Phylogeny of the Diploid Species of Rubus (Rosaceae)

Abstract

Rubus L. (Rosaceae, Rosoideae) contains around 700 species distributed on all continents except Antarctica, with the highest species diversity in temperate to subtropical regions of the northern hemisphere. The taxonomy of Rubus is challenging due to the frequency of polyploidy, hybridization and apomixis. Previous studies mostly sampled sparsely and used limited DNA sequence data. The evolutionary relationships between infrageneric taxa, therefore, remain to be further clarified. In the present study, genotyping by sequencing (GBS) reduced-representation genome sequencing data from 186 accessions representing 65 species, 1 subspecies and 17 varieties of Rubus, with emphasis on diploid species, were used to infer a phylogeny using maximum likelihood and maximum parsimony methods. The major results were as follows: (1) we confirmed or reconfirmed the polyphyly or paraphyly of some traditionally circumscribed subgenera, sections and subsections; (2) 19 well-supported clades, which differed from one another on molecular, morphological and geographical grounds, were identified for the species sampled; (3) characteristics such as plants with dense bristles or not, leaves leathery or papyraceous, number of carpels, instead of inflorescences paniculate or not, aggregate fruits and leaves abaxially tomentose or not, may be of some use in classifying taxa whose drupelets are united into a thimble-shaped aggregate fruit that falls in its entirety from the dry receptacle; and (4) a preliminary classification scheme of diploid species of Rubus is proposed based on our results combined with those from previous phylogenetic analyses.

1. Introduction

Rubus L. (Rosaceae; Rosoideae), a species-rich and economically important genus, is characterized by shrubs, subshrubs and herbs bearing stipules, usually prickles or bristles, and a compound fruit comprising an aggregation of drupelets. Species of Rubus are on all continents except Antarctica [1,2]. Except for a fraction of the species in the southern hemisphere, most species of Rubus occur in temperate to subtropical regions of the northern hemisphere, especially in temperate Eurasia and North America [3,4,5,6,7]. The exact species number of Rubus is unclear. The estimated number has ranged from ca. 200 species [8], ca. 437 species [9,10,11], 250–700 species [2], ca. 700 species [4,6,7] and 600–1000 species [12] to 900–1000 species [13]. Various species of Rubus are economically significant as fruit crops, ornamentals, invasive weeds and as pioneers in early forest succession [1,14].

The taxonomy of Rubus has historically been difficult due to morphological diversity and overlapping characteristics, apomixis, polyploidization and hybridization [1,5,15,16,17,18,19,20,21]. Morphologically, the species of Rubus exhibit tremendous diversity, with plants ranging from woody to semi-woody, trailing, erect or climbing shrubs or subshrubs to perennial creeping dwarf herbs. The leaves range from simple to palmately or pinnately compound with 3–11(–15) leaflets [1,7,15,22]. Rubus has a basic chromosome number of seven, and ploidy levels ranging from diploid to tetradecaploid (or octadecaploid) [5,13,20,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42]. Hybridization in Rubus occurs mostly between closely related species, but also in some cases between species classified in different subgenera [1].

The most recent and the only global taxonomic treatment of Rubus was made by Focke [9,10,11] based on morphological characteristics. Focke divided Rubus into 12 subgenera: Anoplobatus (Focke) Focke, Chamaebatus (Focke) Focke, Chamaemorus (Hill) Focke, Comaropsis (Rich. ex Nestl.) Focke, Cylactis (Raf.) Focke, Dalibarda (L.) Focke, Dalibardastrum Focke, Idaeobatus (Focke) Focke, Lampobatus (Focke) Focke, Malachobatus (Focke) Focke, Orobatus (Focke) Focke and Rubus (subg. Eubatus Focke). Two subgenera, Diemenicus A. R. Bean (including only Rubus gunnianus) [43] and Micranthobatus (Firtsch) Kalkm. [44], were subsequently described. Among them, the three largest subgenera are Idaeobatus, Malachobatus and Rubus, the first two occurring mainly in Asia (especially in China); subg. Rubus is in Europe and North America [5]. The raspberries, one of the economically important crops of Rubus, mainly belong to subg. Idaeobatus, while the blackberries are primarily included in subg. Rubus. The subgenera Anoplobatus, Dalibarda and Idaeobatus are predominantly diploid, whereas subgenera Dalibardastrum, Malachobatus and Orobatus are almost entirely polyploid, and subg. Chamaebatus, subg. Cylactis and subg. Rubus contain a small number of diploid members [13,32].

Molecular data have shown Rubus to be sister to the clade comprising the genus Rosa L., tribe Sanguisorbeae DC., tribe Potentilleae Sweet and tribe Colurieae Rydb. [45]. Further studies found Rubus to be monophyletic, whereas among the long-recognized subgeneric taxa, except for subg. Orobatus, all other subgenera were paraphyletic or polyphyletic [1,42,46]. Previously, although a diversity of molecular markers, including GBSSI-1, GBSSI-2, ITS, LEAFY, ndhF, PEPC, rbcL, rpl16, rpl20-rps12, rpl32-ndhF, trnK, trnL-trnF, trnS-trnG and trnV-ndhC [1,14,21,37,46,47,48,49,50,51,52,53,54,55], were used to infer the phylogeny of Rubus, most of those studies applied to a limited number of species and molecular loci or were restricted to taxa within administrative boundaries, resulting in low phylogenetic resolution and sometimes even giving inconsistent results. The evolutionary relationships between the species of Rubus are in need of further clarification.

The rapid development of next-generation sequencing enables researchers to use genome-wide polymorphisms from different species for phylogenetic analysis. Reduced representation sequencing methods, such as genotyping by sequencing (GBS), can provide thousands of single nucleotide polymorphisms (SNPs) and have been used for phylogenetic analyses of both closely and distantly related taxa [56,57,58,59,60,61,62,63,64,65].

Given that the polyploid species of Rubus have a complex evolutionary history, this paper focuses on the phylogeny of diploid species of Rubus. We inferred a molecular phylogeny of diploid species of Rubus with thousands of SNPs identified by the GBS approach to (1) vigorously test the monophyly of subgenera, sections and subsections of Rubus based on larger sampling; (2) identify major clades within diploid species of Rubus; and (3) build the molecular foundation for future taxonomic revision of Rubus.

2. Materials and Methods

2.1. Taxon Sampling

The taxa were chosen to include as many diploids species of Rubus as possible. In total, 186 accessions, representing 65 species, 1 subspecies and 17 varieties of Rubus, constituted our ingroup. Except for subg. Chamaebatus, our sampling covered all subgenera containing diploid species and the major geographical range of the diploid species of the genus. Diploid representatives of Fragaria L. (one accession representing one species, Fragaria daltoniana) and Rosa L. (three accessions representing two species, Rosa cymosa and Rosa luciae) were selected as outgroups based on previous findings that Rosa, Rubus and three tribes (Sanguisorbeae, Potentilleae (including Fragaria) and Colurieae) constitute the super tribe Rosodae in the subfamily Rosoideae of Rosaceae. Voucher information for the sampled taxon are provided in the Appendix A.

2.2. Genotyping by sequencing (GBS) Dataset

Total genomic DNA was extracted from silica-dried material using the CTAB method [66]. The DNA was quantified, and its quality was evaluated using the following methods: (a) Agarose gel electrophoresis to test DNA purity and integrity, (b) Nanodrop spectrophotometer to test DNA purity (OD260/OD280), (c) Qubit Fluorometer to measure DNA concentration.

A reduced representation GBS library was prepared as described by Elshire et al. [56], using the restriction enzymes MseI and HaeII, and sequenced (paired-end reads) using the Illumina HiSeq platform at Novogene Bioinformatics Technology Co., Ltd., Beijing, China.

The raw data (raw reads) were first processed by fastp version 0.23.1 [67] in a series of quality control (QC) procedures using standards as follows: (a) removal of reads containing adapters; (b) removal of reads with >50% bases having phred quality < 5; and (c) removal of reads with ≥10% unidentified nucleotides (N). The remaining high quality clean reads were mapped to the reference genome (chloroplast genome, Rubus coreanus [68]; whole genome, Rubus occidentalis [69]) using the software BWA version 0.7.8 [70] with the command “mem -t 4 -k 32 -M”. In order to reduce mismatch generated by PCR amplification before sequencing, duplicated reads were removed by the help of SAMtools version 1.3.1 [71]. After alignment, we performed SNP calling on a population scale using a Bayesian approach as implemented in the SAMtools package, and then genotype likelihoods from reads for each individual at each genomic location and the allele frequencies in the sample were calculated with a Bayesian approach. The “mpileup” command was used to identify SNPs with the parameters as “-q 1 -C 50 -t SP -t DP -m 2 -F 0.002”. The resulting SNPs were filtered by script with the parameters “dp 4, miss 0.5 and maf 0.01′” to obtain high quality SNPs for subsequent phylogenetic analysis.

2.3. Phylogenetic Analysis

The chloroplast and nuclear datasets were analyzed separately. Equally weighted maximum parsimony (MP) jackknife (JK) analyses [72] were conducted using 1000 random-taxon-addition replicates and tree-bisection-reconnection (TBR) branch swapping in PAUP* version 4.0a169 [73], with MAXTREES set to 10,000 and the removal probability set to approximately 37%, and “jac” resampling emulated analyses. Insertions and deletions were coded as missing data. Jackknife 50% majority-rule consensus trees were computed.

jModelTest 2 [74,75] was used to test the models of nucleotide substitution for maximum likelihood (ML) [76]. The Akaike information criterion (AIC) [77] was used to select among models instead of the hierarchical likelihood ratio test (hLRT), following Pol [78] and Posada and Buckley [79].

Maximum Likelihood (ML) analyses were performed using RAxML-HPC2 on XSEDE version 8.2.12 [80] on the CIPRES web server [81], with 1000 rapid bootstrap analyses followed by a search for the best-scoring tree in a single run [82]. The nucleotide substitution models “GTRCAT” and “GTRGAMMA” were chosen for the bootstrapping phase for the chloroplast dataset and nuclear dataset, respectively.

3. Results

3.1. GBS Data Analysis Summary

A summary of the sequence data generated from the samples studied is included in Supplementary Material Tables S1–S3. The 190 accessions, which consisted of 186 samples of Rubus and 4 samples of outgroups, were successfully sequenced with the Illumina HiSeq sequencing platform. The mean GC content was 37.85%, which is within the normal range. The sequencing data were high-quality (Q20 ≥ 96.19%, Q30 ≥ 89.54%), and subsequent analyses could be performed. After excluding the outgroups, the average sequencing depth of the Rubus samples ranged from 10.81 to 75.48, with an average of 25.21. The average degree of sequence coverage from the samples of Rubus was 14.51% for at least single-base coverage, while the average degree of coverage of the samples with at least 4-base coverage was 8.68%. A total of 7,293,296 SNPs (nuclear genome) and 8115 SNPs (chloroplast genome) were obtained according to detection with SAMtools software; 256,954 high-quality SNPs (nuclear genome) and 1848 high-quality SNPs (chloroplast genome) were obtained after filtration.

3.2. Phylogenetic Tree Based on SNPs of Nuclear Genome

Both maximum likelihood (ML) phylogeny and maximum parsimony (MP) jackknife (JK) analyses based on high-quality SNPs from the nuclear genome showed Rubus to be monophyletic (Figure 1).

ML bootstrap (BS) support and MP JK support are shown on the phylogenetic tree from the ML analysis. Based on our reconstructed nuclear phylogeny and in consideration of morphological characteristics and distribution information, 19 maximumly supported (ML BS = 100%, MP JK = 100%) clades of Rubus were identified in this study (Figure 1). The relationships among most of the 19 clades were also well supported.

The topologies and support values from ML and MP analyses were mostly similar except for the position of Clade XI. The ML analyses resolved Clade XI as sister to the lineage containing Clade XII–Clade XIX with maximum support values. In contrast, the MP analysis resolved Clade XI as sister to the remaining clades except for Clade I and Clade II; support values were also maximum.

Clade I plus Clade II were resolved as monophyletic with 77% ML BS and 94% MP JK support and as sister to the rest of the ingroup.

Except for Clade XI, the basal Clade I plus Clade II, and the predominately polyploid Clade XII, Clade XIII and Clade XIV, the remaining clades formed two strongly supported superclades. Superclade A contained Clade III–Clade X, and Superclade B consisted of Clade XV–Clade XIX.

3.3. Phylogenetic Tree Based on SNPs of Chloroplast Genome

The topologies from the ML and MP analyses were similar for the chloroplast phylogeny, which also showed Rubus to be a monophyletic group. However, the topologies and support values based on the chloroplast data were not identical to those based on the nuclear data (Figure 2). First, for the topology of the chloroplast tree, 13 (Clades II, III, IV, V, VI, VII, IX, X, XII, XIII, XIV, XV and XVII) of the 19 major clades identified from the nuclear data were reconfirmed, but the monophyly of 6 clades (Clades I, VIII, XI, XVI, XVIII and XIX) was not supported. Rubus lasiococcus did not cluster with Rubus pedatus. The accessions of Clade VIII identified by nuclear data were split into two clusters on the chloroplast tree, one of which (including Rubus eustephanos, Rubus hirsutus, Rubus rosifolius, etc.) was sister to Clade VI. Clade XVI clustered with a portion of the accessions in Clade XVIII (Rubus pseudopileatus var. glabratus, Rubus trijugus, etc.) and Rubus lasiostylus and R. lasiostylus var. villosus of Clade XIX. Species in the remainder of Clade XIX clustered with some of the accessions in Clade XVIII (Rubus biflorus, etc.). Second, the two superclades were not supported by the chloroplast data. Third, Clade III was sister to the lineage containing Clades XVI, XVIII and XIX, instead of the lineage containing Clades IV to X. Clade XIV nested within Clade XI and was not sister to Superclade B as identified by nuclear data. Clade XVII was more closely related to Clade XV than to Clades XVIII and XIX.

4. Discussion

4.1. Phylogenetic Incongruence between the Chloroplast and Nuclear Phylogenies

The monophyly of Rubus in its current circumscription was maximally supported in this study (Figure 1 and Figure 2) and consistent with the results of earlier studies [1,42,46], further confirming Rubus to be a natural group. Phylogenetic incongruence, however, was detected between the chloroplast and nuclear phylogenies as the topologies based on the chloroplast data were not identical to those based on the nuclear data. The discordance between the nuclear and chloroplast phylogenetic relationships may be due to incomplete lineage sorting, genetic introgression and hybridization.

4.2. Major Evolutionary Lineages within Rubus Revealed in This Study

Within the ingroups, the 186 accessions representing 65 species, 1 subspecies and 17 varieties of Rubus included in the current study were assignable to the following 19 well-supported clades (Figure 1). Those clades are also supported by morphological characteristics.

The clades (Figure 1) can be divided into five main groups and thus may represent several major evolutionary lineages within Rubus, which is somewhat consistent with the results of Carter et al. [42]. The first group, found primarily in North America and containing Clades I and II, represents the basal lineage of Rubus. The second group was composed of Clade XI and mainly includes members of Rubus bearing aggregate fruit that falls together with the receptacle. The third group consists of Superclade A, including Clades III to X. The fourth group contains Clades XIII and XIV and represents the polyploid species of Rubus. The fifth group consists of Superclade B, including Clades XV to XIX. Unfortunately, morphological homoplasy is common in Rubus, thereby making it challenging to distinguish Superclades A and B from the other groups. That is, morphological synapomorphies of the two superclades are unclear at present.

Clade I––Clade I, composed of R. lasiococcus and R. pedatus, was shown to be sister to Clade II with high support (ML BS = 77%, MP JK = 94%; Figure 1). R. lasiococcus is native to North America and R. pedatus occurs naturally in North America and NE Asia. Both species were included in R. subg. Dalibarda by Focke [9,11]. They are morphologically similar in their creeping herbaceous habit, unarmed stems, broad stipules and filiform filaments.

Clade II––Clade II contained two species, Rubus odoratus and Rubus parviflorus, and was sister to Clade I (Figure 1). The sister relationship between R. odoratus and R. parviflorus was consistent with the findings by Carter et al. [42]. The two species, both native to North America and included in R. subg. Anoplobatus by Focke [9,11], are distinguished from other species of Rubus by habit (erect, prickleless shrubs) and their simple palmately lobed or divided leaves. Our study, together with previous studies [1,42], provides strong molecular evidence that R. subg. Dalibarda, R. subg. Anoplobatus and R. subg. Chamaemorus occupy basal positions in the phylogenetic trees, which conflicts with Lu’s [5] hypothesis of R. subg. Idaeobatus being the most primitive group.

Clade III––Clade III contained Rubus arcticus subsp. stellatus and Rubus pubescens and was strongly supported (ML BS = 100%, MP JK = 100%) as sister to the lineage containing Clades IV to X (Figure 1). Clade III to Clade X together form the lineage Superclade A, the morphological synapomorphy of which is unclear at present. R. arcticus subsp. stellatus occurs in North America and the Russian Far East; R. pubescens is in North America. Morphologically, they are somewhat similar to the members of Clade I, but differ from the latter in having dilated, laminar filaments.

Clade IV––Clade IV, composed of eigh samples of Rubus ellipticus var. obcordatus and Rubus wallichianus, was strongly supported (ML BS = 100%, MP JK = 100%) as sister to the clade containing Clades V to X (Figure 1 and Figure 2). The species of Clade IV are endemic to Asia [6,7] and were included in R. subg. Idaeobatus sect. Idaeanthi ser. Elliptici sensu Focke [10,11]. The morphological synapomorphies of Clade IV are shrubs with sparse, curved prickles and dense, spreading reddish brown bristles and usually three-foliolate leaves.

Clade V––This well-supported clade contained only one species, Rubus peltatus of China and Japan. Clade V was sister to Clade VI with maximum support (Figure 1). R. peltatus differs from other species of Rubus in having peltate leaves. Our molecular analysis supported the treatment of Focke [10,11], Yu and Lu [83], Lu and Yu [6] and Naruhashi [84], who placed R. peltatus in the monospecific section R. subg. Idaeobatus sect. Peltati (Lu and Yu’s section and subsection, respectively, almost corresponding to Focke’s subgenus and section).

Clade VI––In our samples, Clade VI contained Rubus corchorifolius, Rubus chingii, Rubus glabricarpus var. glabricarpus and R. glabricarpus var. glabratus. The sister relationship between Clade VI and Clade V was well supported (ML BS = 100%, MP JK = 100%) in our analyses (Figure 1). The four taxa of Clade VI are distributed in Asia and were included in R. sect. Idaeobatus subsect. Corchorifolii by Lu and Yu [6]. Morphologically, species of Clade VI share the following features: flowers usually solitary, leaves simple, stipules adnate to base of petiole, stems usually pilose or glandular (except in R. chingii), aggregate fruit nearly globose and hairy or glabrous. The members of this clade are similar in appearance to taxa included in Clade VII, with the main difference being that the latter have three to several flowers in nearly corymbiform inflorescences and glabrous aggregate fruit. Clade VI can be divided into two subclades. The first contains R. corchorifolius and R. chingii. Species of this subclade lack glandular hairs and the fruits are hairy. Members of the second subclade, represented by R. glabricarpus var. glabricarpus and R. glabricarpus var. glabratus, have glandular hairy pedicels and glabrous fruits.

Clade VII––Clade VII comprised two species—Rubus crataegifolius and Rubus conduplicatus—in our sampling and was strongly supported (ML BS = 100%, MP JK = 100%) as sister to a monophyletic lineage composed of Clades VIII, IX and X (Figure 1). Species of Clade VII are morphologically similar to each other in their nearly corymbiform inflorescences with three-to-several flowers, simple leaves, stipules adnate to the base of the petiole, aggregate fruit nearly globose and glabrous.

R. chingii, R. conduplicatus (Rubus trianthus), R. corchorifolius, R. crataegifolius, R. glabricarpus, Rubus grayanus, Rubus microphyllus, Rubus palmatus and Rubus pseudoacer were all included in R. subg. Idaeobatus sect. Corchorifolii by Focke [10,11], Yu and Lu [83] and Lu and Yu [6], a classification different from the taxonomic treatment of Naruhashi [84] and Nakai [85,86], who further segregated those species into a group with solitary flowers (R. subg. Idaeobatus sect. Villosi sensu Nakai and sect. Corchorifolii sensu Naruhashi) and a group with nearly corymbiform inflorescences (R. subg. Idaeobatus sect. Crataegifolii sensu Nakai and sect. Microphylli sensu Naruhashi). Our data (Figure 1 and Figure 2) suggested that R. corchorifolius, R. chingii, R. glabricarpus and R. glabricarpus var. glabratus, all bearing solitary flowers, form a monophyletic lineage (Clade VI). R. crataegifolius and R. conduplicatus, both bearing nearly corymbiform inflorescences, formed Clade VII. However, Clade VI + Clade VII were not shown to be monophyletic; that is, our results supported the above mentioned classification of Naruhashi [84] and Nakai [85,86], but not those of Focke [10,11], Yu and Lu [83] and Lu and Yu [6].

Clade VIII––In our sample, Clade VIII contained R. eustephanos, R. hirsutus, R. rosifolius, Rubus sumatranus and Rubus cf. tsangii, and was strongly supported (ML BS = 100%, MP JK = 100%) as sister to the monophyletic lineage formed by Clades IX and X (Figure 1). Species of Clade VIII are mainly in Asia. Morphologically, they share a shrubby habit, imparipinnate leaves and ca. 100 or more carpels usually inserted on a stipitate torus. Our molecular evidence supported the classification of Focke [10,11], Yu and Lu [83], Lu and Yu [6] and Naruhashi [84], who placed these species in R. subg. Idaeobatus sect. Rosifolii as a natural group.

Clade IX––Clade IX is composed of four endemic Asian species—Rubus delavayi, Rubus macilentus, Rubus simplex and Rubus xanthocarpus. The sister relationship between Clade IX and Clade X was well supported (ML BS = 100%, MP JK = 100%; Figure 1 and Figure 2). Morphologically, species of Clade IX are characterized by subshrubs or herbs’ leaves with three (or five) leaflets, and the abaxial surface of the calyx is pubescent and with straight needle-like or curved minute prickles (except in R. macilentus where the calyx is unarmed). R. simplex and R. xanthocarpus were both previously included in R. subg. Cylactis by Focke [9,11] for their suffruticose or nearly herbaceous habit. Yu and Lu [83], Lu and Yu [6] and Lu and Boufford [7], noting that the two species are morphologically similar to some members of R. subg. Idaeobatus in non-paniculate inflorescences and that the abaxial surface of the leaflets and aggregate fruit were not tomentose, transferred them to R. sect. Idaeobatus sensu Lu and Yu [6]. Our study (Figure 1 and Figure 2) showed that R. simplex and R. xanthocarpus are more closely related to R. macilentus of R. subg. Idaeobatus than to R. pubescens of R. subg. Cylactis, which supported the classification of Lu and Yu [6] and Lu and Boufford [7].

Clade X––Clade X consisted of four samples of Rubus columellaris and two samples of Rubus impressinervus. It was resolved as sister to the monophyletic Clade IX with maximum support (Figure 1 and Figure 2). The two species are morphologically quite different from the other species of Rubus that we sampled in having approximately leathery leaves. Our study suggests that R. sect. Idaeobatus subsect. Leucanthi sensu Lu and Yu (including R. columellaris, R. delavayi, R. impressinervus, etc.) [6] is paraphyletic and implies that the species with leathery leaves in R. subg. Idaeobatus may be a natural group and that R. delavayi might be excluded from R. subg. Idaeobatus sect. Leucanthi.

Clade XI––Clade XI was composed of Rubus allegheniensis, Rubus argutus, Rubus canadensis, etc. ML analyses of nuclear data indicated that this clade is strongly supported (BS = 100%) as sister to the monophyletic lineage formed by Clade XII–Clade XIX (Figure 1). The morphological characteristics of this clade include shrubs being often prickly, leaves usually ternate, pedately or palmately and quinately compound, persistent narrow stipules which are mostly adnate to the base of the petiole, drupelets remaining on the fleshy receptacle at maturity or falling with the receptacle, or falling separately. Geographically, the members of this clade are mainly American and Eurasian. The eight species of this clade and Rubus caesius (4x) of Clade XIV were all included in R. subg. Eubatus (= subg. Rubus) by Focke [11]. Although the nine species clustered together based on chloroplast data (Figure 2), the phylogenetic analyses based on our nuclear data (Figure 1), ITS [1] and nearly one thousand low copy nuclear genes [42] suggested that R. subg. Rubus is not monophyletic.

Clade XII––Clade XII contained three samples of Rubus paniculatus, one of the few doubtfully diploid species reported to occur in the predominately polyploid R. subg. Malachobatus [27]. This clade was strongly supported (ML BS = 100%, MP JK = 100%) as sister to Clade XIII based on the nuclear data (Figure 1). The remarkable morphological characteristics of Clade XII include shrubs that are often prickly, stems erect, arching or climbing and broad, and free stipules that are caducous or persistent on the twig near the base of the petiole.

Clade XIII––Clade XIII, comprising the Asian endemics Rubus pentagonus var. pentagonus (tetraploid [42]) and R. pentagonus var. eglandulosus, was sister to Clade XII with maximum support (Figure 1). Species of Clade XIII are morphologically distinguishable from other sampled taxa by their shrubby habit and palmately compound leaves with three or five leaflets. The close phylogenetic relationship between R. pentagonus of subg. Idaeobatus and R. paniculatus of subg. Malachobatus is consistent with the findings of Wang et al. [46] and Carter et al. [42] and may provide some support to the hypothesis that R. pentagonus may be one of the possible progenitors of the subg. Malachobatus polyploids [42,46].

Clade XIV––Clade XIV consisted of two samples of R. caesius (Figure 1), a tetraploid of subgenus Rubus occurring naturally from Europe and western Asia to western China. Our chloroplast phylogeny showed that R. caesius was nested within Clade XI (ML BS = 100%, MP JK = 100%; Figure 2), indicating that the maternal parent of R. caesius was likely from R. subg. Rubus, which is consistent with the findings of Carter et al. [42].

Clade XV––Clade XV contained two North American species, Rubus leucodermis and R. occidentalis. The nuclear phylogeny indicated that Clade XV was sister to the lineage formed by Clades XVI through XIX (ML BS = 100%, MP JK = 100%; Figure 1). Clade XV to Clade XIX together form superclade B (ML BS = 100%, MP JK = 100%; Figure 1), the morphological synapomorphy of which is unclear at present. Morphologically, the two species of Clade XV share palmately compound or ternate leaves, nearly black aggregate fruit and drupelets separating from the torus. R. leucodermis, R. occidentalis and two species, Rubus eriocarpus and Rubus glaucus, which were not sampled, were all placed in R. subg. Idaeobatus sect. Idaeanthi ser. Occidentales by Focke [9,11]. In the molecular phylogeny the three diploid species, R. leucodermis, R. occidentalis and R. eriocarpus, were closely related, while the tetraploid R. glaucus aligned with some putative blackberry × raspberry hybrids [42], suggesting this series defined by Focke is not monophyletic.

Clade XVI––Clade XVI contained Rubus amabilis, Rubus maershanensis, Rubus cf. ptilocarpus, etc., in our sampling. It was sister to the lineage containing Clade XVII–Clade XIX with strong support (ML BS = 100%, MP JK = 100%; Figure 1). Species included in Clade XVI are mainly distributed in the Himalayan region. Morphologically they share the following features: shrubs or subshrubs (R. maershanensis), flowers solitary or inflorescences with few flowers (2–6-flowered) and aggregate fruit usually red, glabrous or pubescent, not tomentose. Two well-supported subclades were identified. The first subclade was represented by Rubus subornatus var. subornatus, R. subornatus var. melanadenus, Rubus sikkimensis, Rubus stans and R. cf. stans, all usually with three-foliolate leaves. The second subclade contained R. amabilis, R. maershanensis and R. cf. ptilocarpus and was characterized by having 7–11-foliolate leaves.

Clade XVII––Clade XVII, composed of Rubus pungens var. pungens, R. pungens var. ternatus and R. pungens var. oldhamii, was resolved as sister to the monophyletic lineage formed by Clade XVIII and Clade XIX with maximum support (Figure 1). Generally, species of Clade XVII are widely distributed from Kashmir to Japan and exhibit complex morphological variability: prickles dense to sparse, glandular hairs present or absent and size of leaflets unstable. However, they differ in appearance from the other species of Rubus we sampled: shrubs, stems longer, climbing or trailing, with dense or sparse needle-like prickles (R. pungens var. oldhamii sometimes nearly unarmed), vegetative reproduction mainly by rooting at apex of stem, three–nine-foliolate leaves, abaxial surface of calyx with needle-like prickles, inflorescences terminal or axillary, 1-flowered or corymbose 2- to 4-flowered.

Clade XVIII––Clade XVIII included R. biflorus var. biflorus, R. cf. biflorus var. adenophorus, R. biflorus var. pubescens, R. pseudopileatus var. glabratus, R. cf. pseudopileatus var. glabratus and R. trijugus and was sister to Clade XIX with maximum support (Figure 1). Species contained in Clade XVIII occur in Asia and have the following features: shrubs, branchlets usually pruinose, pedicels distally inflated, leaves thick papyraceous or semi-leathery, aggregate fruit yellow or reddish yellow and densely gray tomentose (tomentum deciduous in R. biflorus and its varieties), flowers 1.5–3 cm in diameter and one to several in corymbose inflorescences.

Clade XIX––Clade XIX was sister to Clade XVIII and contained 20 species and seven varieties (Figure 1). All taxa of Clade XIX are shrubs occurring mainly in Asia. They have pink or purplish red petals (Rubus mesogaeus and Rubus eucalyptus have white or pink petals), which might be interpreted as morphological synapomorphies for this clade. Clade XIX can be divided into four subclades (Figure 1). The first contains Rubus cf. cockburnianus, R. cockburnianus, R. eucalyptus and Rubus flosculosus var. etomentosus, etc. with medium support (ML BS = 89%, MP JK = 73%). Most species of this subclade, except R. eucalyptus and Rubus subinopertus, have nearly purplish black aggregate fruit. R. cf. cockburnianus, R. cockburnianus, R. flosculosus var. etomentosus, together with Rubus inopertus var. inopertus, R. inopertus var. echinocalyx, R. subinopertus, all with 5–11-foliolate leaves, formed a lineage which was sister to the lineage composed of R. eucalyptus, R. mesogaeus and Rubus subtibetanus, which have 3- or 5-foliolate leaves. The second subclade contained R. lasiostylus var. lasiostylus, R. lasiostylus var. villosus and Rubus wushanensis with maximum support. Species of this subclade have broader stipules and bracts that are ovate-lanceolate, ovate or suborbicular. The third subclade contained R. coreanus var. coreanus, R. coreanus var. tomentosus, Rubus niveus and Rubus thibetanus with maximum support. The species of this subclade have 5–11 (or 13)-foliolate leaves and corymbose inflorescences. The fourth subclade contained Rubus adenophorus, R. flosculosus and Rubus foliolosus, etc. Members of this subclade exhibit significant morphological variability: three-foliolate leaves or five- or seven-foliolate leaves, inflorescences nearly corymbose, racemose or paniculate and plants with or without glandular hairs.

According to Focke’s [10,11] classification, most of the species in our study belong to R. subg. Idaeobatus. Our study showed that members of R. subg. Idaeobatus sensu Focke [10,11] are distributed among 13 clades (Clades IV–X, XIII and XV–XIX, Figure 1). The results showing that subgenus Idaeobatus is not monophyletic are consistent with the findings of previous studies [1,21,42,46,47,50,52,53]. Species of R. subg. Idaeobatus sect. Pungentes sensu Focke [10,11] occurring in Clade IX (R. macilentus), Clade XVI (R. amabilis, R. maershanensis, R. sikkimensis, R. stans), Clade XVII (R. pungens), Clade XVIII (R. biflorus) and Clade XIX (R. eucalyptus, R. lasiostylus) indicated that this section is not monophyletic. Similarly, R. subg. Idaeobatus sect. Idaeanthi sensu Focke [10,11] is not monophyletic as species of this section occurred in Clade IV (R. ellipticus var. obcordatus, R. wallichianus), Clade XVI (R. subornatus) and Clade XIX (R. adenophorus, R. cockburnianus, R. flosculosus, Rubus idaeopsis, Rubus innominatus, R. inopertus, R. niveus, Rubus parvifolius, Rubus phoenicolasius, R. thibetanus). R. sect. Idaeobatus subect. Idaeanthi sensu Lu and Yu [6] (R. eucalyptus and R. niveus) is paraphyletic, and R. sect. Idaeobatus subect. Pungentes sensu Lu and Yu [6] is not monophyletic as members of the latter subsection occurred in Clades IV (R. wallichianus), IX (R. simplex, R. xanthocarpus), XVI (R. amabilis, R. maershanensis, R. sikkimensis, R. stans), XVII (R. pungens) and XIX (R. inopertus). R. sect. Idaeobatus subsect. Pileati sensu Lu and Yu [6] is not monophyletic as members of this subsection occurred in Clades XVIII (R. pseudopileatus var. glabratus) and XIX (R. subinopertus). R. sect. Idaeobatus subect. Stimulantes sensu Lu and Yu [6] is not monophyletic as species of this subsection occurred in Clades IV (R. ellipticus var. obcordatus), XVI (R. subornatus), XVIII (R. biflorus) and XIX (R. mesogaeus, R. parvifolius, R. phoenicolasius, R. thibetanus). R. sect. Idaeobatus subect. Thyrsidaei sensu Lu and Yu [6] is paraphyletic as species of this subsection were embedded in Clade XIX (R. adenophorus, R. cockburnianus, R. flosculosus, R. idaeopsis, R. innominatus). These non-monophyletic sections and subsections indicate that morphological characteristics, such as whether inflorescences are paniculate or not, the indument of the abaxial surface of leaves and aggregate fruits, are not suitable for the classification of R. subg. Idaeobatus.

4.3. A Preliminary Classification Scheme of the Diploid Species of Rubus

According to our results, combined with previous phylogenetic analyses [1,21,37,42,46,47,49,50,52,53], a preliminary classification scheme of the diploid species of Rubus (Figure 3) is proposed herein. R. paniculatus, representing R. subg. Malachobatus, was excluded since its ploidy level is uncertain and it was not verified in this study. Before making formal taxonomic and nomenclatural decisions on an infrageneric classification, more samples and molecular data are needed to unravel and confirm the relationships and evolutionary history of these clades.

A preliminary classification scheme of the diploid species of Rubus.

• Group 1

Rubus repens (L.) Kuntze (North America)

• Group 2

R. lasiococcus A. Gray (North America)

R. pedatus Sm. (NE Asia, North America)

• Group 3

Rubus deliciosus Torr. (North America)

R. odoratus L. (North America)

R. parviflorus Nutt. (North America)

Rubus trilobus Ser. (Mesoamerica)

• Group 4

Rubus nivalis Douglas (North America)

• Group 5

R. allegheniensis Porter (North America)

R. argutus Link (North America)

R. canadensis L. (North America)

Rubus canescens DC. (SW Asia, S and C Europe)

Rubus coriifolius Liebm. (Mesoamerican)

Rubus cuneifolius Pursh (North America)

Rubus hispidus L. (North America)

Rubus robustus C. Presl (South America)

Rubus sanctus Schreb. (N Africa, W Asia, SE Europe)

Rubus setosus Bigelow (North America)

Rubus trivialis Michx. (North America)

Rubus ulmifolius Schott (N Africa, Europe)

Rubus urticifolius Poir. (Mexico to S Tropical America)

• Group 6

R. arcticus L. (Eurasia, North America)

R. arcticus L. subsp. stellatus (Sm.) B. Boivin (North America, Russian Far East)

R. pubescens Raf. (North America)

• Group 7

Rubus hawaiensis A. Gray (Hawaii)

Rubus spectabilis Pursh (North America)

• Group 8

R. ellipticus Sm. (Asia)

R. ellipticus Sm. var. obcordatus (Franch.) Focke (Asia)

R. wallichianus Wight et Arn. (Asia)

• Group 9

R. peltatus Maxim. (China, Japan)

• Group 10

R. chingii Hu (China, Japan)

R. corchorifolius L. f. (Asia)

R. glabricarpus Cheng (China)

R. glabricarpus Cheng var. glabratus C. Z. Zheng et Y. Y. Fang (China)

R. palmatus Thunb. (Japan, Korea)

• Group 11

R. conduplicatus Duthie ex J. H. Veitch (China, Japan)

R. crataegifolius Bunge (E Asia)

Rubus trifidus Thunb. (Japan, Korea)

• Group 12

R. columellaris Tutcher (China, Vietnam)

R. impressinervus F. P. Metcalf (China)

• Group 13

R. delavayi Franch. (China)

R. macilentus Jacquem. ex Cambess. (Himalaya)

R. simplex Focke (China)

R. xanthocarpus Bureau et Franch. (China)

• Group 14

Rubus croceacanthus H. Lév. (E and SE Asia)

R. eustephanos Focke (China)

R. hirsutus Thunb. (E Asia)

Rubus illecebrosus Focke (Japan)

R. rosifolius Sm. (Asia)

R. sumatranus Miq. (Asia)

R. tsangii Merr. (China)

• Group 15

Rubus idaeus L. (Africa, Eurasia, North America)

Rubus strigosus Michx. (North America)

• Group 16

R. eriocarpus Liebm. (Mesoamerica)

R. leucodermis Douglas ex Torr. et A. Gray (North America)

R. occidentalis L. (North America)

• Group 17

R. amabilis Focke (China)

R. maershanensis Huan C. Wang et H. Sun (China)

R. sikkimensis Hook. f. (E Himalaya)

R. stans Focke (China)

R. subornatus Focke (China, Myanmar)

R. subornatus Focke var. melanadenus Focke (China)

• Group 18

R. pungens Cambess. (Asia)

R. pungens Cambess. var. oldhamii (Miq.) Maxim. (E Asia)

R. pungens Cambess. var. ternatus Cardot (China)

• Group 19

R. biflorus Buch.-Ham. ex Sm. (Asia)

R. biflorus Buch.-Ham. ex Sm. var. pubescens T. T. Yu et L. T. Lu (China)

R. pseudopileatus Cardot var. glabratus T. T. Yu et L. T. Lu (China)

R. trijugus Focke (China)

• Group 20

R. adenophorus Rolfe (China)

R. cockburnianus Hemsl. (China)

R. coreanus Miq. (E Asia)

R. coreanus Miq. var. tomentosus Cardot (China)

R. eucalyptus Focke (China)

R. flosculosus Focke (China)

R. flosculosus Focke var. etomentosus T. T. Yu et L. T. Lu (China)

R. foliolosus D. Don (Asia)

Rubus grandipaniculatus T. T. Yu et L. T. Lu (China)

R. idaeopsis Focke (China)

R. innominatus S. Moore (China)

R. innominatus S. Moore var. aralioides (Hance) T. T. Yu et L. T. Lu (China)

R. innominatus S. Moore var. kuntzeanus (Hemsl.) L. H. Bailey (China)

R. inopertus (Focke) Focke (China, Vietnam)

R. inopertus (Focke) Focke var. echinocalyx Cardot (China)

R. lasiostylus Focke (China)

R. lasiostylus Focke var. villosus Cardot (China)

R. mesogaeus Focke. (Asia)

R. niveus Thunb. (Asia)

R. parvifolius L. (E Asia, Australia)

R. parvifolius L. var. adenochlamys (Focke) Migo (China, Japan)

R. phoenicolasius Maxim. (E Asia)

Rubus subcoreanus T. T. Yu et L. T. Lu (China)

R. subinopertus T. T. Yu et L. T. Lu (China)

R. subtibetanus Hand.-Mazz. (China)

R. thibetanus Franch. (China)

R. wushanensis T. T. Yu et L. T. Lu (China)

Key to the Groups

1a. Flowers of two kinds, essentially petaliferous staminate flowers, usually with pistils abortive, fertile apetalous flowers and with few stamens; drupelets 5–10, nearly dry; low stoloniferous herbs with simple, unlobed, reniform leaves …………….…… Group 1

1b. Flowers of only one kind (in dioecious or polygamodioecious species only slightly dissimilar), mostly petaliferous, sometimes apetalous but bisexual; drupelets many or sometimes several, often fleshy; shrubs, subshrubs or herbs.

2a. Styles club-shaped; stigmas slightly 2-lobed; receptacle flat; unarmed shrubs with exfoliating bark and simple digitately ribbed and lobed leaves ……….………… Group 3

2b. Styles filiform; stigmas capitate; receptacle convex, hemispheric or nipple-shaped; stems unarmed, bristly or prickly; leaves simple or compound.

3a. Stem herbaceous, never prickly, rarely bristly; stipules broad, free or nearly so; floral branches arising directly from rootstock or from stolons.

4a. Filaments filiform, not dilated ……………………………………………… Group 2

4b. Filaments dilated, laminar ……………………………………………..…… Group 6

3b. Stem more or less woody, usually prickly; if unarmed or merely bristly, the stipules narrow and more or less adnate to the petiole.

5a. Stipules broad, nearly free, at junction of stem and petiole; plants with needle-like prickles or bristles; subshrubs, stems creeping ………………………………..…… Group 4

5b. Stipules narrow, linear-lanceolate or subulate, more or less adnate to the petiole; plants with or without needle-like prickles or bristles; shrubs, subshrubs or herblike.

6a. Drupelets remaining on fleshy receptacle at maturity or falling together with receptacle, or falling separately ………………………………………………..………..Group 5

6b. Drupelets united into a thimble-shaped aggregate fruit, falling off entire from dry receptacle.

7a. Flowers solitary or few, large, 2–3.5 cm in diameter; petals pink or magenta, much exceeding sepals; filaments laminar; leaves ternate, papyraceous ………..……… Group 7

7b. Flowers solitary or in inflorescences, small to large, if large (> 3 cm in diameter), the petals white; petals white, pink or purplish; leaves simple or compound, papyraceous or leathery.

8a. Plants with dense, spreading, reddish brown or purplish brown mostly eglandular bristles, 3–7 mm long; aggregate fruit golden yellow or reddish yellow; leaves three-foliolate ……………………………………………………………………………….… Group 8

8b. Plants usually without dense, spreading eglandular bristles; aggregate fruit color varied; leaves simple, 3–11 (or 13)-foliolate.

9a. Leaves leathery ……………………………………………………………… Group 12

9b. Leaves papyraceous or membranous.

10a. Leaves simple.

11a. Leaves peltate; carpels ca. 100 or slightly more; aggregate fruit terete or cylindrical …………………………………………………………………………….………..… Group 9

11b. Leaves not peltate; carpels ca. 10–60, rarely slightly more; aggregate fruit subglobose or ovoid-globose.

12a. Flowers solitary ………………………………………………………….… Group 10

12b. Flowers in nearly corymbiform inflorescences ……………………….… Group 11

10b. Leaves compound.

13a. Carpels ca. 100 or more, usually inserted on stipitate torus…………… Group 14

13b. Carpels 10–70 or slightly more, inserted on sessile torus.

14a. Subshrubs or nearly so; three-foliolate leaves; semi-leathery, terminal leaflet much or slightly longer than lateral leaflets; petals white; fruit orange (R. macilentus with orange or red fruit) …………………………………………………………………… Group 13

14b. Shrubs, if nearly subshrubs, leaflets more than three or petals pink or purplish; leaves 3–11-foliolate; petals white, pink, purplish; fruit red or black or sometimes orange.

15a. Stems scrambling or trailing, 1–3 m long, with dense or sparse needle-like prickles; abaxial surface of calyx with dense, straight needle-like prickles; leaves abaxially pubescent or nearly glabrous ……………………………………….…………….…… Group 18

15b. Stems mostly erect or arching, sometimes scandent or trailing, mostly without needle-like prickles; calyx unarmed or its prickles mostly not needle-like; leaves abaxially tomentose, pubescent or glabrous.

16a. Petals pink or purplish red (R. mesogaeus and R. eucalyptus with white and pink petals); mainly Asian, especially in China ………………………………………… Group 20

16b. Petals white (R. subornatus, R. subornatus var. melanadenus and R. sikkimensis with purplish red petals); occurring in Africa, Eurasia and North America.

17a. Aggregate fruit densely gray tomentose, yellow or reddish yellow; branchlets pruinose; pedicels distally inflated ………………………………………………… Group 19

17b. Aggregate fruit mostly glabrous or pilose, not gray tomentose, dark purplish, black, red, orangish red or sometimes yellow; branchlets pruinose or not.

18a. Aggregate fruit usually dark purplish or black; leaves palmately compound or ternate ………………………………………………………………………………… Group 16

18b. Aggregate fruit red, orangish red, or yellow; leaves pinnately compound or ternate.

19a. Native to Himalayan regions …………………………………………… Group 17

19b. Native to North America (R. strigosus), or Africa, Eurasia and North America (R. idaeus) …………………………………………………………………………….…… Group 15

5. Conclusions

The taxonomy of Rubus is challenging and the phylogenetic relationships within Rubus remain to be clarified. This study inferred the phylogeny of Rubus with emphasis on diploid species based on GBS data with comprehensive taxon sampling. Our results provided useful information for deducing the phylogeny of Rubus, especially providing important insights into the evolution of Rubus in China. We reconfirmed that R. subg. Idaeobatus, recognized by Focke, is not monophyletic. We found that characteristics such as leathery or papyraceous leaves may be of some use in classifying the raspberries. Based on our results, and combined with previous phylogenetic analyses, a preliminary classification scheme of the diploid species of Rubus is proposed here. Further studies, however, including more samples and additional molecular data, are still needed to better unravel and confirm the complicated evolutionary history of Rubus.