New insights into the phylogeny of Sinocarum (Apiaceae, Apioideae) based on morphological and molecular data

Abstract Sinocarum is a Sino-Himalayan endemic genus of Apiaceae and distributed in high-elevations from Nepal to SW China. In this study, morphological characteristics were combined with nuclear internal transcribed spacer (ITS) and two chloroplast DNA (cpDNA) intron sequences (rpl16 and rps16) to determine the phylogenetic placement of Sinocarum and the infrageneric relationships between five Sinocarum species. The results confirmed that Sinocarum was a polyphyletic group separated into two clades, Acronema and East Asia clades. S. coloratum, the generic type of Sinocarum, S. cruciatum, S. vaginatum and S. filicinum are in the Acronema clade. Among them, the first three species are clustered into a subclade and are closely related to the genus Acronema. While S. filicinum has a close affinity with Meeboldia. S. schizopetalum did not ally with its congeners we collected and is allied closely with members of the distantly related East Asia clade. In addition, the fruit of the Acronema clade Sinocarum species is usually oblong-ovoid or ovoid, and the pollen is super-rectangular, while the Sinocarum species in the East Asia clade have broad-ovoid fruit and sub-rhomboidal pollen. This study has furnished cumulative evidence to reduce phylogenetic uncertainty and provide a more comprehensive description of the plant morphology, fruit morphology and anatomy, and pollen morphology of these five Chinese Sinocarum species.


Introduction
Sinocarum H. Wolff ex R. H. Shan & F. T. Pu (1980: 374) was transferred from the genus Carum L. (1753: 263) by Wolff (1927), but formally described by Shan and Pu (1980). Sinocarum encompasses about 20 species, with eight species (four endemic) in China and is distributed at high-elevation in the Sino-Himalayan region from Nepal to SW China (Pu et al. 2005). It is usually classified by a suite of characteristics: elongate rhizome, expanded petiole sheaths, obtuse at apex and clawed at base petals and oblong-ovoid fruit Pu et al. 2005). Despite several easily recognizable characteristics, there remains morphological and taxonomic confusion in the genus, including the lack of morphological description and specimens of mature fruit, unclear intergeneric boundary and excessive use of synonyms. Sinocarum is a taxonomically complex genus that is closely related to Acronema Falcon. ex Edgew. (1846: 51) and sometimes difficult to distinguish (Pu et al. 2005). Thus, further work and more extensive specimen collections are needed to clarify the situation.
Previous studies have shown that fruit characteristics play a key role in the classification of subfamily Apioideae (Kljuykov et al. 2004;Lyskov et al. 2017;Guo et al. 2018;Jia et al. 2019). The fruit characteristics of Sinocarum species are described in Flora Reipublicae Popularis Sinicae and Flora of China. The fruit of Sinocarum is oblong-ovoid with 5 filiform ribs, but only young fruit is involved, and the mature fruit is unknown (Shan and Pu 1985;Pu et al. 2005). Hence, the definition of the fruit's morphological and anatomical characteristics needs to be supplemented to allow for better identification.
Similarly, previous molecular studies have been limited and results ambiguous. Sinocarum was found to be polyphyletic based on ITS, cpDNA sequences and limited specimen materials (S. coloratum, S. cruciatum and S. dolichopodum) and there has been no consensus on its phylogenetic placement (Valiejo-Roman et al. 2002;Zhou et al. 2008;Zhou et al. 2009;Downie et al. 2010). Consequently, there is a gap in our understanding of Sinocarum's phylogeny and infrageneric classification due to insufficient specimen sampling. Together with limited definitions of morphological characteristics, there is a need to study the phylogeny and morphology of this genus based on new, comprehensive materials.
Therefore, the objective of this study was to estimate the phylogenetic placement of Sinocarum and the infrageneric relationships of the five Sinocarum species we collected. This is the first comprehensive phylogenetic analysis of Sinocarum using morphology and three DNA regions data (i.e. ITS, rpl16 and rps16). Given this more comprehensive analysis, we also discuss the significance of using morphology in phylogenic analyses. In addition, we provide more comprehensive descriptions for the plant morphology, fruit morphology and anatomy, pollen morphology and identification of herbarium specimens of five accepted Sinocarum species. We believed that this study will contribute to a better understanding of the phylogenetic status, infrageneric relationships and morphological identification of Sinocarum.

Materials and methods
Field investigation, morphology study and specimen examination  Pu (1998: 70) was obtained from Kunming, Yunnan. Photographs of specimens were made using a Nikon D5600 camera. Fruits were observed and photographed using a stereomicroscope, Nikon SMZ 25 (Japan), and five representative fruit samples were selected to observe characters and measure their size, and then calculate the average value. Pollen grains from the anthers of specimens were directly mounted on copper stubs with conductive carbon adhesive tabs using a needle, sputtercoated with gold, and observed with a Hitachi-SX-450 SEM (Japan). The continuous section of the middle transection of the mericarp was made by the normal paraffin section method. And the section was observed and photographed using stereomicroscope Nikon SMZ25 (Japan). A total of ten pollen grains were selected to measure their length of polar axis (P) and equatorial axis (E), and calculate their average value, ratio of polar axis to equatorial axis (P/E) and size index ( P E × ). The micromorphological characteristics of pollen were described according to Shu and She (2001). Morphological characteristics were measured using Kayotype (Altınordu et al. 2016). Voucher specimens were deposited in the herbarium of Natural History Museum of Sichuan University (SZ) ( Table 1).

DNA extraction, amplification and sequencing
Total genomic DNA was extracted from silica gel-dried leaves and herbarium materials according to the protocols of plant genomic DNA kit (Tiangen Biotech, Beijing, China). Nuclear ribosomal DNA (nrDNA) ITS sequences and two chloroplast DNA (cpDNA) -- intron sequences (rpl16 and rps16) were applied to phylogenetic analyses. The primers ITS4 (5'-TCC TCC GCT TAT TGA TAT GC-3') and ITS5 (5'-GGA AGT AAA AGT CGT AAC AAG G-3'; White et al. 1990) were used for PCR-amplification of a complete ITS fragment. The rpl16 intron region was amplified with primers F71(5'-GCT ATG CTT AGT GTG TGA CTC GTT G-3') and R1516 (5'-CCC TTC ATT CTT CTA TGT TG-3'; Jordan et al. 1996;Kelchner and Clark 1997). The rps16 intron was amplified using primers rps16 5'exon (5'-AAA CGA TGT GGN AGN AAR CA-3') and rps16 3'exon (5'-CCT GTA GGY TGN GCN CCY TT-3'; Downie and Katz-Downie 1999). Amplification was undertaken in a 30 µL mixture of 2 µL plant total DNA, 10 µL ddH 2 O, 1.5 µL forward primer, 1.5 µL reverse primer and 15 µL 2 × Taq Master-Mix (cwbio, Beijing, China). The amplification of the ITS region was obtained by initial denaturation for 3 min at 94 °C, followed by 30 cycles of 45 s at 94 °C, 60 s at 54 °C, and 90 s at 72 °C, and then a final extension of 10 min at 72 °C. The amplification of the rpl16 region was obtained by initial denaturation for 3 min at 94 °C, followed by 36 cycles of 45 s at 94 °C, 70 s at 58.5 °C, and 90 s at 72 °C, and then a final extension of 10 min at 72 °C. Whereas amplification of rps16 region was obtained by initial denaturation for 3 min at 94 °C, followed by 36 cycles of 45 s at 94 °C,70 s at 54 °C, and 90 s at 72 °C, and then a final extension of 10 min at 72 °C. All PCR products were separated using a 1.5% (w/v) agarose TAE gel and sent to Sangon (Shanghai, China) for sequencing. New sequences generated for this study have been deposited in GenBank (Table 1).

Sequence alignment and phylogenetic analysis
We used 53 nrDNA ITS sequences obtained from GenBank, and six sequences newly sequenced for this study (Table 1), to infer the phylogenetic placement of Sinocarum. Seventy-four accessions obtained from GenBank for the nrDNA (ITS) and cpDNA (rpl16 and rps16), and 15 accessions newly sequenced (Table 1) represented 35 species from 21 genera of Apiaceae and were used to reconstruct the phylogenetic tree of the Acronema clade. Tribe Scandiceae was selected as the outgroup (Downie et al. 2000a;Zhou et al. 2008;Zhou et al. 2009). Eighty-three accessions obtained from GenBank for the nrDNA (ITS) and cpDNA (rpl16 and rps16), and three accessions newly sequenced (Table 1) represented 31 species from 15 genera of Apiaceae and were used to reconstruct the phylogenetic tree of the East Asia clade. Tribe Pleurospermeae was selected as the outgroup (Downie et al. 2000b;Zhou et al. 2008;Zhou et al. 2009). Sequence data for the ITS 5.8S region were excluded from the analysis because they were unavailable for several previously published taxa. SeqMan (Burland 2000) was used to assemble DNA sequences and obtain consensus sequences. DNA sequences were aligned with ClustalX ver. 2.1 (Larkin et al. 2007) and then adjusted manually using MEGA7 (Kumar et al. 2016). Phylogenetic analyses of data were conducted by employing Maximum Likelihood (ML) and Bayesian Inference (BI) methods. Maximum Likelihood phylogenetic reconstruction was performed using RAxML-HPC ver. 8.2.10 under the GTR+G nucleotide substitution model and 1,000 rapid bootstraps. The BI analysis was performed in MrBayes version 3.2 (Ronquist et al. 2012). MrModeltest version 2.2 (Nylander 2004) was used for BI analysis to determine a best-fit model of nucleotide substitution. From a random starting tree, the BI analysis was run for 10 million generations and the trees were saved to a file every 1,000 generations. Posterior probabilities were approximated by sampling trees using a variant of the Markov Chain Monte Carlo (MCMC) method. The first 1,000 trees were discarded as "burn-in" and a majority-rule consensus tree was calculated based upon the remaining 9,000 trees resulting from Tracer 1.4 analysis (Drummond and Rambaut 2007).

Palynology
The pollen morphology of the five Sinocarum species was studied by SEM, as shown in Table 5 and Fig. 3. The average ratio of the polar axis to the equatorial axis (P/E) of the pollen grains of S. coloratum, S. cruciatum and S. vaginatum is greater than 2, and the average size index was greater than 19. The pollen grains of these three species are superrectangular in equatorial view, trilobate circular in polar view ( Fig. 3A1-A3, B1-B3, E1-E3). The exine ornamentation of the polar area is cerebroid with a few perforations, and the equatorial area is cerebro reticulate (Fig. 3A3-A4, B3-B4, E3-E4). The pollen grains of S. filicinum are super-rectangular in equatorial view, trilobate circular in polar view ( Fig. 3C1-C3). The exine ornamentation of the polar area is striate reticulate with a few perforations, and the equatorial area is cerebro reticulate (Fig. 3C3-C4). Compared with other Sinocarum species, the pollen size of S. schizopetalum is smaller, and its size index is 15.11(14.15~16.62). And its pollen grains are sub-rhomboidal in equatorial view, obtuse triangled in polar view ( Fig. 3D1-D3). The exine ornamentation of the polar area is cerebroid, and the equatorial area is pitted reticulate (Fig. 3D3-D4).

Sequence characteristics
The characteristics of the three DNA regions are summarized in Table 6. These results indicated that the aligned length of the background tree using 59 ITS sequences from  Apiaceae was 472, containing 73.94% average sequence divergence and 276 parsimony informative characters. In addition, average sequence divergence of ITS was more variable (60.50%; 56.76%) than cpDNA (16.76%; 13.06%) across the Acronema clade, East Asia clade and their outgroups. Overall, ITS was more variable than cpDNA, with greater average sequence divergence and more parsimony informative characters.

ITS trees (Figs 4, 5A, 6A)
After phylogenetic analyses with comprehensive sequence data, we confirmed that the collected Sinocarum materials were from a polyphyletic group and fell into two different clades (i.e. Acronema clade, East Asia clade) according to the ITS tree inferred by ML approach (Fig. 4). The ITS trees of Acronema clade and its outgroups (Fig. 5A) inferred by ML and BI approaches were inconsistent for subclade topology. The ITS tree dem-

cpDNA trees (Figs 5B, 6B)
The cpDNA trees of Acronema clade and its outgroups (Fig. 5B) inferred by ML and BI approaches had consistent topologies. The cpDNA trees indicated that the generic type of Sinocarum, S. coloratum, together with S. cruciatum and S. vaginatum constituted a supported monophyletic group (PP/BS = 1/92%) as sister group to Acronema (PP/BS = 1/58%). Two populations of S. filicinum allied powerfully with the genus Meeboldia (PP/BS = 1/79%). The cpDNA trees of the East Asia clade and its outgroups (Fig. 6B) inferred by ML and BI approaches had consistent topologies and indicated that the position of S. schizopetalum differed from the ITS tree and was located in the East Asia clade.  Zhou et al. (2008Zhou et al. ( , 2009 and Downie et al. (2010).

Morphology
We have studied the plant morphology, fruit morphological and anatomical characteristics, and palynology of five species of Sinocarum, and perfected the mature fruit characteristics of these species. Through the analysis of comprehensive morphological data, the five Sinocarum species can be divided into three groups. Group 1 includes S. coloratum, S. cruciatum and S. vaginatum. They are characterized by slender and glabrous plants, usually ovate or oblong-ovate sheath, mostly absent bracts and bracteoles, typically entire petals, ovoid or oblong-ovoid mature fruit with 1-3 vittae in each furrow and 2-4 on commissure, sub-pentagon transection and flat endosperm concrescence. And the pollen grains of these three species are super-rectangular in equatorial view, trilobate circular in polar view. Group 2 includes S. filicinum, whose morphological characteristics were significantly different from those of other Sinocarum species we collected, and the key identification features were the linear-lanceolate bracts and bracteoles, oblong-ovate blade ultimate segments with serration on the margins, sparsely pubescent petioles, rachides and the abaxial surface of segments. Group 3 includes S. schizopetalum, whose most prominent features are apex palmately 3-4-lobed and white or violet petals, broad-ovoid mature fruits and sub-rhomboidal pollen. Among them, petal characteristics are very special in the whole genus Sinocarum. It is concluded that plant morphology, fruit morphological and anatomical characteristics, and palynology have important taxonomic significance.

Phylogenetic placement of Sinocarum
Previous studies have shown that Sinocarum is not a monophyletic group and the phylogenetic placement remains unclear (Valiejo-Roman et al. 2002;Zhou et al. 2008;Zhou et al. 2009;Downie et al. 2010). S. coloratum is the generic type of Sinocarum, its phylogenetic placement represents the phylogenetic placement of Sinocarum. In this study, we confirmed that Sinocarum is not a monophyletic group and used three sequences of S. coloratum (MN846685, AY328927, FJ385063), our sequenced specimen and two downloaded sequences, to determine the phylogenetic placement of Sinocarum. We found that one of the downloaded (FJ385063) was located in the East Asia clade, while our accession and the other downloaded accession were located in the Acronema clade but these two were not clustered together. The results of our field investigation, morphological study and specimen verification of S. coloratum obtained from the type locality (Mt. Yulong) showed that our collected material is highly consistent with the type specimen and the original literature description of S. coloratum.
In conclusion, the true phylogenetic placement of Sinocarum is within the Acronema clade and the genus has a close affinity with Acronema.

The relationship between Sinocarum and Acronema
This study's phylogeny results indicated that there was a close and complex relationship between Sinocarum and Acronema. Fusiform or elongate roots and apex slightly obtuse or rarely lobed petals are easily recognizable characteristics of Sinocarum, and an apex long-linear or long-aristate petal is the most prominent feature of Acronema. In fact, within each genus there are species that deviate in one or more morphological characteristics from the typical and the generic boundaries are blurred with a few species being easily confused as belonging to the other genus (Watson 1996;Watson et al. 2004;Pu et al. 2005). For example, S. cruciatum has torulose roots and several species of the genus Acronema, A. chienii R. H. Shan & S. L. Liou (1980: 197), A. chinense H. Wolff (1926: 309) have apex acute or obtuse-acute petals, characteristics typically observed in the other genus. Through a literature review, field investigation, morphological study and specimen examination, we found that the plants of Sinocarum and Acronema are all slender. In addition, Sinocarum and Acronema are both distributed in the high-elevation Sino-Himalayan region from Nepal to SW China. The habitat of the two genera is extremely similar as they are distributed in the humid environment of rock crevices, alpine meadows or shady forests. These conditions provide further evidence for the close and complex affinity between the two genera. This study and others have provided cumulative evidence to reduce phylogenetic uncertainty. Despite recent collections, the range of materials for the two genera is still limited and more field specimens will be required to provide a comprehensive revision of the phylogeny of Sinocarum and Acronema across their geographic range.

Infrageneric relationships
Our ITS and cpDNA trees showed that S. coloratum (generic type), S. cruciatum and S. vaginatum clustered together, but S. cruciatum had a closer relationship with S. vaginatum, which was consistent with the results of morphological study. The closer relationship between S. cruciatum and S. vaginatum is supported by the ultimate segments of their blades being more slender than other Sinocarum species and forming a group of narrow-leaved taxa. However, S. vaginatum develops elongate-linear ultimate segments of basal leaves and cauline leaves, and more rays, about 10-12. Whereas S. cruciatum has subequal rays and torulose roots. These two species are recognizable by these major features. In addition, S. cruciatum and S. vaginatum were both collected from the Dali range, Dali, Yunnan, overlapping in their ranges. Consequently, the morphological evidence and geographical distribution are consistent with the phylogenetic analysis results.
Sinocarum filicinum H. Wolff (1929: 182) was originally described by Wolff (1929) based on G. Forrest n. 6863, 11691, 7230, and obtained from the eastern flank of the Dali Range in Yunnan. Since the description of S. schizopetalum, its phylogenetic placement has been controversial. Franchet (1894) originally described it as a new species as Carum schizopetalum Franch., and Wolff (1927) transferred it to Sinocarum, later Wu Zhengyi (1984) transferred this species to Dactylaea H. Wolff (1930: 304) (2017) also accepted Wolff's view that this species belongs to Sinocarum after studying the specimens of Apiaceae, especially the type specimens. In addition, no molecular phylogenetic studies have been carried out on S. filicinum and S. schizopetalum.
The results showed that the two populations of S. filicinum were not related to S. coloratum (Sinocaurm generic type) and allied most closely with the genus Meeboldia, according to the ITS and cpDNA trees. Our morphological results indicated that the morphological characteristics of S. filicinum are distinct from the three other Sinocarum species in the Acronema clade that we collected and are very consistent with the characteristics of Meeboldia. Among them, fruit characteristics play a key role of subfamily Apioideae classification (Kljuykov et al. 2004;Lyskov et al. 2017;Guo et al. 2018;Jia et al. 2019). And the fruit characteristics of S. filicinum are similar to those of Meeboldia yunnanensis, they are all ovoid, with 5 filiform inconspicuous ribs, 2-3 vittae in each furrow and 4 on commissure, semicircle transection of mericarp and cordate concave or sub-cordate endosperm concrescence ( Fig. 2A, D). The molecular data and morphological evidence indicated that S. filicinum is closely related to Meeboldia and should be isolated from Sinocarum, but due to the lack of comprehensive samples, the phylogenetic placement will not be revised at present.
Our phylogenetic results showed that S. schizopetalum was distantly related to the other Sinocarum species we collected (S. coloratum, S. cruciatum, S. filicinum and S. vaginatum). We found that the exact phylogenetic placement of S. schizopetalum was inconsistent between the ITS tree and the cpDNA tree, but was nevertheless located in the East Asia clade. Morphologically, S. schizopetalum has apex palmately 3-4-lobed petals, broad-ovoid mature fruits and sub-rhomboidal pollen, and these features are clearly distinct from other species of Sinocarum. Unlike the other studied Sinocarum species, the plant morphology, fruit and pollen morphology of S. schizopetalum are more similar to species of the East Asia clade. According to the results of phylogeny and morphology studies, it is suggested S. schizopetalum should be isolated from Sinocarum. However, due to the complex taxonomic problems among genera in the East Asia clade, the phylogeny of S. schizopetalum cannot be resolved. Thus, S. schizopetalum needs revision pending expanded sampling and phylogenetic analyses to include more East Asia clade species from the Sino-Himalayan region.