Introduction

The orchid genus Disperis Sw. was described by Swartz in 1800 and encompasses species from the Western Cape of South Africa distinguished by their unique floral morphology. The representatives of the genus have delicate subterranean stems that are tuberoid, erect, or horizontal. Amplexicaul, alternate to opposite leaves (1–3) are located near the middle of the stem and are often similar in size to the floral bract or even smaller. The inflorescence is usually few-flowered, with flowers held horizontally. The lip and the gynostemium are hidden in a saccate to spurred hood (galea) formed by marginally connected petals and the dorsal sepal. The linear lip claw differs in length and is basally united with the gynostemium. The apical part of the claw is usually curved and changes into a small linear limb with simple or two bilobed appendages (Szlachetko and Olszewski 1998). The gynostemium structure is highly variable in different species. The column foot is absent. The anther is covered by a large, conspicuous stigma and peculiar rostellum. The stigma is usually bilobed with lobes clearly separated from each other, located near the opposite sides of the claw. The rostellum is trilobed, with a large middle lobe and lateral lobes of variable size, which are usually slightly apically twisted and transformed into a large viscidium (Szlachetko and Rutkowski 2000).

The genus comprises about 84 species (Pridgeon et al. 2001) of terrestrial, rarely epiphytic or lithophytic herbs that mostly grow in wooded habitats, except the southern African species, which is found in grassland or sclerophyllous scrub. Disperis is distributed mainly in southern and tropical Africa, with a few endemic species recognized from tropical Asia (Pridgeon et al. 2001), which have recently been identified as conspecific (Kurzweil and Manning 2005).

In 1898, Schlechter recognized two groups within the genus based on the arrangement of the leaves; however, his delimitation is of no significance today. Cladistic analyses of Disperis were conducted by Manning and Linder (1992) based mainly on the characters of the lip and its appendage(s) and on pollen sculpturing. The authors recognized two distinct clades, known informally as the “tropical clade” and the “southern African clade,” and their work provided the foundation for Kurzweil and Manning’s (2005) proposal to distinguish two subgenera, which are easily told apart from one another by some morphological characters and geographical distribution. The subgenus Disperis corresponds to the “southern African clade,” while that of Dryorkis Thouars to the “tropical clade.” The authors also report information on preliminary ITS sequence data that suggest that Disperis subg. Dryorkis (Thouars) Kurzweil & J.C.Manning is a well-supported sister to subgenus Disperis, but only five species of the known 44 have been examined in the subgenus.

Szlachetko et al. (2010), unaware of the proposal by Kurzweil and Manning (2005), distinguished the Disperis subg. Disperella Szlach., Mytnik & Rutk. as different from the nominal taxon based on the form of galea. Disperis subg. Disperis is characterized by the cylindrical-conical to clavate shape of the galea, whereas in Disperis subg. Disperella it can be cochleate or concave, and never forms an elongate structure.

Manning and Linder (1992) analyzed speciation patterns within the genus. They conclude that the representatives of subgenus Disperis originated in southern Africa with one major and one minor radiation in the summer rainfall of eastern South Africa, and the third major radiation in the winter rainfall of the Western Cape. Speciation patterns in the subgenus Dryorkis have not been analyzed and little is known about its pollination biology.

In the most recent study (Szlachetko et al. 2010), nine species of Disperis from Cameroon were reported. In this paper, we propose a new species based on morphological and molecular data.

Materials and methods

Morphological study

Materials were collected during a field expedition conducted in 2011 and were preserved in Kew Mixture. We examined 72 herbarium specimens of the Disperis from West–Central Africa. The specimens were either borrowed from or examined at the following herbaria: BM, BR, K, MO, P, WAG, and W-R. The materials examined were prepared according to standard taxonomic procedures. The vegetative and generative characters of the new species were studied and compared with types, protologs, other specimens of Disperis representatives, and the literature. The shape, size, and arrangement of the leaves and the structure of the inflorescence were examined. The flower dimensions, morphology (galea, dorsal sepal, lateral sepals, petals, lips), and surface area were analyzed with stereoscopic microscopy.

The abbreviations of author names followed Brummitt and Powell (1992). The acronyms of herbaria were adopted from Index Herbariorum, which is continuously updated (Thiers 2016).

Molecular data

Taxon sampling for phylogenetic analyses included 39 specimens representing both the genus Disperis and outgroup taxa. Most of the sequences were taken from GenBank (www.ncbi.nlm.nih.gov), and a few were obtained at the laboratory of the Department of Plant Taxonomy and Nature Conservation, University of Gdansk. New sequences were deposited in GenBank. Vouchers and Genbank accession numbers are listed in Table 1.

Table 1 List of samples used for molecular analyses
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The material from the new species (fresh leaves) was collected and preserved in silica gel (Chase and Hills 1991). The DNA was extracted using a DNA Mini Plant Kit (A&A Biotechnology, Poland) following the manufacturer’s protocol. Lysing Matrix A tubes that were pre-cooled at − 45 °C and a FastPrep instrument (MP Biomedicals, USA) were used for sample homogenization. Pelleted DNA was resuspended in 50 µl of dd H2O. Isolates purchased from the Kew DNA Bank (see Table 1) were used for eight samples.

The ITS1-5.8S-ITS2 region and part of the matK gene (approximately 1400 bp) were amplified and sequenced in Eppendorf and Biometra TGradient thermal cyclers. Primers 101F and 102R were used for the ITS region (Douzery et al. 1999). For the plastid region, primers 19F (Molvray et al. 2000) and 1326R were used (Cuénoud et al. 2002). The polymerase chain reaction (PCR) for both markers (ITS and matK) was performed in a total volume of 25 µl containing 1 µl temple DNA (~ 10–100 ng), 0.5 µl of 10 µM of each primer, 12.5 µl Thermo Scientific DreamTaq PCR Master Mix (Thermo Fisher Scientific, USA), water, 1.0 µl dimethyl sulfoxide (DMSO) only for the ITS region, and 0.5 µl of 25 mM MgCl2 only for matK. The amplification parameters for nrITS (ITS1 + 5.8S + ITS2) were 94 °C, 4 min; 30X (94 °C, 45 s; 52 °C, 45 s; 72 °C, 1 min), and 72 °C, 7 min. The parameters for part of the matK gene were 95 °C, 3 min; 33X (94 °C, 45 s; 52 °C, 45 s, 72 °C, 2 min 30 s); and 72 °C, 7 min. Wizard SV Gel and the PCR Clean-Up System (Promega, USA) were used to clean the PCR products in accordance with the manufacturer’s protocol. The purified products of the PCR reaction were cycle sequenced using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Inc., ABI, Warrington, Cheshire, UK). The cycle sequencing parameters were 95 °C, 2 min 40 s and 25X (95 °C, 10 s; 50 °C, 10 s; 60 °C, 4 min). The total volume sequencing reaction was 10 µl containing 1.3 µl of 5X sequencing buffer, 1 µl of Big Dye terminator, 0.4 µl of 10 µM primer (1.6/3.2 pmol), 0.5 µl dimethyl sulfoxide (DMSO), 1 µl of amplified product (30–90 ng/µl), and water. The sequencing reaction products were then purified and sequenced on an ABI 3720 automated capillary DNA sequencer in the Genomed SA (Warsaw, Poland). The DNA sequence chromatograms were inspected and edited using FinchTV and assembled using an AutoAssembler (Applied Biosystems, Inc.).

Phylogenetic analyses

The DNA sequences, from both the ITS region and part of the matK gene, were automatically aligned with SeaView (Galtier et al. 1996) using the MUSCLE algorithm (Edgar 2004). Then alignment was checked and manually corrected with SeaView. Data matrices are available as supporting material (Online Resources 1–2). The data were analyzed using two methods: maximum parsimony (MP) and Bayesian inference (BI). The first method, MP, was performed on two matrices separately using heuristic searches in PAUP*4.0b10 (Swofford 2000) with tree bisection and reconnection (TBR) branch swapping and the MULTREES option in effect, simple addition, and ACCTRAN optimization. All characters were unordered and equally weighted (Fitch 1971). Gaps were coded as “-”, and missing data were coded as “?”. The internal support of clades was evaluated by character bootstrapping (Felsenstein 1985) using 1000 replicates. For bootstrap support levels, we considered bootstrap percentages (BPs) of 50–70% as weak, 71–85% as moderate, and > 85% as strong (Kores et al. 2001).

Bayesian Inference (BI) was performed with MrBayes version 3.1.2 (Ronquist and Huelsenbeck 2003). An evolutionary model for each region (ITS, matK) was calculated with MrModeltest 2.2 (Nylander 2004). The general time reversible model of substitution with gamma distribution (GTR + G) was selected according to the Akaike Information Criterion (AIC) for both matrices. Each analysis consisted of two simultaneous runs of four chains for 10,000,000 generations, sampling one tree for each 100, until the average standard deviation of split ranges was smaller than 0.01 after discarding the initial 25% trees of each chain as the burn-in. A majority-rule consensus tree was generated for the remaining trees in PAUP* to assess the topology and clade posteriors. The values of posterior probabilities (PPs) are not equivalent to BPs, which are generally higher (Erixon et al. 2003).

Results

Molecular analyses

The data matrices for both the ITS and matK markers included 39 taxa representing species of Disperis and outgroup. Statistics for the datasets are separated below by “/” (ITS, matK). The aligned length of the matrix was 800/1746 characters, of which 412/293 were parsimony informative. The maximum parsimony analyses resulted in 162 (ITS) and 270 (matK) most parsimonious trees. All of them were used to build a strict consensus tree (Online Resources 3, 4). Tree length for one of the most parsimonious tree was 1176/703, consistency index (CI) = 0.605/0.752, and retention index (RI) = 0.839/0.862. The topologies of the majority-rule consensus tree (Fig. 1, Online Resource 5) and strict consensus tree are similar for both the ITS and matK matrices analyzed. Therefore, in this paper, we present the majority-rule consensus tree (Fig. 1). However, other trees are available in supplemental materials (Online Resource 3, 4, 5).

Discussion

The new species of Disperis belongs to a group of taxa with a relatively shallow, concave, or cochleate galea that never forms elongate spurs. D. tomaszii Szlach., Grochocka, Dudek & Olędrz. is morphologically similar to D. nitida Summerh. (Fig. 2), reported from Cameroon. Both species share a similar form of petal and the general form of the lip limb. The galea of D. nitida is prominently bilobed when viewed from the side—its basal and apical parts almost touch each other limiting the entrance into the flower. In D. tomaszii, the mouth of the flower is relatively broad. The apical part of the petal lobes of both species is more or less malleolate, but with acuminate terminal ends in D. tomaszii and acute ends in D. nitida. However, the most distinguishing character that easily separates the two species is the lip limb. It is 3 mm long, in total, in D. nitida, and it resembles two letters T joined basally by an elongate, linear, acute, glabrous projection. The T-like lobules are 2 mm long and 2 mm across, shallowly bilobulate, and papillate on the inner surface. The limb of D. tomaszii is larger, over 5 mm long, apically quadrilobulate with two oblong-triangular, obtuse lobules directed forward and two obliquely elliptic-ovate lateral lobules widely spread. The limb is covered very densely by swollen, globular cells, except the lateral lobules and the basal elongate projection (Table 2).

Fig. 1
figure 1

Majority-rule consensus tree of Disperis species obtained from a Bayesian analysis of ITS1-5.8S-ITS2 sequences. Values above branches represent posterior probabilities (≥ 50%) and bootstrap support (≥ 50%) from 1000 replicates (PP/BP)

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Table 2 Comparison of morphological features in Disperis tomaszii, D. nitida, D. thomensis, D. anthoceros, D. dicerochila
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The other species that could possibly be confused with the new species is D. thomensis Summerh. (Fig. 3), which has a very wide distribution range from Sierra Leone through West and Central Africa to Zambia and Tanzania. The lip limb of D. thomensis is up to 2 mm long, linear, subacute, and densely papillate. The appendages are 4–6 mm long and 2 mm across the apex, and apically bilobulate; the lobules are linear, obtuse, divergent, and papillate. The elongate, linear, and acute projection is densely papillate (Table 2). Disperis thomensis possesses a cylindrical spur is distinctly swollen in the apical half. There is no elongate spur in D. tomaszii.

Fig. 2
figure 2

Disperis nitida Summerh. a Flower, b dorsal sepal and petals, c lip with appendage, d details of lip appendage (reproduced from Szlachetko et al. 2010); scale bar a–b = 3 mm, c–d = 1 mm

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Unfortunately, no sequence of either of the aforementioned species was available in GenBank. Analyses of selected fragments of DNA from 28 Disperis species available in GenBank indicate possible affinity between D. tomaszii and both D. anthoceros Rchb.f. (Fig. 4) and D. dicerochila Summerh. (Fig. 5).

Fig. 3
figure 3

Disperis thomensis Summerh. a Habit, b flower, c dorsal sepal and petals, d lip with appendage (reproduced from Szlachetko et al. 2010); scale bar a = 20 mm, b–d = 3 mm

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Fig. 4
figure 4

Disperis anthoceros Rchb.f. a Flower, b dorsal sepal and petal, c lateral sepals with sac-like spur, d–f lip appendage (various views), g gynostemium and lip with appendage (drawn by Olędrzyńska from Schlieben 673, P); scale bar a–c = 3 mm, d–g = 1 mm

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Both MP and BA analyses indicate a close relationship between the new species and D. anthoceros (Fig. 1) with a strong BP (92%). Moreover, the D. anthoceros–D. tomaszii pair is present in the strict consensus tree, which increases the credibility of this clade. Also, D. anthoceros and D. tomaszii combined with D. dicerochila in the clade are supported by high BP and PPs (100/100).

Fig. 5
figure 5

Disperis dicerochila Summerh. a Flower, b petal, c lateral sepal, d dorsal sepal, e gynostemium and lip with appendage (drawn by Olędrzyńska from Hendrickx and Léonard 5050, BR); scale bar a–d = 3 mm, e = 1 mm

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Disperis tomaszii is easily distinguishable from D. anthoceros, mainly in the galea architecture (Table 2). D. anthoceros is characterized by a slender spur, which is absent in D. tomaszii. The galea of D. tomaszii is 6–7 mm long, while in D. anthoceros it is 10–20 mm. The species also differ in limb structure, which consists of four lobules in D. tomaszii and two fimbriate lobules in D. anthoceros. Both D. tomaszii and D. dicerochila are characterized by a cochleate or concave galea that never forms an elongate structure (Table 2). The main difference is visible in the lip construction. The densely glandular limb of D. tomaszii contains two oblong-triangular, obtuse lobules directed forward and two obliquely elliptic-ovate, widely spread lateral lobules. The D. dicerochila limb is divided into two bilobulate appendages. The upper lobule is elongate and narrower toward the papillate apex, and the lower lobule is shorter, recurved, and auriculate. The dimensions of the limbs also clearly differ in the two species—the limb in D. tomaszii is longer (5–5.5 mm) than that in D. dicerochila (2 mm).

Taxonomic treatment

Disperis tomaszii Szlach., Grochocka, Dudek & Olędrz., sp. nov.—HOLOTYPE: Cameroon, Bamenda Highland, Big Babanki, about two hours walking distance from the village Big Babanki to the Czech Field Station, 2150 m a. s. l., ca 6°15′N 10°29′W, epiphyte in secondary forest along stream, up to 1 m above the ground, 11–26 Nov 2011, Szlachetko and Baranow 9460 (YA!; isotypes: UGDA!; MO!, UGDA!—drawings, photos).

Etymology: Dedicated to Prof. dr hab. Tomasz Osiejuk, Adam Mickiewicz University, Poznan, Poland, eminent ornithologist, and organizer and promoter of Polish biological explorations in Africa.

Description: Tuber single, 1–2 cm long, ca. 0.6 cm in diameter, oblong to ellipsoid. Plant 10–25 cm tall, erect, glabrous. Two leaves, subopposite, just above the middle of the stem, widely spread; the lower leaf shortly petiolate, petiole up to 1 cm long, narrow, blade 1.5–2.8 cm long, up to 2.5 cm wide, suborbicular, base subcordate to truncate, acute to subobtuse, green; the upper leaf sessile, blade up to 2.5 cm long and 1.4 cm wide, elliptic, base cuneate, acute. Inflorescence 3-5-flowered, up to 2 cm long, rather lax. Flowers white. Floral bract up to 10 mm long, lanceolate, apex acute. Ovary up to 12 mm long, basally shortly pedicellate, glabrous. Galea (= hood) oblongoid-saccate, 6–7 mm long, the apex prominently trilobed, both lateral lobes (= petals) acute-acuminate, the middle lobe (= dorsal sepal) acuminate. Dorsal sepal 8–9 mm long and 2.5–2.8 mm wide, linear-oblanceolate, acute, distinctly concave below apex. Petals 7–8 mm long and 4 mm wide at the base, in the form of an extended letter B, lower lobe obliquely ovate-elliptic, upper lobe malleolate (hammer-like), truncate at the apex, both ends acuminate. Lateral sepals up to 9 mm long, 4 mm wide, semi-ovate, apex elongate, acuminate, tapering toward the base, with ca. 1.3-mm long, conical sacs near the inner margin. Lip clawed; claw up to 7 mm long, linear, sigmoidally curved; limb 5–5.5 mm long, reflexed, quadrilobulate apically, with two, oblong-triangular, obtuse lobules directed forward, ca. 1.6 mm long and 0.6 mm wide, and two, obliquely elliptic-ovate lateral lobules, ca. 1 mm long and 0.6 mm wide, widely spread. The limb is covered very densely by swollen, globular cells, except the lateral lobules and the basal elongate projection. Gynostemium 2.8 mm long, typical for the genus, rostellum arms untwisted (Fig. 6).

Fig. 6
figure 6

Disperis tomaszii Szlach., Grochocka, Dudek & Olędrz. a Flower, b floral bract, c lip with appendage, d lateral sepal, e dorsal sepal, f petal, g, h leaves (drawn by Baranow from holotype material, Szlachetko and Baranow 9460, YA); scale bar a–f = 3 mm, g–h = 10 mm

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Diagnosis: Disperis tomaszii is characterized by relatively shallow, concave, or cochleate galea. It is morphologically most similar to D. nitida, but the lip limb of D. tomaszii is > 5 mm long, densely covered by swollen, globular cells; it is quadrilobulate apically, with two oblong-triangular, obtuse lobules that are directed forward and two obliquely elliptic-ovate lateral lobules that are widely spread. The limb of D. nitida is 3 mm long in total and is somewhat reminiscent of two letters T joined basally by an elongate, linear, acute, glabrous projection. The T-like lobules are 2 mm long and 2 mm across, shallowly bilobulate, and papillate on the inner surface.

Habitats: Epiphytic in secondary forest in shade, along streams, collected from an inclined moss-covered tree trunk. Growing in clumps counting several specimens. Flowering reported in November.

Distribution: Endemic to Cameroon, Bamenda Highland. Elevation ca. 2150 m (Fig. 7).

Fig. 7
figure 7

Distribution map of Disperis tomaszii, D. nitida, D. thomensis, D. anthoceros, D. dicerochila

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Conservation status: To date, Disperis tomaszii is known from a single locality in Cameroon. The population of this species numbers several specimens. The Bamenda Highlands, where the orchids grow, are under strong human pressure that has resulted in highly fragmented natural plant communities (BirdLife International 2010).

Key to Disperis species reported in Central–West Africa (Szlachetko et al. 2010, modified)

  1. 1a.

    Lip claw ca s6–15 mm long, much longer than appendage … 2

  2. 1b.

    Lip claw up to 4 mm long, as long as … 10

  3. 2a.

    Galea formed by dorsal sepal and petals conical-cylindrical … 3

  4. 2b.

    Galea cochleate … 6

  5. 3a.

    Lip claw 8–15 mm long; lip apex oblong, entire, appendages pendulous, divided irregularly to form a sort of fringe, ca. 1.5 mm long … D. anthoceros

  6. 3b.

    Not the above combination of features … 4

  7. 4a.

    Lip appendage consists of two furculate structures, basally joined into a single, filiform, glandular projection … D. thomensis

  8. 4b.

    Lip appendage lanceolate- or oblong-ovate, with two, small basal projection, glabrous … 5

  9. 5a.

    Claw of lip dilated in middle to form two small rounded projections; apical petal lobes acuminate projecting from front of the hood like two horns, ca. 4 mm long … D. mildbraedii

  10. 5b.

    Claw of lip not dilated in the middle; apical petal lobes up to 2 mm long, acute to rounded … D. kamerunensis

  11. 6a.

    Leaves 2–4, … 7

  12. 6b.

    Leaves 2, (sub) … 8

  13. 7a.

    Lip appendage 2.5–3.5 × 4 mm, reniform … D. katangensis

  14. 7b.

    Lip appendage 1.5 × 0.6 mm, oblong-ovate … D. johnstonii

  15. 8a.

    Petals 12.5 × 4 mm … D. nitida

  16. 8b.

    Petals 7–10 × 4–7.5 mm … 9

  17. 9a.

    Inflorescence 1-3-flowered, subumbellate, lip appendage papillate at the apex only … D. dicerochila

  18. 9b.

    Inflorescence 3-5-flowered, lax, lip appendage densely glandular … D. tomaszii

  19. 10a.

    Stem covered densely by papilla-like hairs … D. pusilla

  20. 10b.

    Stem glabrous … 11

  21. 11a.

    Leaves up to 2, opposite … D. aphylla

  22. 11b.

    Leaves 1–4, alternate … 12

  23. 12a.

    Lip claw with two diverging lobules near the middle … 13

  24. 12b.

    Lip claw simple, without any lobules … 14

  25. 13a.

    Lip appendage transversely elliptic, obtuse to notched at the apex; hood ovate in general outline, acute, with cordate base, widest on the lower part … D. togoensis

  26. 13b.

    Lip appendage cordate-ovate, acute; hood obovate in general outline, obtuse to notched at the apex, widest in the upper half … D. szolc - rogozinskiana

  27. 14a.

    Lip appendage obtriangular or sagittate in general outline … 15

  28. 14b.

    Lip appendage oblong to orbicular-elliptic … 16

  29. 15a.

    Leaf 1, 0.5–1.2 × 0.25–0.6 cm … D. raiilabris

  30. 15b.

    Leaves 1–4, 2.5–4.2 × 0.9–2.5 cm … D. reichenbachiana

  31. 16a.

    Leaves 1–2, scale-like, basal, up to 0.9 × 0.5 cm, inflorescence 1-flowered … D. parvifolia

  32. 16b.

    Leaves 2, alternate, up to 2.5 × 2 cm, inflorescence 2-3-flowered … D. fayi