Phylogenetic placement of Cailliella praerupticola (Melastomataceae), a rare, monospecific lineage from Guinea, West Africa

Abstract: The genus Cailliella includes a single species, C. praerupticola, restricted to a sandstone plateau region in Coyah, Dubreka, Forécariah and Kindia Préfectures, Guinea, West Africa. Until its rediscovery in 2016, this species was known only by the type gathering made in 1937. We provide an emended description, photographs of living material, locality and habitat data, and a discussion of its conservation status. We further designate a lectotype for C. praerupticola. On the basis of morphology, Cailliella had been considered an isolated member of the tribe Melastomateae, possibly sister to Melastomastrum. We tested the classification of Cailliella by means of a phylogenetic analysis of C. praerupticola and 40 other samples of African Melastomateae, based on molecular sequence data from the external and internal transcribed spacer regions of nuclear ribosomal DNA (nrETS and nrITS) as well as the ndhF, psbK-psbL and accD-psaI regions of chloroplast DNA. Our results corroborate the placement of Cailliella in the Melastomateae, where it was resolved as sister to a clade consisting of both Melastomastrum and Tristemma. The hypothesis of C. praerupticola being an evolutionary relict or “palaeoendemic” is supported by our findings. Citation: Veranso-Libalah M. C., Stone R. D., Haba P. M., Magassouba S., Kadereit G. & Burgt X. M. van der 2021: Phylogenetic placement of Cailliella praerupticola (Melastomataceae), a rare, monospecific lineage from Guinea, West Africa. – Willdenowia 51: 47–56. Version of record first published online on 9 March 2021 ahead of inclusion in April 2021 issue.


Introduction
The sandstone plateau region in Coyah, Dubreka, Forécariah and Kindia Préfectures in Guinea is floristically very interesting. The sandstone table mountains and vertical cliffs have been a factor in promoting speciation, and these species have been preserved by the inaccessibility of some of the table mountains. This region was one of the main study areas of the Darwin Initiativefunded Tropical Important Plant Areas (TIPA) Guinea project undertaken by the Kew Herbarium (Darbyshire & al. 2017;Couch & al. 2019) and the Garfield Westonfunded African Tree Seed Project by Kew's Millennium Seed Bank. New species discovered and published during the work on these projects, and endemic to the sandstone plateau, are: Gladiolus mariae Burgt (Burgt 48 Veranso-Libalah & al.: Phylogenetic placement of Cailliella praerupticola & al. 2019), Inversodicraea koukoutamba Cheek and I. tassing Cheek (Cheek & al. 2019b), Keetia susu Cheek (Cheek & al. 2018a), Kindia gangan Cheek (Cheek & al. 2018b), Talbotiella cheekii Burgt (Burgt & al. 2018), Ternstroemia guineensis Cheek (Cheek & al. 2019a) and Trichanthecium tenerium Xanthos (Xanthos & al. 2020). Numerous plant species are endemic or near-endemic to the sandstone plateau region, such as Anisotes guineensis Lindau, Cincinnobotrys felicis (A. Chev.) Jacq.-Fél., Diospyros feliciana Letouzey & F. White, Fleurydora felicis A. Chev., Mesanthemum bennae Jacq.-Fél. and the remarkable Pitcairnia feliciana (A. Chev.) Harms & Mildbr., the only Bromeliaceae occurring naturally outside of the Americas. To protect these and other plant species, parts of the sandstone plateau region, notably the Kounounkan Forest and Plateau, are proposed to be given increased protected status (Couch & al. 2019). Kew Herbarium is currently working with the Guinean government to achieve this.
In this paper, we report an investigation of the monospecific genus Cailliella Jacq.-Fél. (Jacques-Félix 1939: 637), another Guinean endemic first collected in December 1937 by the French botanist, agronomist and explorer Henri Jacques-Félix (1907-2008, who went on to make a huge contribution to our knowledge of African Melastomataceae. The type gathering of C. prae rupticola Jacq.-Fél. (JacquesFélix 2139) was made on sandstone cliffs of the Benna Plateau (west of Gombokori village, in Forécariah and Kindia Préfectures). For almost 80 years, C. praerupticola was known only from the type material (including three sheets housed in the Paris herbarium plus one duplicate in Kew) until September 2016 when collecting efforts led by two of the present authors (X.M.v.d.B. & P.M.H.) rediscovered it in a second locality on the southern part of the Kounounkan Plateau (Rokni & Lopez Poveda 2018). In 2017 to 2019, plants from several additional localities were found; the species is now known from seven table mountains.
Morphologically, Cailliella is distinguished from other genera of Melastomataceae by its solitary flowers and isomorphic stamens, with anthers attenuate toward the apex, recurved, connective with two linear ventral appendages, one acute dorsal appendage and not produced at base into a pedoconnective (Jacques-Félix 1939, 1995Fig. 1). In the protologue, Jacques-Félix (1939) proposed that Cailliella should be placed in the Osbeckieae (= Melastomateae) even though its seeds were said to be cuneate, not cochleate as in the other members of this tribe (Whiffin & Tomb 1972;Renner 1993;Clausing & Renner 2001;Michelangeli & al. 2013). Among the genera of Melastomateae, a close relationship with Melasto mastrum Naudin has also been suggested (Jacques-Félix 1995;Veranso-Libalah & al. 2017).
Although several African Melastomataceae taxa have been recently sampled in a phylogenetic context (see Ve ranso-Libalah & al. 2017), the monospecific Cailliella had remained unsampled until now. With recent collections of C. praerupticola now available, we aimed to test for the first time the phylogenetic placement of Cailliella using molecular data. We further provide an emended description, photographs of living material, locality and habitat data, and a discussion of conservation status. Because the name C. praerupticola was originally published without a holotype, we also designate a lectotype selected from the duplicates kept in the Paris museum.

Material and methods
Taxonomic sampling and DNA sequencing -Sequence data from five regions (nrETS, nrITS, ndhF, psbK-psbL and accD-psaI) were used in this study (see Table 1 for accessions data). DNA extraction, PCR amplification and sequencing of a herbarium specimen of Cailliella prae rupticola (Haba & Burgt 428, sampled from a duplicate in BR) was accomplished following the protocols described in Veranso-Libalah & al. (2017. Also included in our analysis were 40 other species representing 12 genera from across the African Melastomateae, with corresponding sequences downloaded from GenBank. Phylogenetic inference -We conducted phylogenetic analyses using maximum likelihood (ML) and Bayesian inference (BI) methods. We first separately analysed each of the five datasets (nrETS, nrITS, ndhF, psbK-psbL and accD-psaI), then combined the two nuclear (nrETS and nrITS) and three plastid (ndhF, psbK-psbL and accD-psaI) datasets and analysed them with the same settings. With no supported conflict identified between the plastid and nuclear gene trees based on ML methods (see supplementary Fig. S1, S2), all further analyses were based on the combined data including all five regions.
The Bayesian inference was performed using Mr-Bayes v3.2 (Ronquist & al. 2012). The best-fitting model for nucleotide substitutions was determined using Partitionfinder v.2 (Lanfear & al. 2017). The selected substitution models for the plastid and nuclear datasets were GTR + Γ + I and GTR + Γ, respectively. The BI analyses were run for 20 million generations with four chains in two parallel runs sampling every 2000 generations. All analyses were performed via the CIPRES Science Gateway (http://www.phylo.org/; Miller & al. 2010). Proper mixing was determined using Tracer v.1.5 (Rambaut & Drummond 2009), and all parameters had an effective sample size (ESS) value greater than 200. The first 2000 trees (10 %) were discarded as burn-in, and a maximum clade credibility (MCC) tree was con- Table 1. GenBank accession numbers for all species included in this study.

Results
Phylogenetic analysis -The final sequence alignment for the plastid data included 41 sequences 2126 nucleotides in length (ndhF 667, psbK-psbL 485 and accD-psaI 974), while the nuclear data alignment included 41 sequences 1566 nucleotides in length (ETS 649 and ITS 917). All sequence alignments are provided in the Supplemental content online. Summary statistics of genomic regions sequenced for the phylogenetic placement of Cailliella are provided in Table 2. A tree showing the position of C. praerupticola is presented in Fig. 2. All currently recognized clades of African Melastomateae are resolved with C. praerupticola recovered as sister to a clade comprising Melastomastrum and Tristemma Juss. Diagnostic features -Flowers terminal, solitary, closely subtended by an involucre of leafy bracts. Stamens isomorphic, connective not produced at base into a pedoconnective, dorsal appendage short, 2 ventral appendages linear. Seeds cochleate. Differs from Melastomastrum by having isomorphic stamens (versus iso-or dimorphic); connective with two ventral appendages and one dorsal appendage (versus two ventral appendages and no dorsal appendage), pedoconnective not developed (versus usually well-developed and distinctly prolonged). Jacques-Félix (1939) had pre-viously described the seeds of Cail liella as cuneate. However, our observations indicate that the seeds of this genus are indeed cochleate (Fig. 1H, similar in this respect to other members of the tribe Description -Much-branched deciduous shrub, 0.3 -2.5 m tall, half-spherical in shape; juvenile plants with a pachycaulous base, mature plants branched near base, old plants with adventitious roots (Fig. 1B -D). Branches to 3.5 m long; young branches subterete, dilated at nodes, densely strigose; older branches terete, glabrous (Fig. 1A). Leaves opposite, densely strigose; petiole 7 -8 mm long; blade oblong-lanceolate, 50 -60 mm long × 13 -16 mm wide, abruptly cuneate or rounded at base, progressively narrowed then acute at apex; 5-nerved from base with nerves impressed adaxially, prominent abaxially, sometimes also with a pair of faint marginal nerves; transverse veins invisible (Fig. 1E). Bracts 3 pairs, closely subtending hypanthium, 15 -18 mm long × 7 -10 mm wide, with reticulate, non-protruding veins, apex acute or subobtuse; outer bracts strigose, inner bracts pubescent only on adaxial surface (Fig. 1F, 3A2). Flowers 5-merous. Hypanthium oblong, 12 -15 mm long × 6 -8 mm wide, glabrous; sepals persistent, oblong, 8 -11 mm long × 6 -8 mm wide, obtuse at apex, margin ciliate (Fig. 1G, 3B). Petals pink-mauve, c. 20 mm long × c. 16 mm wide. Stamens 10, equal; filament c. 8 mm long; anther 11 mm long, falcate (Fig. 1F); connective not extended at base into a pedoconnective but provided with a short dorsal appendage and two linear ventral appendages c. 3 mm long. Ovary remaining included in hypanthium and adherent to its base by lines, free toward apex, strigose on lines of dehiscence, epigynous crown with 5 ciliate lobes; placentas elongated, stipitate; style c. 13 mm long. Seeds c. 0.8 mm long, cochleate, surface finely echinulate (Fig. 1H).
Distribution -Cailliella praerupticola is endemic to Guinea in the Coyah, Dubreka, Forécariah and Kindia Préfectures (Fig. 4). Table 2. Summary statistics of genomic regions sequenced for the phylogenetic placement of Cailliella. Accessions sampled  40  41  41  39  31  41  Aligned base pairs  649  917  667  485  974  3692  % Missing data  2  0  0  0  24  6  Conserved sites  310  652  506  356  812  2636  Variable sites  331  248  144  94  134  951  % Variable sites  51  27  22  19  14  26  Parsimony-informative sites  273  152  97  59  67  648  % PIS  42  17  15  12  7 (Burgt & al. 2019). This shrubland is remarkably poor in grasses. Three soil samples taken under this vegetation contained no charcoal at all, although many wood pieces were present. These patches of shrubland may have been protected from fire because they are remote and surrounded by vertical rock cliffs and sub-montane forest (Burgt & al. 2019). These five patches, with a few other patches not visited but seen on Google Earth (2020), form the only open vegetation in the sandstone plateau region that is not yet affected by fire. Elsewhere in the sandstone plateau region, annual dry-season fires occur, and C. praerupticola shrubs are only found growing on vertical cliffs out of reach of fires. Rows of shrubs can often be seen at the top of these cliffs, where enough water is available for them to grow and where they are just out of reach of the dry-season grassland fires. Juvenile plants of Cailliella praerupticola have a pachycaulous base. The woody, succulent stem base of a young sapling c. 10 cm tall was elliptic in shape and c. 2.5 cm in diam. (Fig. 1G). A larger sapling c. 23 cm tall had a pachycaulous base c. 5 cm in diam. (Fig. 1E). This is thought to be an adaptation to the long dry season and the low water-retention capacity of the sandstone cliffs. The pachycaulous lower stem of juvenile C. praerupti

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Veranso-Libalah & al.: Phylogenetic placement of Cailliella praerupticola cola plants is remarkable because this seems to be the first report of this feature among African members of the Melastomataceae. In South America, where this family has its highest diversity, pachycauly is also seen in young seedlings of certain species of Pleroma D. Don, notably P. heteromallum (D. Don) D. Don, inhabiting the seasonally dry Cerrado (tropical savanna) region of Brazil (Dr. Frank Almeda, pers. comm., 17 Apr 2020). Another adaptation to the long dry-season is that the foliage of both juvenile and mature plants is strongly deciduous; during several dry-season months, the plants have no leaves, and only the fruits remain on mature plants.
Conservation status -Cailliella praerupticola was assessed in the IUCN category Endangered, EN D (Rokni & Lopez Poveda 2018). This assessment was based on fewer than 250 mature individuals having been seen. Since this assessment was made, many more mature individuals have been found in several new sites. Cailliella praerupticola is currently known from 13 collections, one made in 1937 and the others from 2016 to 2019. It is estimated that 1600 mature individuals were seen by two of the present authors (X. M.v.d.B. & P.M.H.) at the type locality, the other collection sites and a few other nearby sites where no collections were made. Google Earth (2020) imagery was used to find most of these other sites, and shows there are some more patches of fire-free shrubland and much more vertical cliff habitat in the sandstone plateau region, which have not yet been assessed for the presence of this species. We therefore believe that the actual number of remaining individuals is higher than the number currently observed.
The Extent of Occurrence of Cailliella praerupticola is 1160 km 2 ; the Area of Occupancy is 56 km 2 (Bachman & al. 2011;GeoCAT 2020). The species occurs in seven locations. Within and near the Extent of Occurrence, on the table mountains of the sandstone plateau, only a few patches of open shrubland vegetation exist. Most of the vegetation is grassland, which is annually burned. The burned grassland is visible on Google Earth (2020) historical imagery by the grey colour of the vegetation. Before the arrival of farmers and cattle rangers in the area, practising annual burning to support their farming, C. praerupticola was without doubt much more common in these grassland areas. The population reduction may well be 95 -99 % since the start of the fires, but these fires probably started to occur longer ago than three generation lengths of C. praerupticola. The population reduction over three generations is estimated at 75 % over 90 years. There may have been much decline in Area of Occupancy and in extent and quality of habitat, and this is still continuing. The last remaining large populations of C. praerupticola on the table mountains of the southern Kounounkan Plateau and Banga Guemey are very much at risk of being destroyed by fire. A single fire lit by a hunter could destroy hundreds of shrubs. The population reduction over the next 90 years may well be over 50 %.
Cailliella praerupticola is here assessed in the IUCN (2012) Red List category Endangered EN A2c+3c.
Seeds of Cailliella praerupticola have been collected from a population of c. 50 individuals on the southern Kounounkan Plateau (Burgt 2106). These seeds are stored in Kew's Millennium Seed Bank and in the seed bank of the HNG Herbarium in Conakry. The estimated seed quantity at Kew's MSB, adjusted using the results of an X-ray test on a sample of 50 seeds, is 950 000 seeds.
Remarks on typification -Of the original material (JacquesFélix 2139), there are three sheets in P, i.e. three specimens, each of which has been labelled as a "TYPE". However, the Code of nomenclature says that the type of a species name is a single specimen (Turland & al. 2018: Art. 8.1), and the protologue (Jacques-Félix 1939) did not specify which sheet should be considered as the type (in fact he did not even state that this material was kept in Paris, although one has to assume that this was the case). Even in his Fig. 4, which is a photograph of one of the Paris sheets (P00412601), he did not state that this sheet was the type, nor is the "TYPE" label visible in the photograph (presumably having been attached later). The Code does allow a type specimen to consist of more than one herbarium sheet, as long the sheets "are clearly labelled as being part of the same specimen, or bear a single, original label in common" (Art. 8.3). However, this is not true of the original material of Cailliella. In conclusion, because the name C. praerupticola was published without a holotype, our designation of a lectotype is warranted (Art. 9.3, 9.11). Willdenowia 51 -2021 Kindia and Coyah Préfectures: Plateau de Tassing above Fossikouré Village, top of the 270 m high Chute de Kili, 9.70853°N, 13.21375°W, elev. 430 m, 7 Dec 2017, Burgt 2183 (BR, HNG  Sequence alignments for the five regions (in nex format): nrETS, nrITS, ndhF, psbK-psbL and accD-psaI.