Morphology and molecules support the new monotypic genus Parainvolucrella (Rubiaceae) from Asia

Abstract Parainvolucrella R.J. Wang, a new monotypic genus for P.scabra (Wall. ex Kurz) M.D.Yuan & R.J.Wang, new combination, is segregated from the Hedyotis-Oldenlandia complex, based on morphological and molecular evidence. Phylogenetically, the new genus is sister to Scleromitrion, from which it differs by a combination of morphological characters: herbaceous habit, terminal inflorescence with subtended leaves, heterostylous flowers, indehiscent fruits and pollen with double microreticulate tectum. A key to the genera of the Hedyotis-Oldenlandia complex in China is provided for further identification.


Introduction
As one of the largest species groups of the family Rubiaceae, the Hedyotis-Oldenlandia complex contains hundreds of species distributed in the tropical and subtropical region worldwide. Due to morphological intermediacy and homoplasy, systematic studies in herbaceous Rubiaceae are very difficult (Gibbons 2020). The generic delimitation within this complex is complicated and controversial (Neupane et al. 2015) and historically disputed. The commonly shared morphological characters, such as four petals and calyx lobes, 2-celled ovaries with numerous ovules on axile placenta and capsular fruits made some studies treat this complex as one genus, Hedyotis L., in a broad sense (Lamarck 1792; Fosberg and Sachet 1991;Dutta and Deb 2004;Chen and Taylor 2011). Whereas, morphological differences in habit, inflorescence position, homo-or heterostylous flowers, dehiscent or indehiscent fruits, as well as the shape and ornamentation of seeds and pollen, provide unquestionable evidence to separate this complex into several small genera (Bremekamp 1952;Terrell et al. 1986;Terrell and Robinson 2003). Recent phylogenetic analyses, based on multiple nuclear and chloroplast DNA markers, revealed that this complex was polyphyletic and supported its subdivision into small genera (Groeninckx et al. 2009;Neupane et al. 2009;Guo et al. 2013;Wikström et al. 2013;Neupane et al. 2015;Gibbons 2020 (Neupane et al. 2015;Wang 2018).
During our field investigation in Guangxi Zhuang Autonomous Region, we came across the species Hedyotis scabra Wall. ex Kurz, not recorded previously in China (Wei 2018), in bamboo forest nearby the Nonggang National Nature Reserve. This species has arbitrarily been treated as Scleromitrion scabrum (Wall. ex Kurz) Neupane & N.Wikstr. with insufficient morphological and molecular evidence (Neupane et al. 2015). Morphologically, it is similar to Involucrella coronaria (Kurz) Neupane & N.Wikstr. for its terminal inflorescence subtended by four involucral leaves. Our subsequent morphological comparison and phylogenetic analysis, based on multiple DNA markers, support that this species represents a new genus.

Materials and methods
Morphological characters of Hedyotis scabra were scored from living materials and dried specimens. All vouchers which we collected were deposited at the herbarium of South China Botanical Garden, Chinese Academy of Sciences (IBSC). Pollen and seeds were observed using scanning electron microscopy (JSM-6360LV) under 15.00 kV accelerating voltage. Pollen terminology for description followed Punt et al. (2007).
Methods of DNA extraction and PCRs followed Guo et al. (2011). Sequences of all taxa were downloaded from GenBank for molecular phylogenetic analysis, except for the newly added Hedyotis hainanensis, H. ovata, and three samples of Hedyotis scabra (Table 1). Geneious v.11.0.3 (Kearse et al. 2012) was used for sequence alignment and MrModeltest 2.0 was applied for selecting the best-fit nucleotide substitution model (GTR+G+I) on the basis of the AIC criterion (Nylander 2004). Bayesian Inference (BI) was performed using MrBayes v.3.2.7 (Ronquist et al. 2012), with a calculation of posterior probabilities (PP) to each clade. The bootstrap (BS) values were obtained by IQ-TREE v. 2.0 (Nguyen et al. 2015) for Maximum Likelihood analyses based on the best-fit nucleotide substitution model (GTR+F+R3) selected by ModelFinder (Kalyaanamoorthy et al. 2017). Table 1. Taxa, vouchers, localities and GenBank accession numbers of ITS, petD, rps16, trnH-psbA and trnL-F sequences for phylogenetic analysis. Notes: "*" indicates the newly-seqenced fragments, "/" indicates the missing data.

Discussion
The plant habit, stipule shape, inflorescence position, flower distyly and the dehiscent pattern of the fruits are of diagnostic significance in the different genera of the Hedyotis-Oldenlandia complex (Dutta and Deb 2004). Several successive field collections observed that the fruits of Hedyotis scabra are completely indehiscent, which was obscurely diagnosed by Hooker (1880) and incorrectly described by Dutta and Deb (2004). Hedyotis scabra differs from Scleromitrion by the terminal inflorescences with involucral leaves (vs. axillary or terminal and axillary in the uppermost leaf axils in Scleromitrion), the heterostylous flowers (vs. homostylous in Scleromitrion), pollen grains tectum double microreticulate, with psilate suprareticulum and microechinate infrareticulum (vs. rugulate tectum with microechinate muri in Scleromitrion) and indehiscent fruits (vs. loculicidally dehiscent in Scleromitrion). On the other hand, Parainvolucrella scabra is similar to Involucrella coronaria with respect to their terminal inflorescence subtended by involucral leaves, heterostylous flowers and indehiscent fruits, but Parainvolucrella has decumbent habit (vs. erect or ascending in Involucrella coronaria), young fruits with 4 longitudinal projections (vs. smooth surfaces in Involucrella coronaria) and trigonous seeds with no pits on the surface (vs. ellipsoidal and 3-5 pitted seeds in Involucrella coronaria) ( Table 2). Based on the combined nuclear (ITS, ETS) and plastid (petD, rps16) data, Neupane et al. (2015) did not provide a well-resolved phylogenetic tree to support the placement of Hedyotis scabra as sister to the remainder of Scleromitrion in the Hedyotis-Oldenlandia complex, neither did Gibbons (2020). In addition, it seemed that the morphological confliction between the H. scabra and Scleromitrion and the phylogenetic exclusion of H. scabra from Scleromitrion clade were overlooked before making the new combination by Neupane et al. (2015). Our further integrated analysis, based on the morphological incongruence and the robust phylogenetic support (BS = 100, PP = 1), based on nrITS and plastid petD, rps16, trnH-psbA and trnL-F, elucidated the taxonomic and phylogenetic confusions and thus the new monotypic genus Parainvolucrella is proposed here.