Zehneria grandibracteata (Cucurbitaceae), an overlooked new species from western Kenyan forests

Abstract Zehneria grandibracteata, a new species of Cucurbitaceae from western Kenya, is described here, based on morphological and molecular data. It has long been misidentified as the widely-distributed species Z. scabra. However, it differs by its ovate leafy probract at the base of the inflorescences, subglabrous condition of the entire plant, shorter receptacle-tube and filaments, as well as denser and sessile inflorescences. Furthermore, the molecular phylogenetic analysis of Zehneria, based on nrITS sequences, further supports the argument that Z. grandibracteata should be segregated from Z. scabra.


Introduction
Zehneria Endlicher (1833: 69) is a genus of Cucurbitaceae. It contains over 60 species, which are mainly distributed in tropical and subtropical Africa, Madagascar and southeastern Asia (Schaefer and Renner 2011a;Dwivedi et al. 2018). Zehneria is character-ised by male flowers largely with the three stamens all 2-thecate, the thecae ± erect, straight or little curved (Simmons and De Wilde 2000;Schaefer and Renner 2011a). De Duyfjes (2006a, b, 2009a, b) split several genera from Zehneria s.l. (in the sense of Jeffrey), with only the type species, Zehneria baueriana Endlicher (1833: 69) remaining in Zehneria s.s.. Besides, De Wilde and Duyfjes (2006a) proposed morphological characters including leaf drying colour, stamen insertion, presence or absence of staminode, presence or absence of probract and shape of stigmatic lobes, disc and seed, in their circumscription of Zehneria s.s. and the related genera. Nevertheless, this treatment is not supported by the molecular phylogeny inferred by Schaefer et al. (2009), Schaefer and Renner (2011a, b) and Dwivedi et al. (2018), who argued against over-splitting of the group. East Africa has been recognised as a neglected diversity centre for Zehneria (Wei et al. 2017), with several new taxa discovered and named in recent years (Zhou et al. 2016;Wei et al. 2017;Watuma et al. 2019;Ngumbau et al. 2020). Besides, Africa was also referred to as the origin centre (Schaefer et al. 2009;Dwivedi et al. 2018), followed by recent long-distance dispersal to other continents and islands.
During field investigations of the Kenyan flora in 2016, a Zehneria species with evident leafy probracts attracted the authors' attention for the first time. Herbarium specimens had been identified as Z. scabra Sond. in Harvey and Sonder (1862: 486), a widespread species with great morphological variability. In the following years, more specimens were collected and detailed morphological studies were conducted. Measurements of morphological characters, as well as molecular phylogenetic analysis, based on nrITS, all support the segregation of this Zehneria from Z. scabra. Hence, we describe it as Z. grandibracteata below.

Morphology
Specimens of East African Zehneria deposited in the herbaria of K, EA and HIB were studied, as well as relevant digitised specimens from online databases, including specimens from the herbaria B, BR, BM, E and P (herbarium acronyms follow Thiers (2020)). Morphological measurements of the details given in the description are based on living materials during the field trips, except tendrils and seeds confirmed by specimen observations at herbaria. The detailed morphological comparison between Z. scabra and our collection was initially made. Given Z. longiflora G.W. Hu & Q.F. Wang in Wei et al. (2017: 89) has largely overlapped the distribution area with our collection, as well as the great similarity with the latter, Z. longiflora was also included for morphological comparison.

Molecular phylogeny
Aiming to delimitate the phylogenetic position of our Zehneria collections, a total of 63 sequences were used to infer a phylogenetic tree. Amongst these sequences, 60 ac-cessions representing 38 Zehneria species were included and another three accessions from Cucumis, Coccinia, Benincasa were treated as outgroups, according to Schaefer et al. (2009) and Dwivedi et al. (2018). Nineteen sequences of African Zehneria species were newly generated in this study, while the other sequences were downloaded from GenBank. The source of the materials and the corresponding GenBank accession numbers were given in Table 1. Total genomic DNA was extracted from silica gel-dried material using a modified CTAB protocol (Doyle and Doyle 1987) (see Suppl. material 1). The primers of nrITS region were obtained from White et al. (1990). PCR amplification, sequencing and data analysis were performed according to Dwivedi et al. (2018). Forward and reverse sequences were manually checked and edited where necessary. Sequences were aligned by MAFFT v. 7 (Katoh and Standley 2013). Gblocks (Talavera and Castresana 2007) was used to trim with the default setting to remove any ambiguous alignment. Additionally, these alignments were visually inspected in Geneious 8.0.2 (Kearse et al. 2012) and manually adjusted where needed. The best-fit model for Bayesian Inference (BI) and Maximum Likelihood (ML) analyses was estimated by ModelFinder (Kalyaanamoorthy et al. 2017) under the Bayesian Information Criterion (BIC). ML analyses were inferred by IQ-TREE v.1.6.8 (Nguyen et al. 2015) under the Ultrafast bootstrapping algorithm (Guindon et al. 2010) with 1000 bootstrap replicates. BI analyses were performed with MrBayes 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 1000 generations with the initial 25% discarded as burn-in. The remaining trees were then used to construct majority-rule consensus trees. The average deviation of split frequencies was verified by reaching a value below 0.01 at the end of MCMC analyses. The effective sample sizes (ESS) for all parameters and statistics were assessed using Tracer version 1.7.1 (Rambaut et al. 2018). The phylogenetic tree was visualised using the online tool iTOL (Letunic and Bork 2007).

Morphological comparison
The Table 2 distinguishes morphological characters of these three species, mainly based on Jeffrey (1967Jeffrey ( , 1978, Wei et al. (2017) and observations on specimens. Our collection can be readily recognisable by its large leafy probract. Besides, it also differs from the other two species by morphological characters including thick stem, subglabrous leaf blade, sessile inflorescence and size of perianth, pedicel, filament, style and fruit.

Phylogenetic analysis
In total, 60 sequences representing 38 Zehneria species were included in our dataset. Multiple sequences per species were identical as to some species, like Z. grandibracteata, Z. anomala, Z. tuberifera and Z. longiflora. They might, however, be different regard- ing the other species, such as Z. scabra, Z. pallidinervia and Z. minutiflora. The final trimmed alignment of 63 sequences has 721 columns, with 92 parsimony-informative sites. Z. grandibracteata differs in the 71 th position (G vs. A) and 208 th position (A vs. T) of ITS1 alignment from other Zehneria species. HKY+F+G4 was selected as the best-fit model to infer the Maximum Likelihood tree and Bayesian tree. As shown in Figure 1, three accessions of Z. grandibracteata clustered together with robust support (PP = 0.99; BS = 98%). Then, it joined the other three East African taxa group (Z. oligosperma, Z. tuberifera and Z. longiflora), which offers morphological synapomorphies and a conclusive biogeographic scenario of its evolution. These four species formed a monophyly with high support (PP = 0.99; BS = 96%). However, accessions of Z. scabra did not form a monophyly as expected (newly-sequenced accessions are monophyletic, but two previously-published accessions are nested in Z. monocarpa).
Distribution and ecology. Numerous populations of this new species have been documented in the western parts of Kenya's forests, including Morongiot and Kobujoi Conservation status. This new species was found in the western Kenyan forests with numerous localities. It is locally quite common in the wild and frequently grows in forests or at forest margins. Thus, we assess it to be "Least Concern" (LC) based on IUCN Red List Categories and Criteria (IUCN 2001).
Phenology. Flowering and fruiting from April to July and November to January, corresponding to the wet seasons of the bimodal rainfall pattern of this region.
Etymology. The epithet "grandibracteata" refers to the fairly large leafy probract of this new species.

Discussion
Our Z. grandibracteata collections are recognised as monophyletic, separated from the related Z. scabra. The possible reasons to explain the paraphyly of Z. scabra in our phylogeny are 1) the nrITS provides limited phylogenetically-informative sites in Zehneria and mutations on few loci produced inconsistent phylogenetic topology; 2) the two accessions collected by Schaefer here probably should be Z. monocarpa, which was separated from Z. scabra recently (Ngumbau et al. 2020). Furthermore, we also found that species of Neoachmandra in the sense of De Wilde and Duyfjes (2006a) and De Boer et al. (2015), are paraphyly. In line with the conclusion made by Dwivedi et al. (2018), the whole genus tended to be separated into two major clades (clade 1 and clade 2), with African taxa being the basal lineages. Even though the morphological characters proposed by De Wilde and Duyfjes (2006a) are not suitable for splitting groups (Dwivedi et al. 2018), they are still important and helpful characters when identifying at the species level. The ovate leafy probracts in our new species are readily distinguishable, while probracts on other East African taxa tend to be minute linear hooked or even caducous. Geographically, it is only documented in western Kenyan forests (Figure 2), while Z. scabra is widely distributed in the pantropical Old World region. Furthermore, the molecular phylogenetic analysis of Zehneria, based on nrITS sequences, also supports the segregation of Z. grandibracteata from Z. scabra. Combined with morphological and phylogenetic analyses, Z. grandibracteata is confirmed as new to science.
The broadly circumscribed concept of Zehneria may represent a better natural group, while there is no comprehensive classification system for this group until now. Jeffrey (1962) tried to divide Zehneria into two subgenera, namely subg. Zehneria and subg. Pseudokedrostis (Harms 1923: 616) Jeffrey (1962) (largely accord with clade 1 and clade 2 here), mainly based on the position of stamen insertion, the thecae and connective of anther and length of pedicel. Viewing from the phylogenetic tree inferred by Dwivedi et al. (2018), as well our tree here, Jeffrey's morphological summaries mostly work well. Besides, the two fruit shapes, short (sub)globose and long fusiform/ellipsoid, largely fit in with clade 1 and clade 2, respectively, though several taxa with round fruits could also be found in clade 2. All these characters would provide insights into building a classification system within the genus Zehneria. Future biogeographical analysis, based on a robust phylogenic framework, would substantially improve our understanding towards its origin and dispersal history.