Taxonomic revision of Bergera J.Koenig ex L. (Rutaceae) based on the molecular phylogeny and morphology

. Several phylogenetic studies have revealed that Murraya s. lat. is not monophyletic, suggesting that it should be split into two genera, namely Murraya s. str. and Bergera J.Koenig ex L. Here, the genus Bergera is comprehensively revised based on molecular and morphological data. The genus consists of 11 species in total, including all species previously placed in Murraya sect. Bergera and an additional species previously placed in Murraya sect. Murraya . Ten new combinations are proposed, namely Bergera alternifolia (Kurz) F.J.Mou comb. nov., B. crenulata (Turcz.) F.J.Mou comb. nov., B. euchrestifolia (Hayata) F.J.Mou comb. nov., B. glabra (Guillemin) F.J.Mou comb. nov., B. kwangsiensis (C.C.Huang) F.J.Mou comb. nov., B. macrophylla (C.C.Huang) F.J.Mou comb. nov., B. microphylla (Merr. & Chun) F.J.Mou comb. nov., B. siamensis (Craib) F.J.Mou comb. nov., B. stenocarpa (Drake) F.J.Mou comb. nov., and B. tetramera (C.C.Huang) F.J.Mou comb. nov. An identification key of the 11 species and morphological descriptions are provided, as well as illustrations of diagnostic characteristics. Phylogenetic relationships of the species in Bergera are discussed.


Introduction
The genus Bergera J.Koenig ex L. was first published based on Bergera koenigii L., while the genus Murraya J.Koenig ex L. was founded based on Murraya ("Murraea") exotica L. (Linnaeus 1771).Later, Bergera koenigii was transferred to Murraya by Sprengel (1825), which was accepted by most subsequent researchers.Though the generic name Chalcas L. was earlier described by Linnaeus (1767) based upon C. paniculata L. of Caminium of Rumphius (Rumpf 1747), most people accepted the name Murraya and it was proposed to be conserved by Mabberley (2016Mabberley ( , 2017)).Based on several flower characteristics, Chinese Academy of Sciences) and SWFC (Herbarium, Southwest Forestry University).Morphological characteristics were obtained and photographed in the field and laboratory studies, such as paraffin section of ovaries.The measurements were done under a microscope using a vernier calliper and a ruler.In the morphological description, most data are based on at least 20 measurements for the leaf, flower, and fruit characteristics.

Taxa sampling
The sampled species were from the genus Bergera in China and Vietnam, along with five species from Clausena.Some species have multiple accessions that were sampled from different populations due to their prominent morphological differences.Two species from Merrillia and Murraya were selected as outgroups based on the phylogeny of the tribe Clauseneae Wight & Arn.(Mou et al. 2018).The list of taxa and vouchers is provided in Table 2.

DNA extraction, amplification, and sequencing
Fresh leaves or silica-dried leaves collected in the field or greenhouse were used to extract total genomic DNA.Considering high levels of polyphenol in the leaves of species of Bergera, leaf powders were preprocessed five times after being ground in liquid nitrogen before extraction, referring to the modified CTAB method (Doyle & Doyle 1987;Tian 2014).The primers of one nuclear marker (ITS) and five chloroplast markers (trnL-F, atpB-rbcL, matK, psbH-petB, and psbA-trnH) for amplification are listed in Table 3.
The PCR amplification was performed in 25 μl volumes, with the following PCR protocol: an initial denaturation step at 95°C for 5 min, followed by 38-40 cycles of denaturation at 95°C for 1.0-1.5 min, primer annealing at 53-58°C for 1.0-1.5 min, and DNA strand extension at 72°C for 1.5 min, with a final extension of 8 min at 72°C.The PCR products were subjected to electrophoresis in 1.2% agarose gels.The PCR products and the PCR primers were used for cycle sequencing reactions with BigDye ver.3.1 in 10 µl volumes.Cycle sequencing conditions started at 96ºC for 1 min, followed by 30 cycles of 96ºC for 10 sec, 50ºC for 5 sec, 60ºC for 2 min, and a final extension of 60ºC for 2 min.

Sequence assembly, alignment, and phylogenetic analysis
The data set for phylogenetic analysis of ITS contained 29 accessions, including eight species of Bergera, five species of Clausena, Murraya elongata DC. ex Hook.f. and Merrillia caloxylon Swingle downloaded from GenBank; the data set for five cpDNA markers comprised 31 accessions, in addition to B. siamensis (Craib) F.J. Mou (syn. Murraya siamensis Craib).Most sequences in this study were generated anew and a few sequences were downloaded from GenBank, including Bergera siamensis, five species of Clausena, and Merrillia caloxylon (Samuel et al. 2001;Bayer et al. 2009;Morton 2009).A full list of GenBank accessions is provided in Table 4.
Base confirmation and editing of raw sequences were performed using Sequencher ver.4.9 (Gene Codes Corp., Ann Arbor, MI, USA).Sequences were aligned by ClustalW in MEGA ver.7.0.21(Kumar et al. 2016) and adjusted manually where needed.Some accessions are relatively incomplete and have extensive missing data due to the fact that sequencing was not successful for some fragments using the available herbarium specimens, or sequences were not available from GenBank for all six DNA regions.Those were encoded as missing data in the matrix.Five cpDNA alignments were concatenated using the software MEGA ver.7.0.21.The incongruence length difference (ILD) test (Farris et al. 1995) was used to test for incongruence between the genes.
Bayesian inference (BI) and Maximum likelihood (ML) methods were used to reconstruct the phylogenetic trees based on data matrices containing the combination of ITS and five cpDNA markers, using MrBayes ver.7 (Ronquist & Huelsenbeck 2003) and PhyML 3.0 (Guindon et al. 2010), respectively.For BI analysis, two independent Markov Chain Monte Carlo (MCMC) runs were conducted simultaneously, each with four linked chains, for 1 000 000 generations, sampling one tree every 100 generations, and starting with a random tree.PhyML analysis was conducted with automatic model selection by SMS (Lefort et al. 2017), and BioNJ start tree (http://www.atgc-montpellier.fr/phyml/).Bergera macrophylla-550

Phylogenetic analyses of Bergera
The phylogenetic trees inferred from the ITS and five cpDNA data showed that species of Bergera formed a strongly supported monophyletic group, sister to Clausena (Fig. 1

Description
Shrubs or small trees.Unarmed trees; stems and root bark dark-brown.Leaves are odd-pinnate, with alternate leaflets or unifoliolate; leaflet margins entire or crenate.Cymes are axillary or terminal, with many flowers.Flower buds are cylindrical or long-ovoid; flowers small, 4-or 5-merous; calyxes 4-or 5-lobed, and ovate or lanceolate; sepals united at the base or only in the lower third; petals 4 or 5, less than 8 mm long, lanceolate or linear, and imbricate.Stamens are free, 8 or 10, and elongate; filaments dilated, alternately unequal in length; anthers small, broadly elliptic or oval.Disks are short, annular, and cushion-shaped or cylindrical.Ovaries are ovoid, with 2 locules, each with 2 or 1 superimposed or almost collateral ovules; styles rather long and slender, deciduous in fruit or sometimes basal portion persistent; stigmas capitate.Berries are small, ovoid or subglobose, with mucilaginous pulp.Seeds are medium-sized, glabrous with membranous testa; cotyledons with plano-convex oil glands.

Etymology
The specific epithet refers to the leaves with alternate leaflets.

Phenology
Flowering in May, while fruiting from July to August.

Remarks
The species is unique in the genus Bergera and differs from all other species in this genus by deciduous leaves, narrowly winged rachis, pubescent filaments, and the ovary borne on a slender, nearly cylindrical stipe, which looks like the gynophores found in Clausena (Swingle & Reece 1967).A very strong smell comes out from the leaves of this species and is very similar to B. koenigii.It is thought that the fresh juice of this species' leaves can neutralize the toxic action of snake venoms from cobra or viper (Aye et al. 2001;Min et al. 2004Min et al. , 2007)).

Etymology
The specific epithet refers to the leaves with crenulate margins.

Phenology
Flowering between May and July, while fruiting from August to January next year.

Remarks
The In Vanuatu, the fruits are considered edible.The whole plant is also used in folk medicine (Mota Lava, Banks Islands, and north of Vanuatu).In New Caledonia, maceration of the plant is used against migraines and headaches (Lifou Island).The crushed leaves have a very pleasant and characteristic smell; the vernacular name in Lifou means 'scent like honey'.The flowers are worn by traditional dancers to leave a pleasant smell in Anatom, the southernmost island of Vanuatu, and in the past the leaves were used for the confection of scented costumes for men in Maré Island, New Caledonia.On Ouvéa Island (New Caledonia), a mixture of Murraya crenulata bark and grated coconut is used as a shampoo (Hnawia et al. 2007).

Etymology
The specific epithet refers to the glabrous leaves.

Phenology
Flowering between July and August, while fruiting from November to December.

Remarks
This plant most closely resembles Bergera crenulata and B. tetramera in appearance, but the calyx lobes are acutely triangular, and the leaflets are not so oblique, lacking crenulation.In this respect, it approaches B. koenigii more closely, although the number of leaflets is fewer and the leaflets are larger and glabrous.
It differs from both species in having a smaller flower, an obscure stigma, and a slightly constricted ovary.

Etymology
The specific epithet refers to the glabrous leaves.Ovaries are cylindric, warty, glabrous, distinctly constricted at ca ⅓ from the apex; styles longer than ovary, elongated, with prominent dots; stigmas globose, yellow, scabrous, and ca 3 mm long.Berries are subglobose, diameter 7-9 mm, rough, with glands, red when ripe, and containing 1-2 seeds.This species is described based merely on Swingle & Reece (1967) and some images of specimens and plants in Vietnam.

Phenology
Flowering between April and June, while fruiting from July to October.

Distribution and habitat
Vietnam: Tonkin, Annam; Lang Son, Quang Ninh, Ninh Binh, Hoa Binh, Thanh Hoa, Vinh Phuc, Quang Tri, Hai Phong (Cat Ba, the National Park Cat Ba); Nature Reserve Hang Kia-Pa Co (Peace); Ha Nam, Hoa Binh, Quang Tri, Da Nang.It is common among limestone rocks, often grows wild in clumps and drought-resistant.

Remarks
The leaves and leaflets of this species are the longest and widest in the genus Bergera.All parts of the plant, especially the leaves and fruits, are strongly aromatic and dispersed with very prominent oil glands becoming black dots when drying.or crenulate, glabrous, midrib often puberulous.Inflorescences are terminal, many flowered.Flowers are small, white, 5-merous, and ellipsoid in the bud; sepals ovate, less than 1 mm; petals white, oblanceolate to oblong, 5-7 mm long.Stamens are alternately shorter, 10.Ovaries are 2-celled, styles short and thick; stigmas capitate.Fruits are blueish black when mature, ovoid to oblong, 1.0-1.5 cm; 1-or 2-seeded.
Seed coats are membranous.

Phenology
Flowering between March and April, while fruiting from July to August.

Remarks
The natural distribution is somewhat uncertain in that it is widely cultivated and naturalized as the indispensable 'curry leaf' in the Indian subcontinent.Curry leaves are highly aromatic when rubbed or bruised.The species can be distinguished by the more numerous, more pubescent and small leaflets, and more compact corymbose terminal inflorescence and the pistil is much shorter than the stamens.

Etymology
The specific epithet is derived from the type locality, Guangxi in China.

Phenology
Flowering between June and July, while fruiting from October to November.

Remarks
The leaves and fruits of this species have a strong, pleasant scent.

Etymology
The specific epithet refers to the large leaves.

Phenology
Flowering between June and July, while fruiting from August to December.

Distribution and habitat
China, SW Guangxi (Longzhou, Daxin, Jingxi, and Napo); Vietnam.Growing in forestry of limestone hill or valley forests between alt.550 and 1400 m.

Remarks
The species Bergera macrophylla shares few similarities of flowers with B. kwangsiensis, but both are so different in many morphological characteristics (Mou et al. 2020).However, the taxon is more similar to B. glabra from Vietnam in leaf morphological characteristics, and different having fewer oil glands in the mesophyll, smaller cymes, fewer flowers and fruits.

Etymology
The specific epithet refers to the small leaflets.

Description
Shrubs or small trees, up to 6 m.Branches are glabrous; branchlets with a dispersed white pubescence.

Phenology
Flowering between April and May, while fruiting from July to October.

Remarks
This species is very close to B. koenigii on some flower and fruit morphological traits, but with much smaller leaflets.

Etymology
The specific epithet is derived from the type locality, Thailand (namely Siam).

Phenology
Flowering between March and April, while fruiting from May to August

Distribution and habitat
The species is only found in Thailand (Lampang) in forestry between alt.50 and 250 m.

Remarks
Although a few researchers reduced Bergera siamensis (syn.Murraya siamensis) to a synonym of Bergera koenigii (Esser 2021;Govaerts et al. 2021), it is an independent species based on both phylogenetic relationship and morphology.Although this species Bergera siamensis is closely allied with B. koenigii based on some morphological characteristics, the dots on the petals are nearly indistinct, filaments are less subulate, petals much narrower, and fruit much larger and 4-grooved; the pubescence of the plant is more pronounced than B. koenigii, and sometimes the whole plant is covered with white curved hairs.
Many volatile oil glands were seen in its leaves (Inpuron et al. 2013).It has a specific volatile smell and the crushed leaves are aromatic.In Thai folkloric medicine, it has been used for the treatment of blurred vision (eye sores), snake-poison and tuberculosis (Likhitwitayawuid et al. 1999).

Etymology
The specific epithet refers to the narrow fruits.

Description
Small shrub, up to 1.0-1.5 m high.Branches are grayish brown, short pilose.Leaves unifoliate, rarely 2-foliolate; petiole 3-4 cm, with a swelling at the base of the leaf blade; leaflet blades 8-15 × 3-6 cm, subcoriaceous, shining, elliptical or oblong-lanceolate, acute at the base, narrowed at the apex, rounded to obtuse or retuse to subemarginate, and margin evident teeth or serrate; secondary veins 10-12 pairs, pilose at veins in both sides, midvein slightly ridged; oil glands easily visible principally on the margins and in the angles of the teeth; petiolule of leaflet 5 mm, pilose.Cymes are axillary, 2 cm, (2-or) several flowered; pedicel pilose.Flowers are small; calyxes 5-lobed, short, triangular, and 0.5 mm long; petals 5, white, glabrous, oblong, 3-4 mm, and with pellucid dots on the surface.Stamens are 10, filaments free, 5 long and 5 short, dilated at the base and abruptly pointed at the tip, and pilose.Ovaries are subglobose, glabrous, borne on a low disk, 2-ovuled, and each with 1 ovule.Berries are fleshy, oblong, 15 mm long, 1 cm in diam., shortly attenuated at the tip, and with many evident oil glands; reddish or orange when mature.The species is described based merely on Swingle & Reece (1967) and some images of specimens and plants in Vietnam.

Phenology
Flowering between September to December, while fruiting from March to April (next year).

Remarks
This species is characterized by the unifoliate leaves, which is familiar to other species Bergera unifolia.

Material examined
Seed coats are membranous and smooth.

Phenology
Flowering between March and April, while fruiting from July to August.

Remarks
Morphologically, this species is relative to B. euchrestifolia, but the plants are characterized with heavier smell and flowers are 4-merous stably.Two species have different distributions.

Etymology
The specific epithet refers to the unifoliate leaves.

Description
Shrub, up to 1 m high.Spineless, glabrous; oil glands prominent and easily visible principally on the young branches, rachis, and petiolule, as well as along the margins and in the mesophyll.Young branches are green, becoming grey-brown when old.Leaves are unifoliolate; rachis ca 2.5-5.5 cm long and swelling at the base; petiolule more swollen than rachis and 4-5 mm long; leaflets subcoriaceous, shining above and hairy underneath, and elliptical or oblong-lanceolate; leaf blades 7-12 cm long, 3-6 cm wide, acute cuneiform at the base, narrowed attenuate and slightly emarginate at the tip, and obtuse or subemarginate, serrate; with 10-15 pairs of lateral veins and clearly distinct from the veinlets and elevated above the surface below.Cymes are both axillary and terminal and up to 50-flowered.Flower buds are small, 2.5-3.5 × 2.0 mm; calyxes 5-lobed, short, ovate, acute, and 0.8-1.3× 0.5-0.9mm; petals oblong, 2.5-3.5 × 0.5-1.0mm wide; pellucid dots on the surface of the petals and calyx lobes.Stamen filaments are free, 10, alternately unequal in length, ciliate, 2.5 mm and 3 mm long, respectively, dilated at the base and abruptly pointed at the tip; anthers not large, pubescent.Pistils are 1.8-2.5 mm long; ovaries are oblong, yellow, substipitate, and glandular, borne on a low disc, with 2 each with 1 ovule; styles cylindrical, pubescent, and white.Fruits are fleshy, red, oval, and up to 15 mm long.

Phenology
Flowering between May and June, while fruiting from July to August.

Distribution and habitat
The species is only endemic to Dahua (Guangxi Province, China).It grows in limestone hills at alt. 500 m.

Remarks
This species is similar to B. stenocarpa with unifoliate leaves, but is distinguished easily from the latter by having smaller and thinner leaflets.The leaflets of this taxon are also close to B. kwangsiensis due to the hairs under the leaflets, oil glands and smell (Deng et al. 2021).In a previous study by But et al. (1986), the species B. alternifolia (syn.B. alternans) was included in Murraya sect.Murraya, but lacking more information of morphological or biochemical data.However, strong aroma from the leaves and flowers is very similar to that from B. koenigii (formerly placed in Murraya sect.Bergera); meanwhile, the leaves and branches of B. alternifolia get slightly brown after drying in a shady and well-ventilated area.Although a new dihydrofuranocoumarin was found in B. alternifolia (Aye et al. 2001;Min et al. 2004Min et al. , 2007)), it is a special species in Bergera, based on morphological data, and needs further studies.
Our phylogenetic analyses inferred from ITS and cpDNA data reveals that Bergera koenigii, B. siamensis and B. microphylla form a clade, which is in accordance with morphological data.Based on flower and fruit characteristics, Bergera microphylla is very close to B. koenigii, but has much smaller leaflets.Our molecular data also support these morphological affinities between B. microphylla and B. koenigii.Most organs and sometimes the whole plant of B. siamensis are densely covered with the white curved hairs.

Table 1 .
The morphological and biochemical differences between two genera Murraya J.Koenig ex L. and Bergera J.Koenig ex L.

Table 2 .
Voucher information of Bergera J.Koenig ex L. and Murraya J.Koenig ex L. species in phylogenic studies.

Table 3 .
The sequence of all primers in phylogenic studies.

. Fig. 2. Bayesian
tree, inferred from cpDNA markers of representatives of Bergera J.Koenig ex L., Clausena Burm.f., Murraya elongata DC. ex Hook.f. and Merrillia caloxylon Swingle.Branch lengths are proportional to the number of nucleotide changes (indicated above branches with Bayesian posterior probabilities, PP); bootstrap support (BS) values for maximum likelihood (ML) are given below the branches.
species Bergera crenulata is allied to B. koenigii, but has fewer, thicker, and larger leaflets, attached to a glabrous instead of a pubescent rachis.The fruit is subglobose and minutely apiculate, whereas in B. koenigii it is short-ellipsoid and apiculate.Meanwhile, B. crenulata is often confused with Clausena anisum-olens Merr.and Micromelum minutum var.curranii (Elmer) Tanaka in the field, but it has longer flower buds and longer pistils, which are never found in Clausena or Micromelum.