Plant Syst. Evol. 247: 233–239 (2004) DOI 10.1007/s00606-004-0155-x Studies in the tribe Spermacoceae (Rubiaceae-Rubioideae): the circumscriptions of Amphiasma and Pentanopsis and the affinities of Phylohydrax M. Thulin1 and B. Bremer1,2 1 2 Department of Systematic Botany, EBC, Uppsala University, Uppsala, Sweden The Bergius Foundation at the Royal Swedish Academy of Sciences, Stockholm, Sweden Received October 18, 2003; accepted February 24, 2004 Published online: June 11, 2004
Springer-Verlag 2004 Abstract. Phylogenetic analysis of rbcL cpDNA sequences for 34 members of the tribe Spermacoceae s. lat. indicates that the African genera Amphiasma, Conostomium, Manostachya, Pentanopsis and Phylohydrax form a strongly supported clade, characterised by basal placentation and heterostyly. This is a new position for the sea shore genus Phylohydrax that has previously been associated with Diodia and Spermacoce in Spermacoceae s. str. Amphiasma is not monophyletic as the two samples of A. gracilicaule included form a strongly supported clade with Pentanopsis fragrans. A new taxonomy is therefore proposed, where the previously monotypic Pentanopsis is circumscribed as a genus of two species in northeastern tropical Africa, whereas Amphiasma is treated in its original sense as a genus of about eight species confined to south-central tropical Africa. The new combination Pentanopsis gracilicaulis is made. Key words: Rubiaceae, Spermacoceae, Amphiasma, Conostomium, Hydrophylax, Manostachya, Pentanopsis, Phylohydrax, chloroplast DNA, rbcL, morphology, phylogeny, taxonomy. Fifty-two genera were included in the tribe Spermacoceae by Bremer and Manen (2000), 32 of these on the basis of molecular data, and the remaining 20 on the basis of morphological similarity. With this wide circumscription the tribe includes, for example, all investigated taxa of the formerly generally recognised tribes Hedyotideae and Knoxieae. However, in the published cladograms (Bremer and Manen 2000: Figs. 3, 4) only a small part of these genera are shown. In the present study we include a relatively large sample of taxa from the Spermacoceae s. lat. and focus on a clade including the African genera Amphiasma, Conostomium, Pentanopsis (all formerly Hedyotideae), and Phylohydrax (formerly Spermacoceae s. str.). These four genera were included also in the analysis of Bremer and Manen (2000), but they were absent from the published cladograms. Here we add five further sequences from taxa belonging in this clade, with the particular aim of investigating the circumscriptions of Amphiasma and Pentanopsis, as well as the phylogenetic position of the enigmatic Phylohydrax that was segregated from Hydrophylax by Puff (1986). 234 M. Thulin and B. Bremer: Studies in the tribe Spermacoceae (Rubiaceae) Material and methods Results The sampling strategy was to include representative taxa from different parts of the tribe Spermacoceae, but with a concentration on the Amphiasma/Pentanopsis clade, according to the analyses in Bremer and Manen (2000). Based on general morphological similarities and type of placentation we also included genera that we suspected could be close to Amphiasma and Pentanopsis (Dibrachionostylus, Manostachya, and Mitrasacmopsis). Two samples of Amphiasma gracilicaule were included, one from a plant closely similar to the type, and one from a more large-flowered form. We also added a sequence of Hydrophylax. Altogether 33 taxa representing 26 genera of Spermacoceae were sampled. As outgroup we used one taxon from outside the subfamily Rubioideae (Luculia) and five genera representing other tribes within Rubioideae (Bremer and Manen 2000). Silica gel-dried or herbarium material was used in the DNA investigations. DNA was extracted and amplified according to Bremer et al. (1995) and sequencing was performed with a MegaBACE 1000 (Amersham Biosciences) following the protocol of the manufacturer. The sequences of the coding gene rbcL were manually aligned by using the reading frames of the corresponding amino acid sequences. The rbcL matrix comprises 40 sequences. Eight of these have been produced for this study (Fig. 1), and the other sequences have been published before (Bremer et al. 1995, Manen and Natali 1995, Bremer 1996, Bremer and Manen 2000). Phylogenetic relationships were obtained by parsimony analysis using PAUP
(Swofford 1998), version 4.0b10. Only informative characters were analysed. The search method was heuristic with 100 replications of RANDOM stepwise additions of sequences, the TBR branch swapping, and MULPARS options in effect. Support for the nodes was calculated with jackknife analysis with 1000 replicates and TBR branch swapping and MULPARS off. The data matrix comprises 39 species (one species, Amphiasma gracilicaule, is represented by two accessions) and 1402 positions, 187 of which are parsimony-informative. The heuristic analysis resulted in 18 most parsimonious trees 649 steps long with a consistency index CI ¼ 0.604 and a retention index RI ¼ 0.709. A strict consensus tree is shown in Fig. 1. The included taxa of Conostomium, Phylohydrax, Manostachya, Amphiasma, and Pentanopsis, form a strongly supported clade (99%), ‘‘the Pentanopsis clade’’. Further, Conostomium is strongly supported as monophyletic (100%), as is Amphiasma plus Pentanopsis (99%), but the two samples of A. gracilicaule form a strongly supported clade (100%) with Pentanopsis, whereas A. benguellense is strongly supported (94%) as sister to A. luzuloides. Hydrophylax is nested with species of Diodia and Spermacoce in a strongly supported clade (99%) that is sister with strong support (97%) to a clade with other taxa with solitary ovules, these two clades together corresponding to the former Spermacoceae s. str. Spermacoce itself is, according to our analysis, biphyletic with S. hispida in the former and S. laevis in the latter of these clades. Discussion Puff (1986), when erecting the new genus Phylohydrax for P. carnosa (in South Africa and Mozambique) and P. madagascariensis (in Madagascar and Tanzania), suggested that this genus and Hydrophylax (with the remaining species H. maritima in southern Asia) are derived from different portions of ‘‘an ancestral Diodia–Spermacoce-like stock’’. Our results confirm that Hydrophylax maritima is indeed closely related to Diodia and Spermac Fig. 1. Strict consensus tree with jackknife values above branches. The asterisks (
) indicate new sequences (Amphiasma benguellense, Bamps et al. 4489 (UPS); Conostomium longitubum, Thulin 10855 (UPS); Dibrachionostylus kaessneri, Strid 2464 (UPS); Hydrophylax maritima, Lundqvist 8945 (UPS); Manostachya ternifolia, Malaisse & Robbrecht 2063 (K); Mitra-sacmopsis quadrivalvis, Richards 11069 (K); Pentanopsis gracilicaulis (Amphiasma gracilicaule), Thulin et al. 10608 (UPS) & Thulin et al. 10512 (UPS) M. Thulin and B. Bremer: Studies in the tribe Spermacoceae (Rubiaceae) 235 236 M. Thulin and B. Bremer: Studies in the tribe Spermacoceae (Rubiaceae) coce. As Spermacoce comes out as biphyletic, Hydrophylax may indeed even be nested within Spermacoce. The delimitations between Spermacoce, Diodia, Hemidiodia, and various other segregates in this complex, are weak (see, for example, Verdcourt 1976). Our sampling is too small to allow any conclusions to be drawn, but surely the clade corresponding to Spermacoceae s. str. is in need of further study. As pointed out by Puff (1986) several apparently unrelated species of Spermacoce and Diodia grow on tropical sea-shores and Hydrophylax maritima, with its creeping stems rooting at the nodes and indehiscent fruits, may represent an extreme case of adaptation in this respect. The Pentanopsis clade has not previously been detected in phylogenetic analyses. Andersson and Rova (1999) showed a close relationship between Conostomium and Amphiasma, but also Oldenlandia affinis was nested in this clade. We are not able to contradict this position for O. affinis, but regard it as highly unlikely. Despite the strong support for the Pentanopsis clade in our molecular analysis, it is not easy to characterise the group morphologically. All members seem to be heterostylous, but heterostyly is found also elsewhere in Spermacoceae, for example in species of Pentas and Oldenlandia. The number of ovules per locule varies from numerous to one, but a unifying feature is that the placentation always is more or less basal, whereas in Spermacoceae s. str. the solitary ovules are attached near the middle of the septum. Manostachya was originally stated to have ovules attached to the middle of the septum (Bremekamp 1952), and this has been repeated by, e.g. Verdcourt (1989). However, in material studied by us, Manostachya has basal placentation as the other members of the Pentanopsis clade. Basal placentation is otherwise rare in Spermacoceae, and the only other instances may be Carphalea, Chamaepentas, Mitrasacmopsis, Pseudonesohedyotis and Dibrachionostylus. Of these genera Carphalea, Mitrasacmopsis and Dibrachionostylus are included in this study, and they do not belong in the Pentanopsis clade (see Fig. 1). The remain- ing taxa, Chamaepentas and Pseudonesohedyotis, are both monotypic African genera and, judging from morphology, they probably do not belong in the Pentanopsis clade either. Phylohydrax is clearly nested in the Pentanopsis clade, even if its sister group relationship is uncertain. It is also, with its heterostylous flowers, filiform stigma lobes, and nearly basal attachment of the placenta, morphologically quite at home in this clade, although the solitary ovules and indehiscent fruits are unique. The superficially similar Hydrophylax, instead, has isostylous flowers, capitate or shortly 2-lobed stigma, and the placenta attached near the middle of the septum, all characters that are compatible with Spermacoceae s. str. Obviously the various similarities in habit between Phylohydrax and Hydrophylax can be attributed to convergent adaptations to a life on sea shores. For example, the indehiscent fruits of both these genera may be adapted to dispersal with water as proposed by Puff (1986). The two species of Conostomium included, C. quadrangulare (type of genus) and C. longitubum (type of C. subgen. Beckia), form a strongly supported clade (100%). Conostomium is morphologically characterised by its pollen grains. Bremekamp (1952) described them as porate, but Lewis (1965) showed that short colpi are also present. The other members of the Pentanopsis clade, as far as known, all have colporate pollen with long and distinct colpi. A possible exception is Phylohydrax, the pollen grains of which were described as ‘‘plurizonocolpate’’ by Robbrecht in Puff (1986). Amphiasma was proposed by Bremekamp (1952) as a genus in south-central tropical Africa with eight species from southern Tanzania in the north to Namibia in the south. Four of these were described as new, whereas the remaining species were previously placed in Oldenlandia. Since then, one further species has been described, A. gracilicaule (Verdcourt 1981). This was based on a single collection from central Somalia and, on account of its long-tubed, glabrous corolla and 4-colporate pollen it was placed in a subgenus of its own, A. subgen. Stonocomium. The other species of M. Thulin and B. Bremer: Studies in the tribe Spermacoceae (Rubiaceae) Amphiasma have corollas with a very short tube, hairy in throat, and 3-colporate pollen (Bremekamp 1952). Comparisons were also made between A. gracilicaule on one hand and Pentanopsis and Conostomium on the other (Verdcourt 1981). In our analysis two forms of A. gracilicaule have been included, one corresponding to the type specimen in flower size and one with slightly larger flowers. Both these forms group with strong support (100%) with Pentanopsis, a monotypic genus also from the Horn of Africa region and actually partly sympatric with A. gracilicaule. The type of Amphiasma, A. luzuloides from southern Tanzania and Malawi, forms a strongly supported clade (94%) with A. benguellensis from Angola. Morphological support for this clade is 3-colporate pollen, in contrast to the 4-colporate pollen in the clade with P. fragrans and A. gracilicaule. These two clades together have a strong support (99%) in the analysis and all members have dorsiventrally flattened seeds with thin walls of the testa cells. Of the other members of the Pentanopsis clade, Conostomium has angular seeds, Phylohydrax has seeds rounded in section, and Manostachya has dorsiventrally flattened seeds with thick outer walls of the testa cells (Bremekamp 1952). Chromosome numbers are known for all genera in the Pentanopsis clade, except from Pentanopsis itself. Conostomium and Amphiasma have x ¼ 9 (Lewis 1965, Kiehn 1985) and this base number characterises also many related genera, such as Agathisanthemum, Dibrachionostylus, Lelya, Oldenlandia, and Pentodon, and it is the most common number in the previous Hedyotideae. However, Manostachya is exceptional in having x ¼ 11 (Lewis 1965, Kiehn 1985), a common number in other parts of Rubiaceae, and Lewis (1965) suggested its occurrence in Manostachya to be ‘‘a relic’’. In our view it is more likely that x ¼ 11 in Manostachya is secondarily derived from x ¼ 9, although we cannot suggest any particular mechanism for this. Most interesting is the report of x ¼ 14 (2n ¼ 56) in Phylohydrax (Kiehn 1985, Puff 1986). This base number is 237 characteristic for Spermacoceae s. str., and has also been reported for Hydrophylax (Puff 1986). However, Kiehn (1985) gave the number as 2n ¼ 56 ± 2, and we believe that further studies are needed to exclude the possibility that Phylohydrax is a hexaploid with x ¼ 9. Taxonomy of Amphiasma and Pentanopsis With its present circumscription Amphiasma is obviously not monophyletic and some taxonomic change is necessary. We have considered the following three options, under the assumption that the two samples of A. gracilicaule are conspecific: (1) to unite Amphiasma and Pentanopsis to create a genus of about 10 species, two in north-east tropical Africa and eight in south-central Africa (some of the latter are very similar according to Verdcourt 1976). (2) to transfer A. gracilicaule to Pentanopsis to create a genus of two species in north-east tropical Africa. This would differ from Amphiasma not only in having 4-colporate (versus 3-colporate) pollen grains, but also in, for example, its much larger flowers. The corolla tube in such an extended Pentanopsis would be 12–37 mm long, whereas in Amphiasma s. str. it is only 2–4 mm long. (3) to place A. gracilicaule in a genus of its own differing from Pentanopsis in its glabrous corolla. In P. fragrans the corolla-lobes are covered on the inside with short blunt hairs. On balance we prefer the second option. By this we avoid monotypic entities and get two geographically confined genera that can be very easily recognised by the great difference in flower size. Amphiasma can then be circumscribed in the same way as it was before 1981, whereas for Pentanopsis we propose a new taxonomy that is outlined below. PENTANOPSIS Rendle in J. Bot. 36: 28 (1898). Type: P. fragrans Rendle. Amphiasma subgen. Stonocomium Verdc. in Kew Bull. 36: 498 (1981). Type: A. gracilicaule Verdc. 238 M. Thulin and B. Bremer: Studies in the tribe Spermacoceae (Rubiaceae) Slender shrubs or subshrubs. Leaves opposite, linear-subulate to lanceolate; stipules sheathing the stem, united with petiole, with fimbriate margin, becoming ± woody and persistent. Flowers fragrant, heterostylous, solitary or few together terminating short shoots; pedicels short. Calyx-tube obovoid; lobes 4, narrowly triangular to linear, not united at the base. Corolla white, with slender tube; lobes 4, induplicatevalvate in bud. Stamens 4, inserted in corolla-tube; anthers oblong; pollen grains 4-colporate. Ovary 2-locular with numerous ovules on peltate placentas inserted at the base of the septum; style with filiform stigma-lobes shortly exserted in long-styled flowers, included in short-styled flowers. Capsule obovoid or ellipsoid, with a short beak, loculicidally dehiscent. Seeds flat, elliptic; testa thin-walled, reticulate, with lumina of testa cells smooth. Genus of two species in north-eastern tropical Africa. Pentanopsis differs from Amphiasma in its much larger flowers (corolla tube 12–37 versus 2–4 mm long), 4-aperturate (versus 3-aperturate) pollen grains, and by its stipules becoming more or less woody and persistent. Also, the calyx-lobes are not united at the base, whereas this generally seems to be the case in Amphiasma. 1. Leaves ± scabrid; corolla-tube 18–37 mm long; corolla-lobes ± densely covered with short hairs on the inside.1. P. fragrans – Leaves glabrous; corolla-tube 12–14 mm long; corolla-lobes glabrous on the inside. 2. P. gracilicaulis 1. Pentanopsis fragrans Rendle in J. Bot. 36: 29 (1898). Type: Somalia, ‘‘Wagga’’ Mt, Lort Phillips s.n. (BM holotype). Conostomium brevirostrum Bremek. in Verh. Kon. Nederl. Akad. Wetensch., Afd. Natuurk., sect. 2, 48: 129 (1952), syn. nov. Type: Somalia/Ethiopia border, between Dolo (‘‘Doloun’’) and ‘‘Batta’’, Ruspoli & Riva 1094 (FT holotype). Conostomium squarrosum Bremek. in Kew Bull. 11: 169 (1956). Type: Ethiopia, between Gorrahi and Wardere, Ellis 138 (K holotype). Shrub, 0.3–2.5 m tall. Leaf-blades linear to lanceolate, 6–40 · 0.8–5 mm, acute at the apex, ± densely scabrid with short hairs above to subglabrous, usually with revolute margins; stipule sheath 2–7 mm long, with 1–4 up to c. 1 mm long fimbriae. Pedicels 1–4 mm long. Calyx-tube 1.5–2.5 mm long; lobes narrowly triangular to linear, 2–11 mm long, minutely ciliate. Corolla white or tinged purplish outside; tube 18–37 mm long, sparsely hairy inside; lobes 5–15 mm long, acute, sparsely to densely covered with short blunt hairs on the inside. Anthers 1–2 mm long in long-styled flowers, 2.1–3 mm long in short-styled flowers. Style 2lobed, 16–22 mm long in short-styled flowers, 21–30 mm long in long-styled flowers. Capsule 4.5–7 mm long. Seeds 1.5–2 mm long. Deciduous or evergreen bushland, usually on rocky ground; 145–1500 m. Somalia, eastern Ethiopia, northern Kenya. Representative specimens. Ethiopia. Bale Region: 42 km SE of Ghinir on road to Imi, 655¢N, 4057¢E, 31 May 1983, Gilbert, Ensermu & Vollesen 7978 (K, UPS). Somalia. Nugaal Region: 49 km SE of Sinujiif, 810¢N, 4910¢E, 6 May 2001, Thulin, Abdi Dahir, Abdulkadir Khalid & Ahmed Osman 10463 (K, UPS). Kenya. Northern Frontier Prov.: Dandu, 14 Apr. 1952, Gillett 12789 (K). 2. Pentanopsis gracilicaulis (Verdc.) Thulin & B. Bremer, comb. nov. Amphiasma gracilicaule Verdc. in Kew Bull. 36: 498 (1981). Type: Somalia, slopes E of Gawen village, 519¢N, 4818¢E, Gillett, Hemming & Watson 22249 (K holotype, EA isotype not seen). Shrub or subshrub, 0.5–1.5 m tall. Leafblades linear-subulate, 20–70 · 0.5–1 mm, acute at the apex, glabrous, with revolute margins; stipule-sheath 1–3 mm long with 2–4 up to c. 0.5 mm long fimbriae. Pedicels 1.5– 4 mm long. Calyx-tube 1.5–2.5 mm long; lobes narrowly triangular, 1–4 mm long. Corolla white or tinged greyish-brownish outside, gla- M. Thulin and B. Bremer: Studies in the tribe Spermacoceae (Rubiaceae) brous; tube 12–14 mm long; lobes 7–11 mm long, acute. Anthers c. 1.8–2 mm long in longstyled flowers, short-styled flowers not seen. Style 2-lobed, 11–14 mm long in long-styled flowers. Capsule 4–6 mm long. Seeds 1.2– 1.5 mm long. Acacia-Commiphora bushland or open plains, in shallow soil over limestone; 180– 300 m. North-eastern and central Somalia. Remarks. The plants from the northernmost locality (Thulin et al. 10608) have larger corollas and longer calyx-lobes than the other material. However, the difference is not great compared to the variation found within P. fragrans. Also, the rbcL sequences from largeflowered and small-flowered plants are practically identical. However, the rbcL sequence data is also inadequate to distinguish between P. fragrans and P. gracilicaulis, and would be compatible with a taxonomy recognising either one, two or three species of Pentanopsis. The taxonomy with two species proposed here is therefore ultimately based on the clear discontinuities found in the morphological variation. Additional specimens. Somalia. Bari Region: 72 km from Qarxis on track to Marraya, 823¢N, 5008¢E, 15 May 2001, Thulin et al. 10608 (K, UPS). Nugaal Region: 22 km on track from Gaalogod to Garadeen, 743¢N, 4936¢E, 9 May 2001, Thulin et al. 10512 (UPS). Mudug Region: E of Gawen, c. 30 km on road from Hobyo to Wisil, 519N, 4819¢E, 28 May 1989, Thulin & Abdi Dahir 6673 (E, FT, K, UPS). We thank Nahid Heidari for technical assistance, Royal Botanic Gardens, Kew, for DNA of Manostachya ternifolia and Mitrasacmopsis quadrivalvis, and Johan Rova and an anonymous reviewer for comments on the manuscript. The study has been supported by grants from the Swedish Research Council. References Andersson L., Rova J. H. E. (1999) The rps16 intron and the phylogeny of the Rubioideae (Rubiaceae). Plant Syst. Evol. 214: 161–186. 239 Bremekamp C. E. B. (1952) The African species of Oldenlandia L. sensu Hiern et K. Schumann. Verh. Kon. Nederl. Akad. Wetensch., Afd. Natuurk., sect. 2, 48: 1–297. Bremer B. (1996) Phylogenetic studies within Rubiaceae and relationships to other families based on molecular data. Opera Bot. Belg. 7: 33–50. Bremer B., Andreasen K., Olsson D. (1995) Subfamilial and tribal relationships in the Rubiaceae based on rbcL sequence data. Ann. Missouri Bot. Gard. 82: 383–397. Bremer B., Manen J. F. (2000) Phylogeny and classification of the subfamily Rubioideae (Rubiaceae). Plant Syst. Evol. 225: 43–72. Kiehn M. (1985) Karyosystematische Untersuchungen an Rubiaceae: Chromosomenzählungen aus Afrika, Madagaskar und Mauritius. Plant Syst. Evol. 149: 89–118. Lewis W. H. (1965) Cytopalynological study of African Hedyotideae (Rubiaceae). Ann. Missouri Bot. Gard. 52: 182–211. Manen J. F., Natali A. (1995) Comparison of the evolution of ribulose-l,5-biphosphate carboxylase (rbcL) and atpB-rbcL spacer sequences in a recent plant group, the tribe Rubieae (Rubiaceae). J. Mol. Evol. 41: 920–927. Puff C. (1986) Phylohydrax (Rubiaceae-Spermacoceae) – a new genus to accommodate the African and Madagascan ‘‘Hydrophylax’’ species. Plant Syst. Evol. 154: 343–366. Swofford D. L. (1998) PAUP
. Phylogenetic analysis using parsimony. Sinauer, Sunderland, Massachusetts, USA. Verdcourt B. (1976) Rubiaceae (part 1). In: Polhill R. M. (ed.) Flora of Tropical East Africa. Crown Agents for Oversea Governments and Administrations. Verdcourt B. (1981) Notes on African Rubiaceae. Kew. Bull. 36: 493–557. Verdcourt B. (1989) Rubiaceae (Rubioideae). In: Launert E. (ed.) Flora Zambesiaca 5(1). Flora Zambesiaca managing committee: London. Addresses of the authors: Mats Thulin (e-mail: mats.thulin@ebc.uu.se), Department of Systematic Botany, EBC, Uppsala University, Norbyvägen 18D, SE-752 36 Uppsala, Sweden. Birgitta Bremer, The Bergius Foundation at the Royal Swedish Academy of Sciences, Box 50017, SE-104 05 Stockholm, Sweden.