incongruence be- PAUP* version 4.0b (Swofford, 2000), was applied. Given the extensive tween phylogenetic analyses of the ITS and plastid dataset respectively incongruence in Satyrium (Van der Niet and Linder, 2008), only the (P = 0.858). T. Van der Niet / South African Journal of Botany 111 (2017) 126–133 129

A C

E B

F G

Fig. 2. Satyrium liltvedianum. (a) Entire plant (scale bar = 20 mm); (b) sheathing leaf (scale bar = 10 mm); (c) ﬂower side view (scale bar = 4 mm); (d) ﬂower oblique view (scale bar = 4 mm); (e) pollinarium (scale bar = 0.5 mm); (f) column front view (scale bar = 1 mm); (g) column side view (scale bar = 0.5 mm).

Both the ITS and plastid sequences of the Kogelberg accession are rel- and therefore adds considerable phylogenetic diversity to the other atively dissimilar to any sequences of ‘Satyrium clade’ members. The most known species of Satyrium. Based on these phylogenetic results, the hy- similar ITS sequence was that of S. muticum, which differed by 6.7%. Only pothesis that the accession represents an intraspeciﬁc variant can be con- two species had more dissimilar ITS sequences to any other ‘Satyrium clusively rejected. Hybrid status of the Kogelberg population seems clade’ member, whereas 26 species had an ITS sequence that was more unlikely: in contrast to typical Satyrium hybrid zones, no other Satyrium similar to other ‘Satyrium clade’ members. Results for the plastid se- species which could be putative parental species were recorded from quences were similar: the most similar plastid sequence, of the area encompassing the Kogelberg population (cf. Ellis and Johnson, S. outeniquense, differed by 0.5%. Only two species had more dissimilar 1999). Furthermore, ITS sequence polymorphism, which may be indica- plastid sequences to any other ‘Satyrium clade’ member, whereas 24 spe- tive of hybrid status (cf. Gravendeel et al., 2004), was absent in the cies had a plastid sequence that was more similar to other ‘Satyrium clade’ Kogelberg accession. Even though the Kogelberg population does not members. seem to represent an F1 hybrid, an origin from an ancient hybridisation event may still be considered (e.g. Allan et al., 1997). However, the obser- 4. Discussion vation of congruent phylogenetic patterns for the two gene trees lends further support to the hypothesis that the population did not originate The DNA sequences of the Kogelberg accession are highly distinc- from ancient hybridisation, although this needs to be conﬁrmed with tive. Indeed, the accession occupies an isolated phylogenetic position more detailed population genetic analyses. 130 T. Van der Niet / South African Journal of Botany 111 (2017) 126–133

Table 1 Proportion of specificidentified and unknown scent compounds in the floral bouquet of plants from the Kogelberg population. Compounds are listed by compound class, in the order of increasing Kovats Retention Indices (KRI). Compounds that were also found in the scent of other Satyrium species analysed in Van der Niet et al. (2015) are indicated with ‘yes’.Abbre- viation of species names: S. acu = S. acuminatum, S. lig = S. ligulatum, S. pal = S. pallens, S. sit = S. situsanguinum, S. ste = S. stenopetalum ssp. stenopetalum, S. out = S. outeniquense.

Compound CAS # KRI % ± SD in blend S. acu S. lig S. pal S. sit S. ste S. out

Aliphatic Alcohol 1-Hexadecanol 36653-82-4 2372 0.02 ± 0.01 yes Aldehyde Nonanal 124-19-6 1409 0.05 ± 0.01 Hexadecanal 629-80-1 2147 0.00 ± 0.00 yes yes Ester Hexadecyl acetate 629-70-9 2301 0.04 ± 0.03 yes Benzenoid and phenylpropanoid Alcohol Phenylethyl alcohol 60-12-8 1929 0.00 ± 0.00 yes yes yes yes yes Diterpene Isophytol 505-32-8 2283 0.00 ± 0.00 Irregular terpene 6-Methylhept-5-en-2-one 110-93-0 1352 0.27 ± 0.07 yes yes yes yes yes yes 6-Methylhept-5-en-2-ol 1569-60-4 1465 0.06 ± 0.03 yes yes yes yes Monoterpene Acyclic β-Myrcene 123-35-3 1193 2.44 ± 1.15 yes yes yes yes (Z)-Ocimene 3338-55-4 1255 0.90 ± 0.35 yes yes (E)-Ocimene 502-99-8 1275 0.08 ± 0.02 yes yes yes yes (E)-Linalool oxide 23007-29-6 1455 0.01 ± 0.00 yes yes (Z)-Linalool oxide 1365-19-1 1482 0.06 ± 0.01 yes yes β-Linalool 78-70-6 1558 94.95 ± 1.07 yes yes yes yes yes Hotrienol 29957-43-5 1618 0.05 ± 0.02 yes yes Geraniol acetate 141-12-8 1739 0.00 ± 0.01 yes Nerol 106-25-2 1810 0.01 ± 0.00 yes Geraniol 106-24-1 1856 0.03 ± 0.01 yes yes yes 2,6-Dimethylocta-3,7-dien-2,6-diol 13741-21-4 1945 0.02 ± 0.01 yes yes Cyclic Limonene 138-86-3 1225 0.32 ± 0.08 α-Terpineol 98-55-5 1712 0.11 ± 0.03 yes yes yes Sesquiterpene Acyclic (E)-Nerolidol 40716-66-3 2048 0.28 ± 0.26 yes yes Miscellaneous cyclic compound 1-Methylcycloheptanol 3761-94-2 1599 0.20 ± 0.13 Lavender lactone 1073-11-6 1694 0.04 ± 0.02 yes yes yes Unknown m/z: 79, 81, 67, 53, 93, 94 1638 0.01 ± 0.01 m/z: 71, 43, 99, 57, 41, 85 1804 0.01 ± 0.00 m/z: 191, 121, 109, 119, 135, 91 2088 0.00 ± 0.00 m/z: 58, 43, 71, 109, 69, 95 2134 0.01 ± 0.02 m/z: 71, 93, 119, 67, 43, 81 2267 0.01 ± 0.01 m/z: 82, 96, 81, 95, 67, 55 2522 0.01 ± 0.01 m/z: 149, 150, 104, 57, 223, 167 2544 0.00 ± 0.00 m/z: 82, 96, 81, 55, 83, 67 2612 0.00 ± 0.01

The size, shape, and orientation of the sepals and petals of plants and S. ligulatum, both members of the ‘S. acuminatum subclade’.Itis from the Kogelberg population are distinctive characters (Fig. 2)and,to- therefore hypothesised that the Kogelberg accession belongs to the gether with the results from phylogenetic analyses, warrant its descrip- ‘S. acuminatum subclade’. tion as a new species. However, plants from the Kogelberg population The floral scent of the Kogelberg individuals was dominated by β- have several traits in common with other white-flowered, long- linalool. This compound is one of the most common floral volatiles spurred Satyrium species within the CFR. Given the phylogenetic posi- (Knudsen et al., 2006), which makes its function difficult to interpret. tion of the Kogelberg accession, the sharing of traits could best be However, the combination of long-spurred, white flowers and high explained by convergent evolution, for instance in response to selection emission rates of β-linalool is consistent with the moth-pollination syn- by similar functional pollinator groups (Johnson et al., 1998), or by drome as found in Satyrium and other plant groups (Raguso and shared retention of ancestral character states. Pichersky, 1995; Van der Niet and Johnson, 2013; Van der Niet et al., The phylogenetic position of the Kogelberg accession is not conclu- 2015). Indeed, moth antennae are known to respond to β-linalool sively resolved. Only in the analysis based on nuclear DNA sequences (Raguso et al., 1996). Pollinator observations are required to confirm was there weak support for membership of the ‘S. acuminatum whether the Kogelberg individuals are in fact moth-pollinated, but subclade’ (cf. Fig. 3). However, the sideways projection of the lateral se- this seems highly likely. pals is a morphological trait that is relatively unusual in Satyrium, but It is in general not uncommon for plant species within the CFR, and shared by individuals from the Kogelberg population and members in particular the orchids, to be known from only a single population of the ‘S. acuminatum subclade’, as a whole. Furthermore, the largest (Linder and Kurzweil, 1999; Turner and Oliver, 2006; Galley et al., number of compounds present in the floral scent of individuals 2009; Liltved and Johnson, 2012; Manning and Goldblatt, 2014). The from the Kogelberg population was shared with S. acuminatum new Satyrium is one such species and currently known from one T. Van der Niet / South African Journal of Botany 111 (2017) 126–133 131

ITS PLASTID

S. monadenum 0.55 S. sceptrum Malawi 1.0 S. sceptrum Kenya 1.0 1.0 1.0 S. sceptrum Tanzania 0.97 1.0 S. neglectum Tanzania S. neglectum Malawi 1.0 1.0 S. buchananii 1.0 1.0 S. longicauda var. jacottetianum S. longicauda var. longicauda 1.0 1.0 S. neglectum South Africa 1 1.0 S. neglectum South Africa 2 1.0 1.0 S. fimbriatum S. membranaceum 1.0 1.0 S. princeps S. lupulinum S. candidum S. pallens 0.96 1.0 S. situsanguinum 0.87 1.0 S. erectum Swartberg 1.0 1.0 S. erectum Gifberg 0.96 S. longicolle 0.66 S. humile 0.76 S. bicorne 0.71 S. eurycalcaratum S. acuminatum 0.65 S. carneum 0.92 0.89 1.0 S. emarcidum 1 1.0 1.0 S. emarcidum 2 1.0 1.0 1.0 0.98 S. ligulatum 2 1.0 S. ligulatum 1 0.50 1.0 1.0 S. rupestre 1.0 S. muticum S. liltvedianum 1.0 0.87 S. coriifolium 1.0 S. stenopetalum ssp. brevicalcaratum 1.0 1.0 1.0 S. stenopetalum ssp. stenopeatlum 1.0 S. outeniquense

Fig. 3. Majority-rule consensus trees from Bayesian phylogenetic analysis of nuclear ITS (left) and plastid (right) DNA sequences. The posterior probability of clades is indicated by numbers at nodes. Shading is provided to facilitate the identiﬁcation of clades. In the analysis of ITS sequences S. lupulinum was used as outgroup (indicated by dotted line). The outgroup for the analysis of plastid sequences was removed prior to graphing. Similarly, clones and some multiple accessions of the same species have been reduced to a single terminal for graphical representation. This did not affect the phylogenetic position of the Kogelberg accession.

population comprising c. 50 individuals. The possibility that the fre- population (Johnson and Kurzweil, 1998), which is phylogenetically quent occurrence of range-restricted species reflects failure to ade- nested inside S. erectum (Van der Niet, unpublished results). However, quately sample intraspecific variation of widespread species, a given its isolated phylogenetic position, the new species from the situation typical for poorly explored regions, can be rejected for two rea- Kogelberg Mountains more likely represents a palaeoendemic. While sons in this case. Firstly, the new Satyrium species clearly does not rep- palaeoendemics are usually thought to occupy ecological niches that resent an intraspecific variant and secondly, the southwestern part of have for instance become marginal due to long-term environmental the CFR has for centuries been botanically well explored, its orchid changes, they may also represent species in the late stage of the taxon flora having been particularly well-studied (Bolus, 1893–1896; Liltved cycle, which are prone to extinction even in the absence of a common and Johnson, 2012). Species may indeed have a restricted range due to extrinsic trigger (cf. Ricklefs and Bermingham, 2002). Indeed, the habi- habitat loss induced by human intervention. The Kogelberg sandstone tat in which the Kogelberg population was found, a rocky southeast- fynbos is, however, least threatened and statutorily well conserved facing slope in shallow peaty soil derived from Table Mountain (Mucina and Rutherford, 2006). Rather, it could be argued that the Sandstone, is not marginal within the CFR. Relative long-term climatic new species is naturally rare. Range-restricted endemism from natural stability in the history of the CFR may have allowed for taxon cycle pro- causes may result from recent speciation (neoendemism), for instance cesses such as speciation and extinction to play out, undisturbed by by having ‘budded-off’ through peripatric speciation from a more wide- large scale ecological catastrophes, such as glaciations, that elsewhere spread progenitor species (cf. Grossenbacher et al., 2014). This is likely lead to mass-extinctions (Cowling et al., 2015). This may therefore to be the case for S. pulchrum, known from only a single help explain such great species richness and diversity within the CFR 132 T. Van der Niet / South African Journal of Botany 111 (2017) 126–133 and its relatively large number of range-restricted species, representing 9.0–9.2 mm × 2.3–3.3 mm. Free part of lateral petals oblanceolate, ob- an assemblage of neo- and palaeoendemics. tuse, crisped, concealed by lateral sepals for about half of their width, projecting forward for 4/5 of their length, then abruptly reflexed, 5. Conclusion 8.3–9.2 mm × 2.9–4.4 mm. Gynostemium filling back of galea. Column part slightly curved away from rachis, 5.4–6.8 mm × 1.0–1.1 mm, There are many reasons why efforts to discover and describe spe- white. Stigma projecting slightly away from rachis, obovate, emargin- cies should be ongoing. Only recognised taxa can be adequately con- ate, 2.1–2.4 mm × 1.7–2.2 mm. Rostellum very weakly triangular, served; the success of scientific research often depends on sound 1.4–1.6 mm × 1.5–1.8 mm; viscidia terminal, circular, plate like. taxonomic classification; beneficial properties of species can only Anther reflexed and aligned with column, 1.4–1.8 mm × 1.9–2.1 mm, be communicated if a species has a formal name; and recently connective broadly retuse, not extending beyond individual anther the- Dijkstra (2016) argued that because naming species is inherent to cae. Lateral anther appendages placed at anther base, 0.7–1.2 mm in human nature, continued exploration for the vast majority of species diameter. that are still unknown will improve our consciousness of the natural Habitat and Ecology: recently burnt fynbos vegetation, on a rocky world. Much of Earth's biotic diversity is currently highly threatened southeast-facing slope in shallow peaty soil derived from and many species are on the brink of extinction, or have recently Table Mountain Sandstone (847 m above sea level). Many inflores- gone extinct (Wake and Vredenburg, 2008; Barnosky et al., 2011; cences in the population had been removed, presumably chewed off McCallum, 2015), which provides a great sense of urgency to taxo- by small antelope. nomic enterprise. Several studies, including this study, have shown Flowering time: November, in the first and second years after fire. that new species are regularly still discovered in the field (e.g. Distribution: S. liltvedianum is known from a single population in the Linder and Hitchcock, 2006; Steiner, 2011). Therefore, taxonomy Steenbras Catchment area of the Kogelberg Mountains. cannot solely rely on historical herbarium or museum collections, Etymology: This species is named in honour of William Rune Liltved even from within relatively well-botanised areas such as the (1960–) who, over the past two decades, has made an invaluable contri- Kogelberg Biosphere Reserve of the CFR, only 70 km from the city bution to recording the orchids of the Cape Floristic Region. This work of Cape Town. culminated in publication of the book, The Cape Orchids (Liltved and Johnson, 2012). 6. Species description Conservation status: Similar to several other orchids from the CFR, S. liltvedianum is known only from a single localised population of Satyrium liltvedianum Van der Niet sp. nov. is morphologically simi- about 50 individuals (Linder and Kurzweil, 1999). The species is there- lar to S. candidum, but differs from this species by having lateral sepals fore considered highly vulnerable. that overlap with the outside surface of the labellum and project at a ninety-degree angle from the median sepal instead of projecting from the same plane, and lateral sepals and petals that are approximately 7. Diagnostic key to the white-flowered, long-spurred Satyrium equal in size instead of smaller lateral petals than lateral sepals, and a species of South Africa median sepal that is narrower and longer than the lateral sepals instead of more or less similarly-sized. 1. Leaves on a sterile shoot, populations occurring in the summer- TYPE.—Western Cape Province, 3418 (Somerset West): Steenbras rainfall region of South Africa…………………….………S. longicauda Catchment area near Rockview Dam, Kogelberg Mountains (–BB), 18 1. Leaves wilted at the time of flowering or on the flowering shoot, Nov 2009, W.R. Liltved 120 (NBG, holo.). populations occurring in the Cape Floristic Region of South Terrestrial herbaceous plants with erect stems. Stem: above-ground Africa....………………………………………………………….….………2 portion excluding inflorescence 80–135 mm tall, 3.4–6.0 mm in diame- 2. Rostellum with terminal viscidia………………………………………...3 ter at base, vinaceous. Leaves 2, borne close to ground, frequently wilted 2. Rostellum with lateral viscidia……………………………...………...…5 at anthesis, 21–38 × 20–36 mm, broadly ovate, obtuse, entire, with 3. Spurs down-curved, following the line of the ovary…………………4 prominent striation on underside, glabrous, pale yellowish-green to 3. Spurs standing away from the ovary………………….………S. humile green; transition between leaves and cauline leaf sheaths abrupt. Leaf 4. Lateral sepals more or less equal in size to lateral petals, sheaths 3–4, leaving a portion of stem exposed, 22–43 × 11–23 mm, projecting at a 90° angle from median sepal, distal portion strongly amplexicaul cupuliform, sometimes filled with water, obtuse to attenu- reflexed…..………………………………………….…….S. liltvedianum ate, apex sometimes reflexed, yellowish-pale green with maroon spots, 4. Lateral sepals larger than lateral petals, projecting in the same plane especially dense at base. Inflorescence 42–119 mm long, 27.3–34.5 mm from median sepal, not reflexed at tips……………………S. candidum wide, densely congested with 6–20 flowers; bracts reflexed or partly re- 5. Bracts erect………………………………………….……S. stenopetalum flexed at anthesis, 13.5–24.0 mm × 6.7–13.4 mm, lanceolate, acute to at- 5. Bracts (partly) reflexed.……………………………………………………6 tenuate, glabrous, entire to undulate, green with maroon spots, 6. Spurs following the line of the ovary……………….…………………7 especially around the margins and apex, and more pronounced on abax- 6. Spurs standing away from the ovary………………S. eurycalcaratum ial side. Flowers non-resupinate, white, spur tips and base of sepals and 7. Viscidia lunate shaped, sepals and lateral petals equally-sized, lateral lateral petals green; sweetly scented. Ovary 5.4–7.5 mm × 3.7–5.0 mm, sepals projecting at a 90° angle from the median longitudinally ridged, minutely papillate on ridges, maroon, standing sepal……………………………………………………….S. acuminatum away at an acute angle from rachis. Labellum galeate, globose, with a 7. Viscidia globular-shaped, lateral sepals larger than the median prominent dorsal ridge, margins crisped, reflexed from apex to 90° sepal and lateral petals, but all projecting in the same down on each side for 0.9–1.5 mm, side of labellum forming a loose plane…………………………………………….………S. situsanguinum tube with sepals and lateral petals, base of labellum glabrous to sparsely papillate inside; galea aperture 3.8–4.3 mm wide and 4.1–5.7 mm tall, Supplementary data to this article can be found online at http://dx. forward facing. Spurs 17.4–18.2 mm long, parallel to ovary. Sepals and doi.org/10.1016/j.sajb.2017.03.018. median petals fused for 2.9–3.7 mm. Free part of lateral sepals lanceolate, acute, entire, projecting forward for 4/5 of their length, then abruptly reflexed, 9.0–10.7 mm × 4.1–4.9 mm. Free part of median Declaration of interest sepal oblong, acute, entire to undulate, projecting forward for ¾ of its length, then abruptly reflexed or only weakly projecting downward, I declare no conflict of interest. T. Van der Niet / South African Journal of Botany 111 (2017) 126–133 133

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