Phylogenetic Relationships Between Sedum L. and Related Genera (Crassulaceae) Based on ITS Rdna Sequence Comparisons

Home , Aeonium undulatum, Lenophyllum, Monanthes, Petrosedum, Prometheum, Sedella (plant)

Flora 224 (2016) 218–229

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Phylogenetic relationships between Sedum L. and related genera

(Crassulaceae) based on ITS rDNA sequence comparisons

a,∗ a b

Vyacheslav Yu. Nikulin , Svetlana B. Gontcharova , Ray Stephenson ,

Andrey A. Gontcharov

Institute of Biology and Soil Science FEB RAS, 100-letia Vladivostoka Prosp., 159, Vladivostok, 690022, Russia

Percy Gardens 8, Choppington, Northumberland, NE62 5YH, England, United Kingdom

a r t i c l e i n f o a b s t r a c t

Article history: Sedum is the most species-rich and taxonomically complex member of the family Crassulaceae. The genus

Received 26 May 2016

comprises ca. 420 species, which display notable diversity and homoplasy of growth forms, vegetative

Received in revised form 12 August 2016

and reproductive features traditionally used to delineate crassulacean genera therefore it is impossible

Accepted 12 August 2016

to characterize Sedum phenotypically. Artiﬁcial nature of Sedum was recognized long time ago and it

Edited by W. Durka

was characterized as a “catch-all” taxon. The phylogenetic structure of Sedum and its relationship with

Available online 23 August 2016

allied genera are poorly understood. Two hundred twenty three sequences of nuclear ribosomal internal

transcribed spacer (85 were obtained in this study) were used to address this question and provide a

Keywords:

Crassulaceae phylogeny-based framework for further taxonomic revisions. To identify positional homology between

Sedum divergent sequences, secondary structure models were generated and used to guide the alignment. Our

ITS rDNA molecular phylogenetic data corroborated results of previous studies based on other markers and datasets

Secondary structure and support the monophyly of the tribes Aeonieae, Semperviveae, and Sedeae. Our study is the ﬁrst to

Phylogeny resolve the clade of the tribe Semperviveae based on sequence comparisons, and the clade of the tribe

Sedeae with nrDNA. Sedeae accommodated majority of Sedum species either in the robust Acre clade

or paraphyletic Leucosedum cluster. Besides Sedum, these lineages comprised a number of genera that

were mostly embedded among Sedum species. The highly polyphyletic nature of Sedum and its relatives

corroborated in this study using a large taxon set, calls for re-evaluation of the genus concept in the

family Crassulaceae and the tribe Sedeae in particular.

1. Introduction One of the reasons for the poor status of Sedum taxonomy is

its extreme morphological diversity and homoplasy of phenotypic

The genus Sedum L. is the most species-rich member of the features traditionally used to delineate crassulacean genera. In fact

stonecrop family Crassulaceae DC., as well as the morphologically artiﬁcial nature of this genus was recognized long before the advent

diverse and taxonomically complex. It has signiﬁcantly reduced in of molecular phylogenetic era, and Sedum was characterized as a

the last 50 years, but it still encompasses ca. 420 species that con- “catch-all” taxon (Moran, 1942; Spongberg, 1978; Uhl, 1963). The

stitute one third of the family diversity (Thiede and Eggli, 2007). genus concept has changed signiﬁcantly since then by separating

Molecular data unambiguously demonstrate a polyphyletic nature a number of infrageneric taxa into genera, but it is still impossible

of the genus with species placed in four major crown clades of to characterize Sedum phenotypically (’t Hart and Bleij, 2005; Mort

the crassulacean tree, Acre, Aeonium, Leucosedum, and Sempervivum et al., 2001, 2010; Thiede and Eggli, 2007). Some of these segregates

(Gontcharova et al., 2008; van Ham, 1995; van Ham and ’t Hart, (e.g., Graptopetalum Rose, Dudleya Britton et Rose, Prometheum

1998; Mort et al., 2001, 2010). Moreover, at least nine mostly mor- (Berger) Ohba, Sedella Britton et Rose, etc.) show a close relationship

phologically distinct crassulacean genera are nested within Sedum. with Sedum species in phylogenetic analyses, thus refuting their

independent status or, more plausibly, the current Sedum concept.

There is no agreement between specialists regarding the infra-

generic structure of Sedum. Up to 30 sections have been recognized

in the genus (Alexander, 1942; Berger, 1930; Clausen, 1943; Fu,

∗

Corresponding author. 1965, 1974; ’t Hart, 1991; Jacobsen, 1974; Praeger, 1921; Uhl, 1980),

E-mail addresses: [email protected] (V.Yu. Nikulin),

of which some have been raised to the subgenus level (Clausen,

[email protected] (S.B. Gontcharova), [email protected]

1943, 1979). Fröderström (1930, 1931) proposed seven informal

(R. Stephenson), [email protected] (A.A. Gontcharov).

http://dx.doi.org/10.1016/j.ﬂora.2016.08.003

V.Yu. Nikulin et al. / Flora 224 (2016) 218–229 219

groups based on geographic distribution and the type of fruit. At more than one traditional genus. Monophyly was conﬁrmed for

least two of these were later described as sections, Filipes (Fröd.) S. only three genera: Lenophyllum, Thompsonella, and Pachyphytum

H. Fu and Oreades (Fröd.) K. T. Fu (Fu, 1965, 1974). Two subgenera (Carrillo-Reyes et al., 2009). Thus, Sedum remains poorly studied

are currently recognized: Gormania (Britton) Clausen and Sedum (’t phylogenetically. The branching pattern between its clades and lin-

Hart and Bleij, 2005; Thiede and Eggli, 2007). Gormania includes 110 eages remains largely unresolved, and only the Acre/Leucosedum

species found in the Aeonium, Sempervivum, and Leucosedum clades sistership is undisputable.

and mostly distributed in Europe, the Mediterranean region, and ITS rDNA data have been frequently used to access relationships

North America (van Ham and ’t Hart, 1998; Mort et al., 2001; Thiede in Crassulaceae at different taxonomic scales, from family-level

and Eggli, 2007). The subgenus Sedum accounts for 320 species phylogeny to subsection (Acevedo-Rosas et al., 2004; Carrillo-Reyes

comprising the Acre clade and occurring mostly in Asia (ca. 120 et al., 2008, 2009; Gontcharova et al., 2008; Kozyrenko et al., 2013;

spp.) and the Americas (ca. 170 spp.; van Ham and ’t Hart, 1998; Mayuzumi and Ohba, 2004; Nikulin et al., 2015), and a great deal

Mort et al., 2001). Gormania taxa are usually glandular-pubescent of sequences have been accumulated. However, despite consider-

with broadly sessile sepals of equal length and costate seeds, while able interest in the phylogenetic utility of the spacers, relatively

Sedum representatives are mostly glabrous with sepals that are free little is known about ITS molecular evolution in Crassulaceae in

at the base and spurred (unequal in length when broadly sessile), general and the genus Sedum in particular. High substitution rates

as well as reticulate-papillate to reticulate testa. However, there in the spacer may lead to saturation and homoplasy, and fre-

are discrepancies in the subgeneric assignment of some taxa (e.g. S. quent length mutations hinder homology assessment. Despite a

sedoides (Decaisne) Pau ex Vidal y Lopez, S. hispanicum L., S. gracile relatively rapid rate of primary sequence divergence, conserved

C.A. Meyer, etc.; ’t Hart and Bleij, 2005; Thiede and Eggli, 2007). structural elements within the secondary structures of ITS aid the

The phylogenetic structure of Sedum and its relationship with accurate alignment of homologous positions (Alvarez and Wendel,

allied genera are poorly understood. The genus was generally con- 2003; Coleman, 2003, 2007; Gottschling and Plotner, 2004; Mai

sidered as the most primitive and ancient member of the family and Coleman, 1997; Wolf et al., 2005). To clarify the structural evo-

(Schönland, 1891), but molecular phylogenetic studies refuted this lution of the ITS region in Sedum and address its phylogeny in a

hypothesis by placing its species in a crow assemblage of the more comprehensive way, the ITS regions for 85 accessions were

crassulacean tree recognized as the subfamily Sempervivoideae newly sequenced for this study and analyzed together with 138

(Thiede and Eggli, 2007). There, Sedum taxa comprise a bulk of sequences from Genbank. This widely and densely sampled dataset

the Acre and Leucosedum clades and are intermixed with mem- facilitated the process of alignment by providing many intermedi-

bers of other genera (tribe Sedeae). Several North African species ary sequences. The objectives of this study are 1) to test previously

branched paraphyletically at the base of the Aeonium clade (tribe proposed taxonomic and phylogenetic schemes of the crown cras-

Aeonieae) comprising Macaronesian genera Aeonium Webb et sulacean assemblage using ITS rDNA sequence variation and 2) to

Berth., Aichryson Webb et Berth., and Monanthes Haw. Sedum ser. derive an accurate RNA secondary structure model for this spacer

Rupestria Berger (7 species recognized as the genus Petrosedum region for Sedum.

Grulich by some students) forms a distinct subclade (or inde-

pendent clade) in the Sempervivum clade (tribe Semperviveae; 2. Materials and methods

Carrillo-Reyes et al., 2009; Gontcharova et al., 2008; van Ham and

’t Hart, 1998; Mayuzumi and Ohba, 2004; Mort et al., 2001, 2002). 2.1. Plant material

Leucosedum was one of the best supported clades in the anal-

ysis of chloroplast DNA restriction site data (van Ham, 1995; van The plant material was collected from the ﬁeld or received

Ham and ’t Hart, 1998). However, in all further studies based on from several private collections. The nomenclature of the taxa is

nucleotide sequence comparisons, it was resolved with no boot- according to ’t Hart and Bleij (2005). Supplementary material 1 lists

strap support (Mort et al., 2001) or as a paraphyletic assemblage the plant samples analyzed, including classiﬁcation, localities, and

(Gontcharova et al., 2008; Mayuzumi and Ohba, 2004). Other mem- EMBL accession numbers.

bers of this assemblage include species of Sedum subgen. Gormania

and some N. American (Dudleya and Sedella) and Eurasian genera 2.2. DNA extraction, ampliﬁcation and sequencing

(Rosularia (DC.) Stapf, Prometheum, and Pistorinia DC.). Leucosedum

is expected to accommodate ca. 200 species from ﬁve to seven tra- Total genomic DNA was extracted from fresh leaves using a

ditional genera that are still poorly studied with molecular tools. In DNeasy Plant Mini Kit (QIAGEN, Maryland, USA) following the man-

addition to Gormania species, Leucosedum may include members of ufacturer’s instructions. To amplify the complete ITS1–5.8S–ITS2

the subgenus Sedum as well (Gontcharova et al., 2008; Mayuzumi region, we used universal primer pairs 1400F (Elwood et al., 1985)

and Ohba, 2004; Mort et al., 2001), so its composition and structure and ITS055R (Marin et al., 2003) for the ﬁrst round of ampliﬁcation

remain tentative. and internal primers 18Sm10 (5 -AGGAGAAGTCGTAACAAGG-3 ;

With good support in all analyses, the Acre clade is expected modiﬁed from Wen and Zimmer, 1996) and ITS4R (White et al.,

to comprise ca. 500 species traditionally classiﬁed in seven gen- 1990) for the second round (if necessary) and cycle sequencing.

era (Thiede and Eggli, 2007). These are representatives of Sedum The PCR products were sequenced using a BigDye terminator v.

subgen. Sedum and most endemic American genera (e.g. Echev- 3.1 sequencing kit (Applied Biosystems, USA). Sequences were ana-

eria De Candolle, Graptopetalum, Lenophyllum Rose, Pachyphytum lyzed on an ABI 3130 genetic analyzer (Applied Biosystems, USA)

Link, Klotzsch et Otto, Villadia Rose, and Thompsonella Britton et and assembled with the Staden Package v. 1.4 (Bonﬁeld et al., 1995).

Rose). Sequence comparisons reveal a split between Eurasian and

American taxa in this clade (Carrillo-Reyes et al., 2009; Gontcharova 2.3. Sequence alignments and tree reconstructions

et al., 2008). The latter forms a strongly supported subclade with

Macaronesian S. farinosum Lowe as a sister but to the exclusion Initially, sequences were manually aligned using the SeaV-

of Eurasian Sedum. Asian and European species were arranged iew program (Galtier et al., 1996). The alignment was guided by

into several signiﬁcant lineages with unresolved relationships, and primary and secondary structure conservation (Artiukova et al.,

their common branch gained no support. Five lineages were iden- 2005; Goertzen et al., 2003; Gontcharova and Gontcharov, 2004;

tiﬁed within the American clade. Of these, the groups “Echiveria” Kozyrenko et al., 2013; Mai and Coleman, 1997). The mfold web

and “Villadia” were polygeneric and included representatives of server (http://mfold.rna.albany.edu; Zuker, 2003) was used with

220 V.Yu. Nikulin et al. / Flora 224 (2016) 218–229

default conditions to elucidate the folding pattern of secondary Thus, ITS1 and ITS2 varied signiﬁcantly in length and GC content

structure elements in divergent sequences. This procedure was while showing very few areas of primary sequence conservation.

iterative with ongoing comparison and reﬁnement for each taxon. Identiﬁcation of positional homology in spacers was hardly pos-

Once the conserved structural models of ITS1 and ITS2 in Sedum sible without considering the secondary structure generated for

were established, compensating base changes (CBCs) were also all sequences and used to guide the global alignment. Applica-

examined. tion of secondary structure models to reﬁne sequence alignment

and improve gap positions has been advocated by several authors

(Grajales et al., 2007; Keller et al., 2010; Letsch et al., 2010;

2.4. Phylogenetic analysis

Wolf, 2015; Zhang et al., 2015) because identiﬁcation of ﬂanking

sequences involved in helix formation has the potential to locate

Phylogenetic trees were inferred with ML-optimality crite-

the boundaries for the region domains.

ria using PAUP 4.0b10 (Swofford, 2002) and Bayesian inference

According to our predictions, the crassulacean ITS1 and ITS2 are

(BI) with MrBayes 3.1.2 (Huelsenbeck and Ronoquist, 2001). To

characterized by presence of four helices and ﬁve single-stranded

determine the most appropriate DNA substitution model for our

areas each. Fig. 1 illustrates the proposed base pairing in ITS1 and

datasets, the Akaike information criterion (AIC; Akaike, 1974),

ITS2 of Sedum and its relatives using the sequence of S. acre L. (the

Bayesian information criterion (BIC; Schwarz, 1978), and decision

type species of the genus) as an example. Approximately 65% of

theory performance-based selection (DT; Minin et al., 2003) tests

nucleotides were involved in the formation of the hairpin loops in

were applied with jModelTest 2.1.1 (Darriba et al., 2012). ML anal-

ITS1, while ca. 80% were involved in ITS2.

ysis was done using heuristic searches with a branch-swapping

In ITS1, GC-rich (67–93%) helix III was almost invariant in length

algorithm (tree bisection-reconnection). In BI, two parallel MCMC

(15 nt) and structure. Homoplasious substitution of G → A dis-

runs were carried out for ten million generations, sampling every

rupted the basal pair formation in only Aeonium undulatum Webb

1000 generations for a total of 10,000 samples. Convergence of the

& Berthelot, Sedum trichromum R. T. Clausen, and Thompsonella

two chains was assessed, stationarity was determined according to

xochipalensis Gual & al. Base insertions extended its terminal loop

the ‘sump’ plot (the ﬁrst 1000 samples were discarded as “burn-

in S. moranense and S. uniﬂorum ssp. oryzifolium (Makino) H. Ohba.

in”), and the posterior probabilities were calculated from the trees

Spacers ﬂanking helix III were also conserved across the dataset.

sampled during the stationary phase. The robustness of the trees

Therefore, this domain (corresponding to the Angiosperm Univer-

was estimated by bootstrap percentages (BP; Felsenstein, 1985)

sal Core motif; Liu and Schardl, 1994) and the relatively conserved

and posterior probabilities (PP) in BI. BP < 50% and PP < 0.95 were

35–42 nt long spacer preceding helix I were used as benchmarks for

not taken into account. ML-based bootstrap analysis was inferred

ITS1 sequence alignment. Remaining portions of ITS1 were rather

using the web service RAxML version 7.7.1 (http://embnet.vital-it.

variable in both primary and secondary structure.

ch/raxml-bb/; Stamatakis et al., 2008). MEGA v.6.06 (Tamura et al.,

Helix I had two to three 1–3 nt side bulges, and point mutations

2013) was used to estimate intraspecies and total pairwise dis-

and indels altered the bulges location in many cases, even between

tances (p-distances) between sequences.

closely related species (Fig. 2). This domain was the most variable

portion of this spacer and the most difﬁcult to align. It was signiﬁ-

3. Results cantly shorter and had fewer unpaired nucleotides in all members

of the Aeonium clade (21–25 nt vs. 38–48 nt typical for other taxa). A

We generated 85 new ITS rDNA sequences for mostly Eurasian lack of extensive primary sequence conservation in helix I implied

members of the largest but poorly sampled crassulacean genus alternative scenarios of its evolution in the Aeonium clade (Fig. 3a).

Sedum (71) and its allies (14). In addition, all available sequences Therefore, the homology of the helix I sequence between members

from this region for the tribes Sedeae and Semperviveae (January 1, of this clade and other species remained somewhat questionable.

2015) were retrieved from Genbank. Keeping in mind the distinct- Another case of the helix shortening was observed in Dudleya

ness of Macaronesia genera (Aeonium, Aichyson, and Monanthes) spp. and related American Sedum species (S. debile, S. oreganum

forming the robust Aeonium clade (Fairﬁeld et al., 2004; Mes and Nuttall, S. spathulifolium Hooker, and S. ternatum Michaux). In these

’t Hart, 1996; Mes et al., 1997), Sempervivum s. l. (the Semper- sequences, helix II was formed by 20–23 nt only, in contrast to the

vivum clade), and well-resolved relationships in this clade based on 33–38 nt typical for other species. Our foldings suggested that the

ITS sequence comparisons (Jorgensen and Frydenberg, 1999; Klein former taxa have lost the central part of the helix (ﬁve to seven

and Kadereit, 2015; Mort et al., 2002), we retained only the most nucleotide pairs) but likely retained the loop sequence (Fig. 3b).

divergent sequences to represent respective Aeonium and Sem- ITS2 was generally more conserved than ITS1, making secondary

pervivum/Jovibarba lineages in the dataset. Removal of redundant structure prediction more consistent. It displayed a number of

(2 < nt difference) and partial sequences resulted in an alignment structure features shared by many eukaryotic groups (Caisová et al.,

comprising 223 accessions (Supplementary material). Of these, 140 2013; Coleman, 2003, 2007, 2015; Mai and Coleman, 1997; Schultz

sequences represented the genus Sedum. et al., 2005): four standard helices (or a four-ﬁngered hand); a

All sequenced accessions yielded a single ITS fragment, and pyrimidine–pyrimidine mismatch at the base of relatively con-

all electropherograms were readable over their entire length. The served helix II (U–U and C–A pairings); A-rich conserved spacer

length of the ITS region (ITS1 + 5,8S + ITS2) included in the ﬁnal data between helix II and the longest (84–100 nt) helix III; and a highly

matrix ranged from 576 bp in Monanthes minima (Bolle) Christ to conserved 20 nt harboring angiosperm UGGU motif near the apex

614 bp in Sempervivum spp., with a mean length of 601 ± 6 bp and of helix III (5 side). Helix I displayed signiﬁcant length (from 16

GC content of 56.0 ± 3.3%. The average length of ITS1 was 225 ± 6 bp (S. treleasei) to 30 nt (S. rupestre L.)) and sequence variability, as

(56.3 ± 4.4% GC), except for all members of the Aeonium clade, Grap- well as the C- to G-rich transition (2–6 bp) from 5 to 3 typi-

topetalum fruticosum Moran, S. moranense Kunth, S. debile S. Watson, cal for angiosperms (Hershkovitz and Zimmer, 1996). The region

and Dudleya spp., for which this spacer was 208 ± 2 bp long. The 5.8S of the highest sequence variability in ITS2 was the distal part of

exon did not vary in length (161 bp; 50% to 55% GC) except for A. helix I, the CU-rich 3 end of helix III, and the following G-deﬁcient

palmense Webb ex Bolle (160 bp, likely due to sequencing error). spacer (Fig. 1). Helix II was nearly invariant in sequence and length

ITS 2 was somewhat shorter and less variable in length than ITS1, (30–31 bp) with only a number of single-base indels in the terminal

ranging from 197 bp in S. treleasei Rose to 226 bp in most members loop, mostly in the genus Sedum.

of Sempervivum L. (215 ± 3 bp and 58.4 ± 4.5% GC on average).

V.Yu. Nikulin et al. / Flora 224 (2016) 218–229 221

Helix III A 13-16 nt G A C G A U A GC=67%-93% C G G C C G Helix IV G C 42-65 nt G C A 6-19 nt 8-20 nt C C C AG GC=36%-68% C GA GC=19%-46% U U A A G G GC=25%-75% A U G A CCC G CA C A C CUC GGG U A A CUU U CC A A G GAG C A A A GG A UU U C A AG C Helix II AA U C 20-45 nt C G UC GU U U U G U GC=40%-77% CU C U A A U U C U UC GA ITS1 G U C U C C U U G U C U C G G CU 225±6 nt A G C U G G G G A A C G GU U 7-15 nt G A C U C A A C AGC=0%-40% G U UG G AA U CU U G C C UC GC A 3´ U G C GG U U U A CG GG A C A G G A A G C C UG C A C C C U G UAU UG G AG G U C U C GA U GU AA U U GC A G U G G AGUAAGC A U A G GUG G G CC 35-42 nt CA A C A A U C GC=36%-57% U A Helix I CC C U 21-48 nt G A G AA G U GC=43%-88% C GC C G U G U 5´ UC G G C C G C Helix II G C 28-35 nt U Helix III A A UU 84-100 nt GC=51%-76% A G U GC=44%-68% C C G C C A C C A C C C G C U G U C A A A U U G CG C U C U G G U U C G A C U C U C U C G G G U C G A C C C U AAAAAAG U G U 3-14 nt C G UG GC=0%-42% A 4-10 nt A C GC=0%-66% C A 6-21 nt U A Helix I A A GC=17%-65% 16-30 nt A G GC=43%-90% U CG A GG G ITS2 C U G U U GG CC GU 215±3 nt U U C A C A C C A C G U A A C C G A U 0-12 nt U Helix IV CA G U 6-18 nt CG UAC A G 12-30 nt Ua uGC C G GC=33%-80% c g C A GC=32%-85% u a U G g c U c UC G g c A C 5.8S g c 28S C U 5´ 3´ G G U C C

U A A

Fig. 1. Secondary structure models of ITS1 and ITS2 of Sedum acre based on Mfold predictions. The grayed nucleotides were conserved in 90% of the sequences. Nucleotides

conserved in 90% of the sequences, but differing from those in S. acre are shown in the callouts. Dotted frame marks the most conservative regions of the secondary structure.

Domains length range and GC content are given for each domain.

3.1. Phylogenetic analyses most sequence-rich and genus-rich clade (155 and 8, respectively),

attained maximal support in both methods. The Sempervivum clade

Primary sequences of ITS regions contained many variable (17 sequences) was marginally supported only in ML (50%; Fig. 4).

regions that were difﬁcult to align unambiguously. The alignment The latter lineage was split into two robust subclades comprising

was improved based on adjustment of the secondary structures. 10 Sempervivum species and members of Sedum ser. Rupestria (7

Our ﬁnal dataset of 223 sequences was 590 sites in total (ITS1: accessions), respectively. The Sempervivum clade showed a weak

228 sites, 5.8S: 161 sites and ITS2: 201 sites) and included 382 afﬁnity to the Aeonium clade (72/0.98) but to the exclusion of Acre

MP-informative characters (196, 26, and 160 sites, respectively). and Leucosedum.

ML phylogenetic analysis of the genus Sedum and related genera In our tree, 42 accessions belonging to the Leucosedum clade

(tribes Sedeae, Semperviveae, and Aeonieae) based on the GTR+I+ were distributed between two unequal lineages branching para-

best-ﬁt model yielded the tree shown in Figs. 4 and 5. The acces- phyletically. The larger clade (55/0.99) comprised 39 sequences

sions analyzed were placed into the Aeonium, Sempervivum, and of the genera Sedum and Dudleya, and three more taxa (Rosularia

Acre clades, and the paraphyletic Leucosedum cluster. Acre, the serrata (Linné) A. Berger, Sedum sp. and S. cepea L.) formed an unsup-

222 V.Yu. Nikulin et al. / Flora 224 (2016) 218–229

U ported cluster (Fig. 4). The branching pattern within Leucosedum

a) C C

was only partially resolved. Its largest signiﬁcant subclade (87% BP

and 1.00 PP) comprised American taxa, nine Dudleya spp. (95/1.00),

C G UC

and four species of Sedum (100/1.00). Of the latter, S. oreganum, S.

C G C G

spathulifolium, and S. ternatum comprised a robust clade (100/1.00),

20 C G C G while the afﬁnity of S. debile remained unclear (Fig. 4b). The well-

supported Prometheum clade (98/0.99) showed no relationship to

G C 20 C G

Rosularia, to which it formerly belonged. Instead, it had S. hispan-

G U 30

G C icum as a sister (90/1.00), while R. serrata was placed in a distantly

A C G A related and only weakly supported clade (67/0.99) with an uniden-

A G tiﬁed Sedum representative (Fig. 4b). Seven more Sedum species

G C

G C were placed in a clade supported only by posterior probabilities

A U

A G (0.99; Fig. 4b). Of these, S. brevifolium De Candolle and S. pallidum

G A G C M. von Bieberstein were sisters (0.95–0.99 PP) to S. album L./S. hir-

sutum Allioni (73/0.98) and S. gracile/S. subulatum (C. A. Meyer)

C A C G

Boissier (83/0.95) pairs, respectively. The afﬁnity of S. stefco Ste-

U A U G fanoff remained unresolved.

G C G C The robust Acre clade accommodated most Sedum accessions

(100). It was split into two lineages: an unsupported cluster of

10 C G 10 C G

Eurasian Sedum taxa (42 sequences) and a strongly supported

G C G C clade (98/1.00) of American Sedeae members (113 sequences from

the genera Sedum, Echeveria, Graptopetalum, Pachyphytum, Thomp-

G A 40 A A

sonella, Cremnophyla Rose, Lenophyllum and Villadia; Figs. 4 and 5).

A A 40

A A The only exception in this geographic grouping was a position of

G C G C highly similar and long-branched S. nudum Aiton and S. brisse-

moretii Hamet from Madeira nested within the American subclade G C G C

(Fig. 5).

G U G U The relationship pattern in the Eurasian cluster generally sup-

G C G C ported the grouping of taxa according to their origin. There were

two robust clades of Asian species showing no afﬁnity to each other,

U C G U U C G C

5’ 3’ 5’ 3’ with 9 and 18 sequences, respectively (informally called A1 and

Sedum lineare Sedum alpestre A2; Fig. 4b). There was also a paraphyletic assemblage of 12 Euro-

dG = -24.70 dG = -25.40 pean accessions. In addition, two sequences of S. sexangulare L.

and highly similar long-branched S. anglicum Hudson and S. fari-

b) U A G A nosum were members of the Eurasian lineage. Branching patterns

in Asian Sedum clades were mostly well supported, in contrast to

U A U A unresolved relationships between European species (Fig. 4). The

latter mostly represented ser. Alpestria Berger (9 species) but did

G C 20 G C 20

not form a respective clade.

A U A U The American subclade comprised representatives of seven gen-

era beside Sedum. Members of respective genera tended to group

A U A U together, although none were established as a monophyletic lin-

eage. Generally, the branching pattern in the American subclade

U U U G

was poorly resolved. The largest supported clades were the “Villa-

dia” (98/1.00) and “Echeveria” (89/1.00) groups (Fig. 5), matching

10 G U 10 A U

those established by Carrillo-Reyes et al. (2009). Sedum species

C G C G were scattered across the American subclade, and many of them

showed afﬁnity to representatives of Echeveria, Graptopetalum,

C G C G

Pachyphytum, Thompsonella, Cremnophyla, and Villadia (Fig. 5b).

C U C U We analyzed two more datasets to test whether the support

for clades or lineages established in our analyses was affected by

U U U U highly divergent ITS1 sequences. One had only the most conserved

portions of ITS1, 5.8S and ITS2 (400 positions), and the other was

C G C G

limited to 5.8S and ITS2 sequences (294 positions; Table 1). Accord-

C G 30 C G 30 ing to the results of bootstrap analyses, support for the Aeonium and

Acre clades remained almost unchanged with the reduced datasets

G C G C (99–100%). For the subclades comprising the clade Sempervivum,

the decrease in support was more noticeable (from 100 to 89–91%).

U G C U U G U U

5’ 3’ 5’ 3’ The robust American subclade of the clade Acre had no support with

Sedum emarginatum Sedum hakonense

the reduced datasets (Table 1).

dG = -13.40 dG = -15.60

3.2. Sequence variation and relative rates

Fig. 2. Examples of the secondary structure alterations between closely related

species in ITS1 (helix I; a) and ITS2 (helix II; b) caused by point mutations and indels.

Bias in the length of branches across the phylogenetic tree sug-

Arrowhead marks insertion. Changed nucleotides are in white circles. Homologous

nucleotides in the apical loops are shown by gray color. gested unequal evolutionary rates between accessions and clades.

Pairwise sequence divergence (uncorrected-p) in the spacer region

V.Yu. Nikulin et al. / Flora 224 (2016) 218–229 223

U C a) C G U C U G U C C G 20 U A C G C G A U 20 C G 20 A C U C C G A A 30 A U G C A U A G 30 A A G C A C A A A U A A UA G C G G C GA U A C G C G C C G 10 G C G U G C U U G U A U 10 G C G C U U G C U G U C G C G U 10 C G 10 C C G C A U U G G C A U 20 10 G C G U G C G C A U 40 G U U 20 A U A U 40 C A U C G A C G C U G C G C G C G A C G C A U A U G C A U G U

C G C G A U 40 G C C G

C G 5’ G C A3’ 5’U C C3’ 5’C G C A3’ 5’U C G C3’ 5’A G C A3’ Sedum jaccardianum Monanthes muralis Sedum rupestre Sempervivum Sedum cepaea dG = -19.60 dG = -10.60 dG = -14.90 marmoreum dG = -22.20 ssp. reginae-amaliae dG = -25.50

20 b) U U 20 C C 20 C G U U C G C G A U C U G C C G C G U A G C G C U U G U G C G C G G C A

G U G C A C U U 10 C C

G C A U A U C G C G 10 U G G C G C 10 G C G C G C 30 10 C U 30 10 C U 30 G C G C G C G C G U A U A U A U G C G C A U A G C G U A U U A U A U A U G C G C G C G U G C G C G C C G G C G C

C G C G 20 U G C G C G

A U A A C U A A G G C A C U A C C U AA 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ Sedum debile Dudleya pulverulenta Sedum stefco Sedum villosum Sedum hispanicum

dG = -13.70 dG = -14.00 dG = -27.00 dG = -21.40 dG = -18.80

Fig. 3. Helix I of ITS1 shortened in the Aeonium clade (a) and helix II of ITS1 shortened in the Dudleya-group (b). The grayed nucleotides show identical motives. The arrows

indicate likely homologous stem segments.

was generally high across the whole dataset (0.164 ± 0.007). Within lineages comprising these clades. P-distances between Semper-

the Sempervivum, Acre clades, and the cluster Leucosedum sequence vivum spp. did not exceed 0.018 ± 0.002 and were almost four times

divergence exceeded 0.12. However, in the case of the well- smaller than in Sedum ser. Rupestria clade (0.068 ± 0.006). In the

structured Sempervivum and Acre clades, such a high divergence Acre clade, the Eurasian Sedum species were characterized by high

was mostly due to differences between generally more uniform

224 V.Yu. Nikulin et al. / Flora 224 (2016) 218–229

Sedum modestum b) S. surculosum var. luteum HE999690 S. surculosum var. luteum LM993288 58/- S. jaccardianum 96/0.99 Aichryson palmense Aeonium undulatum Aeonium Monanthes muralis M. minima M. laxiflora Sedum ochroleucum S. cf. montanum HE999685 S. montanum HE999667 Sedum ser. S. sediforme S. amplexicaule ssp. tenuifolium 92/1.00 S. rupestre Rupestria 50/- S. forsterianum Sempervivum globiferum ssp. allionii S. ciliosum S. montanum Sempervivum S. altum S. ossetiense 92/1.00 S. caucasicum Sempervivum s.l. S. dzhavachischvilii S. armenum S. marmoreum S. marmoreum ssp. reginae-amaliae Sedum album LM993277 73/0.98 S. album HE999638 S. hirsutum ssp. baeticum S. hirsutum HE999661

S. cf. hirsutum HE999681

S. brevifolium HE999644

S. brevifolium LM993278 -/0.99 S. stefco S. sp. 4 LM993287 S. pallidum -/0.99 S. gracile 83/0.95 S. subulatum S. sedoides S. lagascae S. villosum S. debile 55/0.99 Dudleya pulverulenta D. attenuata JX960495 D. edulis a) D. linearis 63/1.00 D. viscida 87/1.00 D. virens Leucosedum Aeonium 95/1.00 D. variegata Dudleya D. attenuata AY54568 100/1.00 D. greenei Sempervivum s.l. S. oreganum 50/- Sempervivum -/0.98 100/1.00 Sedum ser. S. spathulifolium ssp. purdyi 95/1.00 S. spathulifolium Rupestria 96/1.00 S. ternatum S. fragrans Prometheum chrysanthum 100/1.00 98/0.99 P. sp. 90/1.00 P. tymphaeum 55/0.99 S. hispanicum HE999663 S. hispanicum HE999686 Leucosedum S. dasyphyllum ssp. glanduliferum S. dasyphyllum LM993279 99/0.99 S. dasyphyllum HE999682 97/0.99 S. dasyphyllum HE999684 S. cepaea 72/0.98 S. sp. 5 HE999691 72/0.98 67/0.99 Rosularia serrata 67/0.99 Eurasian subclade S. sexangulare S. farinosum S. anglicum S. tosaense 96/1.00 S. zentaro-tashiroi S. uniflorum ssp. japonicum S. bulbiferum JQ954567 98/0.99 S. sp. 2 LM993285 A1 S. uniflorum ssp. oryzifolium S. mexicanum S. sp. 3 LM993286 S. lineare FJ980313 97/1.00 S. alpestre S. grisebachii var. horakii S. multiceps HE999670 92/1.00 S. multiceps LM993282 S. ursi 100/1.00 -/0.97 S. apoleipon S. annuum 93/0.99 S. tuberiferum

S. urvillei Eurasian S. acre HE999635

S. acre LM993284 American subclade Acre S. acre HE999636 subclade Acre S. multicaule 53/- S. oreades S. bergeri S. trullipetalum 76/0.95 98/1.00 S. triactina 94/1.00 S. bulbiferum AB088628 S. makinoi 88/0.99 S. baileyi S. emarginatum S. hakonense A2 81/- S. morissonense S. polytrichoides 98/1.00 S. satumense S. subtile S. sarmentosum AB088624 S. sp. 1 LM993283 99/1.00 S. lineare AB088623 S. sarmentosum EU592003

0.06

Fig. 4. (a) Overview of phylogeny of the tribes Sedeae, Semperviveae, and Aeonieae (Crassulaceae) based on comparisons of 223 ITS rDNA sequences (590 nt). The tree

topology was inferred by maximum likelihood in PAUP 4.0b10 (Swofford, 2002) using the GTR+I+ model. Major lineages are reduced to small triangles. (b) Enlarged

portion of the tree showing structure and composition of Aeonium, Leucosedum, Sempervivum clades, and Eurasian subclade of the Acre clade. Support [bootstrap percentages

(BP) ≥ 50% and posterior probabilities (PP) ≥ 0.95: ML/BI] are given above/below the branches. Branches with 100% BP and 1.00 PP are shown in boldface. Accession numbers

are provided for taxa represented by more than one sequence in the data set. Sequences obtained for this study are shown in boldface.

V.Yu. Nikulin et al. / Flora 224 (2016) 218–229 225

Sedum oaxacanum b) S. versadense 0.06 S. nussbaumerianum S. confusum 87/1.00 S. hultenii S. dendroideum Pachyphytum viride 88/1.00 Echeveria elegans E. colorata S. corynephyllum Graptopetalum pachyphyllum S. craigii AY545693 89/0.99 S. craigii HE999649 G. macdougallii G. pentandrum ssp. superbum 56/0.99 G. pentandrum G. filiferum G. rusbyi 81/0.99 78/1.00 G. marginatum G. fruticosum 98/1.00 G. bellum JX960526 E. gibbiflora E. juarezensis E. fulgens 93/1.00 S. commixtum 55/0.99 E. amoena S. clavatum 88/0.99 Cremnophila linguifolia C. nutans “Echeveria” G. grande 87/0.99 G. saxifragoides group 69/1.00 G. pusillum G. bellum AY545718 89/0.99 G. bartramii 98/0.99 S. suaveolens G. amethystinum -/0.97 G. mendozae G. paraguayense ssp. bernalense Thompsonella garcia-mendozae T. colliculosa T. spathulata T. xochipalensis T. minutiflora EF632179 T. minutiflora AY545719 T. platyphylla T. sp. T. mixtecana 98/1.00 E. racemosa a) 88/1.00 E. pringlei E. coccinea E. purpusorum E. megacalyx Aeonium E. setosa 100/1.00 S. adolphii Sempervivum s.l. 65/0.99 E. pulvinata 50/- Sempervivum S. treleasei 100/1.00 Sedum ser. S. pachyphyllum Rupestria S. pacense Acre 99/1.00 S. catorce 100/1.00 S. gypsophilum American 69/- Lenophyllum acutifolium S. guatemalense subclade 55/0.99 58/0.98 99/1.00 S. mocinianum S. ebracteatum Leucosedum S. chazaroi 99/1.00 S. hemsleyanum S. greggii ssp. angustifolium 59/- S. oxypetalum 71/0.98 S. frutescens 72/0.98 S. longipes

67/0.99 Eurasian subclade S. palmeri S. batesii 97/1.00 S. fuscum S. vinicolor S. nudum S. brissemoretii 99/1.00 S. obcordatum S. compactum S. retusum S. reptans S. bourgaei S. chloropetalum S. quevae Villadia pringlei V. cucullata V. aristata 100/1.00 V. misera 78/0.97 S. wrightii S. liebmannianum S. moranense HE999668 S. moranense FJ753954 S. griseum American subclade Acre V. minutiflora V. nelsonii

V. diffusa V. sp . “Villadia” V. recurva 98/1.00 65/0.99 V. imbricata group 87/0.99 S. grandisepalum V. albiflora S. jurgensenii S. jurgensenii ssp. attenuatum S. goldmanii 75/0/99 98/1.00 97/1.00 V. incarum S. andinum S. sp. 6 HE999669 97/1.00 S. plicatum S. grandyi S. alexanderi S. allantoides S. cockerellii 71/0.99 S. alamosanum

S. trichromum

Fig. 5. (a) Overview of ITS rDNA-based phylogenetic tree of the tribes Sedeae, Semperviveae, and Aeonieae (Crassulaceae). (b) Enlarged portion of the tree showing structure

and composition of the American subclade of the Acre clade. See the legend of Fig. 4 for details.

226 V.Yu. Nikulin et al. / Flora 224 (2016) 218–229

Table 1

Angiosperm Universal Core motif GGCRY-(n)4-7-GYGYCAAGGAA

ML bootstrap support for the clades/subclades/groups (Figs. 4 and 5) based on dif-

(Liu and Schardl, 1994) forming helix III and its following spacer

ferent ITS rDNA datasets (223 sequences).

in our model (Fig. 1). The second conserved motif covers the 5 -end

Clade/Subclade/Group 590 bp 400 bp 294 bp

of the spacer but has less sequence homology with representa-

Aeonium 100 100 99 tives of other angiosperm groups. Numerous compensatory and

Sempervivum 50 Para Para hemi-compensatory base changes revealed in pairwise secondary

Sedum ser. Rupestria 100 100 91

structure comparisons (Figs. 2 and 3) provided support for our pre-

Sempervivum s.l. 100 85 89

dictions and veriﬁed the homology searches. Although our models

Leucosedum Para <50 Para

may overestimate some secondary structure elements, we consider

Dudleya 87 67 Para

Acre 100 98 100 the predictions as adequate to guide alignments. This conclusion is

Eurasian <50 Para Para

supported by the lack of discrepancies between our ITS tree and

A1 100 87 Para

topologies based on chloroplast markers regarding clade composi-

A2 100 85 <50

tion, their signiﬁcance, and relationships.

American 98 <50 Para

Our matrix of 223 aligned ITS sequences (809 sites) was almost

−

590 bp ITS1 + 5.8S + ITS2, 590 bp (Figs. 4 and 5). 400 bp − the most conserved por-

35% longer than the mean sequence length, indicating that the

tions of ITS1 + 5.8S + ITS2, 400 bp; 294 bp − 5.8S + ITS2, 294 bp. Para − paraphyletic.

length mutations play an important role in the spacer evolution.

However, most of the length mutations were non-informative

sequence divergence (0.154 ± 0.007), in contrast to the more simi-

autapomorphic 1–2 nt indels. Extensive deletions in ITS1 were

lar American subclade members (0.088 ± 0.005).

synapomorphic in only two instances. The most notable was the

shortened helix I in ITS1 that characterized Macaronesian genera

4. Discussion (Aeonium clade) and likely originated in a North African Sedum

ancestor of the clade (Figs. 3a, 4). Another structural synapomor-

Analyses of a large sample of crassulacean ITS rDNA sequences phy was found for Dudleya, which was allied with American Sedum

allowed us to build the ﬁrst comprehensive phylogeny of the most species to form a distinct lineage in Leucosedum (short helix II of

species-rich and taxonomically complex genus Sedum and related ITS1; Fig. 3b). It appears that partial deletion of ITS helices is a rare

genera (Sempervivoideae, Crassulaceae). Our particular empha- event that does not seem to affect the spacer transcript functional-

sis was on the least studied phylogenetically Eurasian species of ity.

the tribe Sedeae. The topology presented here is generally con-

gruent with previous studies based on limited sets of taxa or 4.1. Phylogeny of Sedum and related taxa

the plastid-encoded matK gene and trnL-trnF intergenic spacer as

phylogenetic markers (Acevedo-Rosas et al., 2004; Carrillo-Reyes ITS rDNA sequence comparisons resolved a complex pattern of

et al., 2009; Gontcharova et al., 2006, 2008; Mayusumi and Ohba, relationships between representatives of the largest crassulacean

2004; Mort et al., 2001, 2002). Comparison of 223 ITS sequences genus Sedum and its allies, with Sedum taxa being found in all

resolved three major lineages: the Aeonium, Sempervivum, and Acre major clades of the tree (Figs. 4 and 5). Monophyly of the tribes

clades (Figs. 4 and 5). Our results support the monophyly of the Aeonieae (Aeonium clade) and Semperviveae (Sempervivum clade)

tribes Aeonieae, Semperviveae, and Sedeae, comprising a crown to the exclusion of Sedeae (Leucosedum and Acre; 72%/0.98; Fig. 4a)

assemblage of the crassulacean tree and accommodating the genus strongly suggests that Sedum species that are members of the for-

Sedum (Thiede and Eggli, 2007). To date, the Sedeae clade (Leucose- mer lineages should be removed from the genus. Although not

dum + Acre; 72/0.98; Fig. 4a) has only been supported in cpDNA data reﬂected by any taxonomic treatment (’t Hart and Bleij, 2005;

and with less representative taxon sampling (Mayusumi and Ohba, Thiede and Eggli, 2007), a distant relationship was asserted 20

2004; Mort et al., 2001). Our study is the ﬁrst to resolve the clade years ago for North African Sedums of the series Caerulea Fröd.,

of the tribe Semperviveae based on sequence comparisons, albeit Pubescens Mes, and Monanthoidea (Batt.) Fröd. with the rest of

with only marginal support. the genus (Mes, 1995; Mes and ’t Hart, 1994), as well as afﬁn-

Relatively high divergence of primary ITS sequences often ity to Macaronesian genera (tribe Aeonieae/Aeonium clade). This

associated in Crassulaceae with significant ITS length variation affinity was confirmed by subsequent phylogenetic analyses for S.

hampered sequence alignment. However, this problem was solved modestum Ball, S. surculosum var. luteum (Emberger) Maire, and S.

by using ITS transcript secondary structure information, which jaccardianum Maire & Wilczek comprising ser. Monanthoidea, and

facilitated the search of homologies between species. Models pre- it is supported by synapomorphic indels in matK and ITS rDNA

dicted for other plant groups (Coleman, 2015; Goertzen et al., 2003; sequences (Gontcharova et al., 2008; Mort et al., 2001; present

Liu and Schardl, 1994; Schultz et al., 2005) were conﬁrmed by our study). The relationship of S. caeruleum L. and S. pubescens Vahl

putative model of the ITS1 and ITS2 secondary structures generated with the Aeonium clade and particularly Monanthoidea have yet to

with the thermodynamic-based folding for Sedeae and related to its be analyzed. It should be noted that monophyly of the latter series

tribes Semperviveae and Aeonieae (Fig. 1). In both ITS1 and ITS2, is also questionable. Mes and ’t Hart (1994) resolved Monanthoidea

the transcripts have four characteristic helical domains. The ITS2 as a clade, but it was paraphyletic in matK and ITS rDNA sequence

model also revealed features typical for most eukaryotic organisms, comparisons (Mort et al., 2001; present study).

namely four domain structures with the third hairpin loop domain In contrast to the uncertainties with relationships between

being the longest, pyrimidine–pyrimidine mismatch at the base of Sedum species in Aeonieae, the composition and structure of the

helix II, and a UGGU site at the distal part of helix III (Schultz et al., Sempervivum clade is better understood and generally taken into

2005). account by the most recent taxonomic treatment. Conﬁrmation

While this core structure of ITS2 persists throughout eukary- is needed for only the afﬁnity of S. assyriacum Boiss., S. mooneyi

otes (Coleman, 2003; Goertzen et al., 2003; Schultz et al., 2005; Gilbert, and Middle East species that are probably related to them

Wolf et al., 2005), the general pattern of the ITS1 architecture has (van Ham and ’t Hart, 1998; ’t Hart, 1995; Thiede and Eggli, 2007).

yet to be established. Often, it has four or fewer relatively short The Sempervivum lineage persistently attained weak or no support

helices with a major part of the spacer being single stranded, a pat- in molecular phylogenetic studies (Gontcharova et al., 2008; van

tern that is followed in Crassulaceae as well. Of the two conserved Ham and ’t Hart, 1998; Mort et al., 2001; present study), while com-

motifs revealed in crassulaceaen ITS1, one corresponds to the posing it Sempervivum s.l. (for more details see Klein and Kadereit,

V.Yu. Nikulin et al. / Flora 224 (2016) 218–229 227

2015) and Sedum ser. Rupestria clades were strongly supported in Glauco-rubens Fröd. (S. hispanicum) and Prometheum. However, the

all phylogenies. Grulich (1984) raised Sedum ser. Rupestria to a sep- afﬁnity of S. fragrans to the Prometheum/S. hispanicum clade in

arate genus, Petrosedum, but despite being corroborated by quite our tree was supported only topologically. Different datasets and

a number of phenotypic traits (Mauritzon, 1933; Stevens et al., markers also placed S. inconspicum Hand.-Mazz., S. ince ’t Hart &

1994, 1996), his suggestion was adopted only recently (Thiede and Alpinar, and American S. commixtum Moran & Hutchison as the

Eggli, 2007). Petrosedum was almost exhaustively sampled for our closest relative of Prometheum (van Ham, 1994; ’t Hart and Alpinar,

analyses, which fully conﬁrmed its distinct nature. The branching 1999; Mort et al., 2001). Keeping in mind the insufﬁcient taxon

pattern within Petrosedum presented here generally corroborated sampling in Leucosedum, we can expect alternative hypotheses on

the results of the study based on cpDNA restriction site variation Prometheum afﬁnity, but its distant relationship with Rosularia,

and phenotypic characters (van Ham and ’t Hart, 1994). It conﬁrmed which has included some of its species, is more or less evident.

the close relationship between S. (Petrosedum) ochroleucum Chaix In our tree, a single Rosularia sequence was placed outside

and S. (Petrosedum) montanum L. and the distinct position of S. (Pet- the larger Leucosedum clade together with long-branched S. cepea

rosedum) sediforme (Jacquin) Pau (Fig. 4b). Apart from relatively and an unidentiﬁed Sedum accession (Fig. 4b). ITS rDNA and matK

high divergence in the ITS rDNA sequence between Petrosedum sequence comparisons could resolve only a weakly supported sis-

species (ca. 7%), they almost lack reproductive barriers, and retic- tership of Rosularia serrata with unidentiﬁed Sedum species and

ulate speciation may play an important role in this group (Gallo, S. lydium Boissier, respectively (Mort et al., 2001; present study).

2012; ’t Hart, 1978). Available molecular data are unable to assess monophyly of the

Sedum species allocated outside the tribe Sedeae clade (see genus Rosularia and its afﬁnity. Although a number of species were

above) comprise only a small fraction of the genus diversity. Most of moved from Rosularia to Prometheum and Sedum, the former still

its species belong to the clade Sedeae and were distributed between accounts for ca. 20 species classiﬁed into two sections with pheno-

the paraphyletic Leucosedum cluster and robust Acre clade. Besides typic data, suggesting a distant relationship between the sections

Sedum, Leucosedum and Acre accommodate a number of genera that (Thiede and Eggli, 2007).

are mostly embedded among Sedum species (Figs. 4 and 5). Gen- In contrast to the uncertain phylogenetic status of Leucosedum,

erally, high ITS rDNA sequence variation likely resulted in some its sister Acre clade consistently attains moderate to strong sup-

unresolved relationships within the tribe clade. In Leucosedum and port (Gontcharova et al., 2008; van Ham, 1995; Mayuzumi and

the Eurasian subclade of Acre, which were supported only topo- Ohba, 2004; Mort et al., 2001). Acre is characterized by the largest

logically, sequence divergence was particularly noticeable at 13.8% diversity of genera (7), species (>500 spp.), and phenotypes among

and 15.4%, respectively. However, a majority of Leucosedum taxa crassulacean lineages. Unlike Leucosedum, where American taxa

formed a weakly supported clade (55%/0.99), and only three long- (Dudleya + Sedum spp.) occupied an unresolved position among

branched sequences (S. cepea, Sedum sp. and Rosularia serrata; Eurasian species (Fig. 4b), ITS rDNA data provided support for a sis-

Fig. 4b) were placed outside this lineage. It remains unclear whether ter relationship in Acre between Eurasian and American species and

the lack of support for Leucosedum was caused by somewhat accel- forming respective lineages. Placement of S. nudum and S. bresse-

erated evolutionary rates, insufﬁcient taxon sampling, or distant moretii from Madeira in the American subclade (98%/1.00; Fig. 5b)

relationships. This phenotypically and biogeographycally diverse was the only exception, but an important one that suggests either

assemblage was established as a well-supported clade by van Ham long-distance dispersal of these species from America to Macarone-

and ’t Hart (1998) based on cpDNA restriction site data, but it is sia or Macaronesian ancestry of the American subclade in Acre.

consistently resolved paraphyletically in all sequence-based anal- Strong afﬁnity of S. nudum to several Macaronesian and African

yses (Gontcharova et al., 2008; Mayuzumi and Ohba, 2004; Mort Sedum species in matK sequence comparisons (Mort et al., 2001)

et al., 2001; present study). may indicate that further Old World Sedum taxa could be a part

Our study established only a few signiﬁcant subclades in Leu- of the American subclade and this intriguing hypothesis requires

cosedum, and neither a morphological nor a lucid biogeographical further scrutiny.

pattern could be inferred from the phylogenetic pattern. Never- Notably, high phenotypic diversity and morphological distinct-

theless, the extended taxon sampling conﬁrmed the afﬁnity of the ness of American genera (recognized as subfamily Echeverioideae

North American genus Dudleya to Leucosedum and revealed its by Berger (1930)) is not reﬂected by ITS rDNA sequence divergence.

close relationship with Sedum subgen. Gormania members from the Although the American subclade comprised the largest number of

same continent (S. oreganum, S. spathulifolium, S. ternatum, and S. sequences (113) representing eight genera, it was characterized

debile; Fig. 4b). This afﬁnity was supported not only by bootstrap by signiﬁcantly lower sequence divergence than 42 Sedum acces-

and posterior probability thresholds, but also molecular synapo- sions in its Eurasian sister lineage and 42 accessions in Leucosedum

morphy in ITS rDNA sequence secondary structure, namely the (0,088 ± 0.006, 0,154 ± 0.008 and 0,139 ± 0.008, respectively). Rela-

shortened helix II of ITS1 shared by all American species of Leucose- tively low sequence divergence was observed in datasets including

dum (Fig. 3b). Earlier, Sedella (Parvisedum R.T. Clausen) was resolved chloroplast markers as well (Carrillo-Reyes et al., 2008, 2009), and

as a sister to Dudleya (Mort et al., 2001) but it was not sampled here. this fact may corroborate recent and fast speciation in the Ameri-

Therefore, the existence of this synapomorphy in ITS1 of Sedella and can subclade of Acre (Mort et al., 2001). However, this process was

its relationship to American Sedum needs conﬁrmation. Unlike the likely accompanied by an accelerated rate of morphological trait

Aeonium clade, we have no clue yet about the possible ancestry evolution and polyploidization (Thiede and Eggli, 2007; Uhl, 1992).

of the American Leucosedum lineage and the origin of its molec- The important role of hybridization in shaping the current diversity

ular synapomorphy. Mort et al. (2001) resolved S. gracile, which of Crassulaceae in general and the American lineage of Acre in par-

is distributed in Turkey and the Caucasus, as a sister to Dudleya ticular was also hypothesized (Banares,˜ 1990; ’t Hart et al., 1993;

and Sedella (Parvisedum). However, no European/Mediterranean Mort et al., 2001; Uhl, 1961, 1992), but it has yet to be detected by

Leucosedum species showed afﬁnity to any American taxon our molecular tools.

analyses (Fig. 4b). Phylogenetic relationships in the American lineage of the Acre

One more polygeneric clade in Leucosedum that was resolved in clade remain only partially resolved. Our data generally corrob-

our study comprises Prometheum spp. and S. hispanicum (90%/1.00; orated the results of multilocus analyses that grouped American

Fig. 4b). Thus, ITS rDNA data corroborated the results of cpDNA taxa into ﬁve lineages (“Villadia” group, “Echeveria” group, two lin-

restriction site analysis, which suggested a sister relationship eages of Sedum spp. and Sedum spp.+Lenophyllum; Carrillo-Reyes

between Sedum ser. Alsenifolia Berger (S. fragrans ’t Hart)/ser. et al., 2009) and in addition to that placed two Sedum species from

228 V.Yu. Nikulin et al. / Flora 224 (2016) 218–229

Madeira in the American subclade (see above). Sedum was a mem- small monophyletic genera as unpractical and untimely, arguing

ber of three polygeneric lineages where its taxa were positioned that relationships between its species are poorly known. Unfortu-

basally and/or in terminal clades often comprising species of more nately, our understanding of Sedum phylogeny has not advanced

than one genus (Fig. 5b). This branching pattern refuted the mono- signiﬁcantly over the last 20 years, and we are still at the beginning

phyly of Echeveria, Graptopetalum, Villadia, and Cremnophila but of shaping the phylogenetic tree of the crassulacean crown group.

resolved generic clades of Lenophyllum, Pachyphytum, and Thomp-

sonella (Acevedo-Rosas et al., 2004; Carrillo-Reyes et al., 2008, Acknowledgments

2009). Of the lineages mentioned, only the “Echeveria” group could

be characterized phenotypically by lateral inﬂorescence and basally

We thank Marko Dobosˇ and Milan Hornát for sharing plants

fused corals (Carrillo-Reyes et al., 2009), while the remaining

from their personal collections. Constructive comments by an

groups are morphologically heterogeneous.

anonymous reviewer helped to improve an early version of the

The Eurasian subclade of Acre was the only large lineage in the

manuscript. This research was partially supported by the Russian

tree that accommodated no other genera except Sedum and pre-

Foundation for Basic Research grants (16-34-00176 and 15-29-

dominantly distributed in Asia species. Early analyses established 0250515).

two clades encompassing Asian Sedum species in Acre. This was

seen as an indication of their possible phylogenetic distinctness, to

Appendix A. Supplementary data

the exclusion of European and American counterparts (Mayuzumi

and Ohba, 2004; Mort et al., 2010). However, in our analyses, all

Supplementary data associated with this article can be found,

but two European/Mediterranean/Near East Sedums allocated to

in the online version, at http://dx.doi.org/10.1016/j.ﬂora.2016.08.

the Acre clade showed afﬁnity to Asian taxa, and this assemblage

003.

formed a sister lineage to the American subclade (Fig. 4b).

Limited taxon sampling and high sequence divergence could

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