Euphorbiambuinzauensis, a new succulent species in Kenya from the Synadenium group in Euphorbiasect.Monadenium (Euphorbiaceae)

Abstract Euphorbiambuinzauensis, a succulent new species of the Synadenium group in Euphorbiaceae from Makueni County, Kenya, is described and illustrated. Morphologically, it is most similar to E.pseudomollis, but differs mainly by its shrubby habit (up to 4 m), abaxial leaves surfaces with densely stellate hairs, 2–4-forked cymes, smaller bracts (ca. 2.5 × 3.0 mm), smaller cyathia (6 mm wide), crimson glands without narrow smooth margin, smaller fruits (ca. 8 × 7 mm) and ovoid seeds (ca. 1.8 × 2.2 mm). Furthermore, we performed a molecular phylogenetic analysis of the Synadenium group in Euphorbiasect.Monadenium, based on complete nuclear ribosomal DNA (nrDNA) datasets. This phylogenetic inference also supports it to be a distinct species. The new species is assessed as Endangered using the IUCN criteria.


Introduction
Synadenium Boiss. (in de Candolle (1862: 187)), was a previously recognized genus of Euphorbiaceae, confined to east and southern tropical Africa, with 14 closely related species (Carter and Leach 2001). Synadenium is readily distinguishable from other genera in Euphorbieae subtribe Euphorbiinae by the unique ring-shaped fused glands in the cyathium (Jones and Smith 1969;Carter 1988;Carter and Leach 2001). It has been regarded as a taxonomically difficult genus (Govaerts et al. 2000), since the differences between the species appear to be slight, especially when observing dried herbarium specimens only. Moreover, Brown et al. (1909) assumed that Synadenium species are locally endemic and probably more numerous than previously known (13 spp. at that time), given their great resemblance to one another, which may have resulted in some of them being overlooked.
Besides emphasizing leaf and cyme-branching characters, Carter and Leach (2001) suggested that emphasis should also be placed upon the habit, the shape and size of the cyathium, the color and furrowing of the involucral glands, and features of the capsules and seeds. Unfortunately, relatively few specimens have been prepared and deposited in herbaria, especially fruiting ones (Carter 1988). Although regional taxonomic treatments have been done, Synadenium has never been comprehensively monographed, and was regarded as a poorly known group. Webster (1967) and Croizat (1972) questioned the delimitation of Synadenium and Monadenium as distinct genera and suggested, based on morphological similarities, that they were closely related to Euphorbia sect. Tirucalli. Analysis of molecular data over the past two decades (Steinmann and Porter 2002;Bruyns et al. 2006Bruyns et al. , 2011Zimmermann et al. 2010;Horn et al. 2012;Dorsey et al. 2013;Wei et al. 2021), have all shown that the genera Endadenium, Monadenium, and Synadenium were deeply nested in Euphorbia. Consequently, they were merged under Euphorbia and treated as subgenus Euphorbia sect. Monadenium, to maintain the monophyly of Euphorbia (Bruyns et al. 2006). However, the Synadenium group has been poorly sampled to date (e.g., three species in Dorsey et al. 2013).
In a field investigation targeting Kenyan Euphorbia in 2018, a shrubby Euphorbia (Synadenium) species attracted the authors' attention. At first sight, it was roughly identified as E. pseudomollis Bruyns in Bruyns et al. (2006: 414), due to its densely pubescent leaves. More specimens of this Euphorbia were collected during the following field trip at the same area, and then detailed morphological studies were conducted. Based on the floral records and the examination of Synadenium specimens deposited in herbaria worldwide, we found that this Euphorbia differs from E. pseudomollis by a combination of several morphological characters. Furthermore, the molecular phylogeny of the Synadenium group based on complete sequences of the nuclear ribosomal DNA (nrDNA) revealed that our Euphorbia collection is sister to E. bicompacta Bruyns var. bicompacta Bruyns in Bruyns et al. (2006: 412). Hence, both morphological studies and phylogenetic analyses indicate that our Euphorbia collections represent a hitherto undescribed species, which we now proceed to describe and name as Euphorbia mbuinzauensis.

Morphological observations
The morphological description of the new species is based on measurements on living specimens collected during the field trips, except for the seeds which were obtained by observations of specimens in herbaria. Comparisons with other relevant Euphorbia species were based on previous regional floristic accounts (Brown et al. 1909;Carter 1988;Carter and Leach 2001) as well as the examination of herbarium specimens and digitized images which are mainly from AMD, BR, BM, EA, HIB, K, P, S, and WAG (herbarium acronyms following Thiers (2021)).

Sampling, genomic DNA extraction, and sequencing
To delineate the phylogenetic placement of our Euphorbia collection, a total of 17 sequences, which were all newly generated in this study, were used to infer the phylogenetic tree for the Synadenium group in Euphorbia. Amongst these sequences, 14 accessions representing nine accepted species of the Synadenium-group were included. The remaining three accessions from traditionally recognized Euphorbia, Monadenium and Endadenium, were treated as outgroups, according to Dorsey et al. (2013) and Wei et al. (2021).
Sources of DNA were silica-dried leaves collected from field trips, or from dried voucher specimens in herbaria. Total genomic DNA was extracted with the Magic-Mag Genomic DNA Micro Kit (Sangon Biotech Co., Shanghai, China) following the instructions of the manufacturer's protocol. DNA quality was assessed by 1% agarose gel electrophoresis. Short inserts (350 bp) were used to construct paired-end 150-bp sequencing libraries using the NEBNext Ultra DNA Library Prep Kit for Illumina (NEB, United States). Libraries were sequenced at Beijing Genomics Institute (Shenzhen, China) using the Illumina HiSeq 2000 Platform (Illumina, San Diego, CA, United States).

Molecular phylogenetic analyses
The complete nrDNA sequences were aligned by MAFFT v. 7 (Katoh and Standley 2013) with the default setting. TrimAl v.1.2 (Capella-Gutierrez et al. 2009) was used to trim the alignment sequence with automatd1 mode to reduce potentially poorly aligned regions. Besides, the trimmed alignments were also visually inspected in Geneious 8.0.2 (Kearse et al. 2012) and manually adjusted if necessary. Partition-Finder 2 (Lanfear et al. 2012(Lanfear et al. , 2016 was used for best-fit substitution model selection for each region under the Akaike Information Criterion: the general time reversible model with a gamma distribution of substitution rates (GTR+G) was chosen for the ETS region; the GTR+I+G model with a proportion of invariant sites was selected for the 5.8S and 28S regions; the Hasegawa-Kishino-Yano model with a proportion of invariant sites (HKY+I) was selected for the ITS1, ITS2, and IGS regions; and the HKY+I+G model with a gamma distribution of substitution rates was selected for the 18S region. The ML tree was inferred by IQ-TREE v.1.6.8 (Nguyen et al. 2015) with 10,000 bootstrap replicates. The BI phylogenetic analysis was performed with MrBayes v.3.2.7 (Ronquist et al. 2012). Two independent Markov Chain Monte Carlo analyses (MCMC) were run with four simultaneous chains of 10 million generations, sampling one tree every 100 generations, and setting the burnin fraction as 0.25. The remaining trees were then used to construct a majority-rule consensus tree. The average deviation of split frequencies was verified by reaching a value below 0.01 at the end of the MCMC analyses. The effective sample sizes (ESS values > 200) for all parameters and statistics were also assessed using Tracer v.1.7.1 (Rambaut et al. 2018). The final phylogenetic tree was shown using the online tool iTOL (Letunic and Bork 2007).

Phylogenetic relationships
The 17 complete nrDNA repeat sequences have average coverage ranging from 430.5 to 524.9 (Suppl. material 1). The aligned length of the seven concatenated nrDNA constituent loci dataset prior to trimming is 11,671 bp, whereas the trimmed alignment dataset consisted of 10,605 bp (Suppl. material 2) with 293 parsimony-informative sites. The ML and BI trees are identical, and the ML tree with both posterior probabilities and ML bootstrap values for each clade is shown as Figure 1. For the ML analysis of the combined seven loci of nrDNA, likelihood score (-lnL) is 23609.7. Overall, the 14 accessions of the Synadenium group clustered together and formed a strongly supported monophyletic group (BS = 100%, PP = 1). The new species, Euphorbia mbuinzauensis, is sister to the clade that consists of two accessions of E. bicompacta var. bicompacta with robust support (BS = 100%, PP = 1). Despite the new species being morphologically closest to E. pseudomollis, they were not sister taxa in our phylogenetic tree. In addition, the accession of E. bicompacta var. rubra did not form a clade with E. bicompacta var. bicompacta as expected. Instead, it is sister to another species, E. pseudomollis, with robust support (BS = 100%, PP = 1).

Morphological comparisons
Morphologically, Euphorbia mbuinzauensis is closest to E. pseudomollis, a species that occurs in Kenya and Tanzania, but differs by a shorter habit (up to 4 m), abaxial surfaces of leaves densely covered with stellate hairs, 2-4-forked cymes, smaller bracts (ca. 2.5 × 3.0 mm) with dentate margin, smaller cyathia (6 mm wide), crimson gland without narrow smooth margin, smaller fruits (ca. 8 × 7 mm) and smaller ovoid seeds (ca. 1.8 × 2.2 mm). Based on the results of our phylogenetic analyses, the sister taxon of Euphorbia mbuinzauensis is E. bicompacta var. bicompacta. However, E. bicompacta var. bicompacta can be easily distinguished from E. mbuinzauensis by its glabrous leaves, shallowly and minutely grooved yellow to greenish red gland, half fused style, and larger seeds (ca. 2.8 × 2.5 mm). Further detailed morphological differences among the three species are presented in Table 1.

Discussion
Euphorbia mbuinzauensis belongs to the Synadenium group of subg. Euphorbia, which is characterized by tree-like or shrubby habit, fleshy leaves with prominent midrib, pseudo-umbels of 2-5 cymes on peduncles, cymes that are forked several times, involucral [cyathial] glands that fused in a ring-like structure, funnel-shaped involucres, and rudimentary caruncles (Brown et al. 1909;Carter 1988;Carter and Leach 2001). Euphorbia mbuinzauensis is distinct, however, from other species in Synadenium group with strong morphological and phylogenetic support.
We show the monophyly of the previously segregated genus Synadenium, using phylogenetic inference based on a nrDNA dataset. Nevertheless, a more representative sampling of sect. Monadenium, especially of those species that belonged traditionally to Monadenium, is needed to draw this conclusion with greater confidence. It is worth mentioning that the phylogenetic relationships among the species in Synadenium group exhibited extremely short branches ( Figure 1B), indicating that this lineage is likely to have radiated very recently. Interestingly, E. bicompacta var. rubra did not form a clade with E. bicompacta var. bicompacta as expected, suggesting a problem in its taxonomic placement. A comprehensive monograph based on an extensive study of specimens and a broad phylogenetic sampling is needed before the Synadenium group can be fully understood. isotypes EA!, HIB!, K!) Diagnosis. Euphorbia mbuinzauensis is most similar to E. pseudomollis, from which it differs by its shorter habit, up to 4 m (vs. to 9 m), abaxial leaves surfaces with densely stellate hairs (vs. simple hairs), 2-to 4-forked cymes (vs. 1-to 2-forked), bracts ca. 2.5 × 3.0 mm (vs. ca. 3.5 × 3.5 mm), dentate margin on bract (vs. entire or with a few teeth), cyathia 6 mm wide (vs. 8 mm wide), gland without narrow smooth margin (vs. with a narrow smooth margin), fruits ca. 8 × 7 mm (vs. ca. 10 × 10 mm), and ovoid seeds ca. 1.8 × 2.2 mm (vs. subglobose, ca. 2.5 × 2.5 mm).
Distribution and ecology. Only one population of the new species was found at the foot of Mbuinzau hill, Makueni County, Kenya (Figure 4). Here it grows in open deciduous woodlands covered by lava outcrops at an elevation of ca. 970 m.
Conservation status. Only one population of the new species was found in an isolated woodland covered by lava outcrops ( Figure 3A) in Mbuinzau area. We assess the species as Endangered (criteria D1) according to the IUCN Red List Categories and Criteria (IUCN 2001), due to the number of mature individuals (< 250) in a very small and restricted population. Unfortunately, the habitat is threatened by human activities. For instance, its habitat has been fragmented by the Mombasa-Nairobi Railway and Mombasa-Road, which are the busiest traffic routes in Kenya. Moreover, the population is under threat from habitat loss, due to the exploitation of lava rocks in its habitat for construction.
Phenology. Euphorbia mbuinzauensis was found with flowers in September and with fruits from September to December.
Notes. The conspicuous latex is extremely poisonous to cattle as well as human beings, according to the comments from local farmers. We observed ants visiting the flowers and they are a possible pollinator to this species ( Figure 3G).
Additional specimens examined (Paratypes