Journal of Plant Research

, Volume 130, Issue 1, pp 107–116 | Cite as

Molecular phylogeny of the cosmopolitan aquatic plant genus Limosella (Scrophulariaceae) with a particular focus on the origin of the Australasian L. curdieana

  • Yu Ito
  • Norio Tanaka
  • Dirk C. Albach
  • Anders S. Barfod
  • Bengt Oxelman
  • A. Muthama Muasya
Regular Paper

Abstract

Limosella is a small aquatic genus of Scrophulariaceae of twelve species, of which one is distributed in northern circumpolar regions, two in southern circumpolar regions, two in the Americas, one endemic to Australia, and six in tropical or southern Africa or both. The Australasian L. curdieana has always been considered distinct but its close phylogenetic relationships have never been inferred. Here, we investigated the following alternative phylogenetic hypotheses based on comparative leaf morphology and habitat preferences or floral morphology: (1) L. curdieana is sister to the African L. grandiflora; or (2) it is closely related to a group of other African species and the northern circumpolar L. aquatica. We tested these hypotheses in a phylogenetic framework using DNA sequence data from four plastid DNA regions and the nuclear ITS region. These were analyzed using maximum parsimony and Bayesian inference. We obtained moderately resolved, partially conflicting phylogenies, supporting that accessions of L. grandiflora form the sister group to the rest of the genus and that L. curdieana groups with the African taxa, L. africana and L. major, and L. aquatica. Thus, the molecular evidence supports the second hypothesis. A biogeographic analysis suggests an out-of-southern Africa scenario and several dispersal events in the Southern Hemisphere. Past dispersal from southern Africa to Australasia is suggested, yet it cannot be excluded that a route via tropical Africa and temperate Asia has existed.

Keywords

Aquatic plants Biogeography Dispersal Lamiales Phylogenetic inference 

Introduction

Limosella L. (mudworts) is a genus in Scrophulariaceae with twelve species (Cook 2004; Glück 1934) that are either aquatic or amphibious in wetlands. The genus is distributed in temperate and subtropical regions. Their habitat and small seeds may have facilitated the global distribution of the taxon by migrating birds (Darwin 1872). The worldwide but discontinuous distribution includes one species in northern circumpolar regions, two in southern circumpolar regions, two in Americas, one endemic to Australia, and six in tropical or southern Africa or both (Cook 2004; Glück 1934; Fig. 1). Given the species richness of the genus and the distribution patterns of the related genera, such as Jamesbrittenia Kuntze, Lyperia Benth., Manulea L., Selago L. (Kornhall and Bremer 2004; Oxelman et al. 2005), it is reasonable to postulate that Limosella originated in southern Africa (Kornhall and Bremer 2004).
Fig. 1

Map of sampling localities of Limosella species. The main distribution areas of Limosella are roughly shaded with circles and letters referring to those used in Fig. 5. Species not included in this study are shown by area in grey font

While many of the species are either distributed in Africa or geographically close to Africa (Limosella aquatica L.), a few species are distributed in distant regions, i.e., L. americana Glück, L. curdieana F. Muell., and L. subulata E. Ives. Among these, L. americana described by Glück (1934) is sometimes recognized as a distinct taxon in Central and South America but was recently merged into L. aquatica (Brako and Zarucchi 1993; Cook 2004). Limosella subulata from North and South America has been recognized in some regional Floras (Brako and Zarucchi 1993; Crow and Hellquist 2000), but occasionally treated as a synonym of L. australis R.Br. (Cook 2004). Limosella curdieana, an Australasian endemic species, in contrast, is remarkable as it has never been synonymized, nor has its phylogenetic origin been inferred (Barker 1986, 1999; Harden 1992; Moore 1961).

Glück (1934) proposed an infrageneric classification of Limosella based on leaf morphology and habitat types, in which L. curdieana and L. grandiflora Benth (including its synonym, L. capensis Thunb.: Hilliard and Burtt 1986) are grouped together. On the basis of floral characters, however, L. curdieana appears to have affinities to the tropical African species, L. africana Glück and L. major Diels, as well as the circumpolar L. aquatica by having “petal lobes equal to or shorter than the sepals” (Figs. 2, 3; Barker 1986, 1999; Cook 2004; Godfrey and Wooten 1981; Gorshkova 1997; Harden 1992; Hong et al. 1998; Ivanina 2001; Moore 1961; Webb 1972; Yamazaki 1993).
Fig. 2

Comparison of key morphological features of the petiolate-leaved Limosella species. Usual morphological variation is indicated with boxes; maximum and minimum values extracted from published taxon descriptions are indicated with bars. a Lengths of petioles; b length of sepals; c length of petals; d length of capsules; e the ratio of petal length to sepal length; f the ratio of capsule length to sepal length. A vertical line show traits (mm) in ad and ratios in e, f. A: L. africana (Cook 2004); B: L. africana (Ghazanfar et al. 2008); C: L. major (Cook 2004); D: L. major (Philcox 1990); E: L. major (Ghazanfar et al. 2008); F: L. grandiflora (Cook 2004); G: L. infata (Cook 2004); H: L. vesiculosa (Cook 2004); I: L. aquatica (Hong et al. 1998); J: L. aquatica (Yamazaki 1993); K: L. aquatica (Gorshkova 1997); L: L. curdieana (Barker 1986, 1999; Harden 1992)

Fig. 3

Limosella curdieana in its natural habitat. Photos courtesy of the South Australian Seed Conservation Centre, Australia (http://www.saseedbank.com.au)

We aim to test these two competing hypotheses for Limosella curdieana in Australasia and examine whether the species is phylogenetically related to an African species, L. grandiflora, or to a Northern circumpolar species and other African species, i.e., L. africana and L. major. For that purpose, we employed simultaneous molecular phylogenetic analyses of plastid DNA (subsequently referred to as ptDNA) and nuclear ITS (subsequently referred to as nrITS) DNA sequences based on our worldwide taxon sampling of Limosella.

Materials and methods

Taxon sampling

Samples of Limosella were collected in the field or obtained from herbaria (Table 1). We follow a broadened taxonomic concept of twelve species in the genus. Because the only comprehensive revision made by Glück (1934) does not provide a key to the species, regional treatments were used for African (Cook 2004), Australasian (Barker 1986, 1999; Cook 2004; Harden 1992; Moore 1961), European (Webb 1972), North American (Crow and Hellquist 2000), and South American species (Cook 2004; Crow and Hellquist 2000). Yamazaki (1993) was consulted to confirm if our collection from Japan corresponded to the only species in the flora. Except for L. grandiflora with characteristic elongated stems (Cook 2004), positive identification of samples without flowers was problematic and these samples were treated as Limosella sp. Our taxon sampling, including four samples used in previous molecular phylogenetic studies (Kornhall and Bremer 2004; Oxelman et al. 2005), covers eight species: L. africana (3 specimens); L. aquatica (5; two from Europe, two from North America, and one from temperate Asia); L. australis (3; two from the Falklands and one from New Zealand); L. curdieana (2); L. grandiflora (4); L. macrantha R.E. Fr.(1); L. major (2); L. subulata (1); and Limosella sp. (1). The specimen UPS:BOT:V-120091, initially identified as L. grandiflora, was re-identified to be L. africana. In the present study we did not include specimens corresponding to L. americana in Central and South America, L. inflata Hilliard & B.L. Burtt and L. vesiculosa Hilliard & B.L. Burtt, both confined to small regions of South Africa, and L. longiflora, a widespread, close relative of L. australis (Cook 2004). Outgroup taxa were chosen following Kornhall and Bremer (2004) and Oxelman et al. (2005): species of Lyperia and Jamesbrittenia from the tribe Limoselleae Dumort., representing the sister genus to Limosella (Lyperia) and the basal genus of the tribe (Jamesbrittenia) for initial molecular phylogenetic analysis; those of Barthlottia, Chenopodiopsis, Cromidon, Dischisma, Glumicalyx, Hebenstretia, Jamesbrittenia, Lyperia, Manulea, Melanospermum, Microdon, Phyllopodium, Polycarena, Pseudoselago, Reyemia, Selago, Sutera, Tetraselago, Trieenea, and Zaluzianskya for species tree analysis (Table 1; Online resource 1). Glekia is not included due to the lack of nrITS data.
Table 1

Specimen and voucher information for the taxa included in this study

Species

No.

Locality

Voucher

ndhF

rbcL

rps16

trnL-trnF

nrITS

Jamesbrittenia megadenia

 

South Africa

UPS:BOT:V-152759

AJ401404

n/a

n/a

AJ296511

AJ550584

Jamesbrittenia foliolosa

 

South Africa

Goldblatt P. & Porter L. 12488 (NBG)

n/a

AM235139

n/a

n/a

n/a

Lyperia antirrhinoides

 

South Africa

Hagstroem & Acock 1162 (S)

AJ401405

n/a

n/a

AJ296521

AJ616324

Lyperia tristis

 

South Africa

Manning J.C. 2854 (NBG)

n/a

AM235140

n/a

n/a

n/a

Limosella africana

α

South Africa: Eastern Cape; Elandsberg

UPS:BOT:V-120091

AJ550552

LC132983

LC133001a

AJ550525

AJ550587

Limosella africana

β

Namibia: Hunsberge; Nuobrivier

W. Giess & M. Muller 14313 (PRE)

n/a

LC132985

LC133003

LC133018

LC133035

Limosella africana

γ

South Africa: Central Cape; De Kom/Aarfontein

N.H. Helme 1541 (NBG)

n/a

LC132984

LC133002

LC133017

LC133034

Limosella aquatica

α

Hungary: South

YI1617 (TNS)

LC132973

LC132991

LC133009

LC133024

LC133041

Limosella aquatica

β

Sweden: Östergötland

UPS:BOT:V-155230

AJ550547

n/a

n/a

n/a

AJ550588

Limosella aquatica

γ

Japan: Saitama; Koshigaya

TD4036 (TNS)

LC132972

LC132990

LC133008

LC133023

LC133040

Limosella aquatica

δ

USA: Nebraska

NEB:295749

LC132974

LC132992

LC133010

LC133025

LC133042

Limosella aquatica

ε

USA: Nebraska

NEB:289070

LC132975

LC132993

LC133011

LC133026

LC133043

Limosella australis

α

UK: Falklands

K:14895

n/a

LC132986

LC133004

LC133019

LC133036

Limosella australis

β

UK: Falklands

K:39593

LC132969

LC132987

LC133005

LC133020

LC133037

Limosella australis

γ

New Zealand: Canterbury; Lake Forsyth

YI1769 (TNS)

LC132970

LC132988

LC133006

LC133021

LC133038

Limosella curdieana

α

Australia: Victoria; Gunbower Isl.

MEL:2371937

LC132976

LC132994

LC133012

LC133027

LC133044

Limosella curdieana

β

Australia: Queensland; South Glen

CANB:00703245

LC132977

LC132995

LC133013

LC133028

LC133045

Limosella grandiflora

α

South Africa: Northeast Cape; Springbok

A. Le Roux 2355 (PRE)

n/a

LC132981

LC132999

n/a

LC133032

Limosella grandiflora

β

South Africa: Western Cape; Riebeeckasteel

NBG:0272166-0

LC132967

LC132980

LC132998

LC133015

LC133031

Limosella grandiflora

γ

South Africa: Western Cape; Knolfontein

NBG:0230228-0

n/a

LC132978

LC132996

n/a

LC133029

Limosella grandiflora

δ

South Africa: Western Cape; Swartruggens

NBG:0249071-0

LC132966

LC132979

LC132997

LC133014

LC133030

Limosella macrantha

 

Ethiopia: Bale Prov.; Bale Mts. Natl. park

UPS:BOT:V-152745

AJ550553

n/a

n/a

AJ550526

AJ550586

Limosella major

α

Ethiopia: Begemdir Prov.; Simien

UPS:BOT:V-152744

AJ550548

n/a

n/a

n/a

AJ550585

Limosella major

β

Ethiopia: Arsi-Robe/Sertu

K:46167

LC158860

LC158859

LC158861

LC158862

LC158858

Limosella subulata

 

Ecuador: Napo; Río Chalupas

Lagaard 101769 (AAU)

LC132971

LC132989

LC133007

LC133022

LC133039

Limosella sp.

 

South Africa: Western Cape; Swartruggens

YI1991 (TNS)

LC132968

LC132982

LC133000

LC133016

LC133033

Sequences generated in the present study are underlined. Herbarium acronyms are in accordance with Index Herbariorum (http://sciweb.nybg.org/science2/IndexHerbariorum.asp)

aThis replaces AJ609170

DNA extraction, amplification, and sequencing

Total genomic DNA was extracted from silica gel-dried leaf tissues or from herbarium specimens using the CTAB method described in Ito et al. (2010). Parts of the plastid DNA regions, ndhF, rbcL, rps16 and trnT-trnF, and nrITS were PCR amplified with the following primers: ndhF-F2 (Oxelman et al. 1999) and ndhF-1955R.re (5′-CGATTATAKGACCAATTATATA) modified from Olmstead and Sweere’s (1994) ndhF-1955R for ndhF; rbcL-F1F (Wolf et al. 1994) and rbcL-1379R (Little and Barrington 2003) for rbcL; rps16-1F and rps16-2R (Oxelman et al. 1997); “a” and “b” for trnT-trnL and “c” and “f” for trnL-trnF (Taberlet et al. 1991); ITS-4 and ITS-5 for nrITS (Baldwin 1992). PCR amplification was performed using TaKaRa Ex Taq polymerase (TaKaRa Bio, Shiga, Japan), and PCR cycling conditions were 94 °C for 60 s; then 30 cycles of 94 °C for 45 s, 52 °C for 30 s, 72 °C for 60 s; and finally 72 °C for 5 min. The PCR products were cleaned using ExoSAP-IT (GE Healthcare, Piscataway, NJ, USA) purification and amplified using ABI PRISM Big Dye Terminator ver. 3.1 (Applied Biosystems, Foster City, CA, USA) with the same primers as those used for the PCR amplifications. DNA sequencing was performed with an ABI PRISM 377 DNA sequencer (Applied Biosystems). Automatic base-calling was checked by eye using Genetyx-Win ver. 3 (Software Development Co., Tokyo, Japan). The sequences generated and their metadata were submitted to the DNA Data Bank of Japan (DDBJ), which is a GenBank data provider (Table 1).

Data analysis

We assembled two datasets of Limosella: (1) ptDNA (ndhF, rbcL, rps16, and trnT-trnF); and (2) nrITS. Sequences were aligned using Mafft ver. 7.058 (Katoh and Standley 2013) and then inspected manually. We used “leave gappy regions” option in Mafft to code gaps found in ndhF, rps16, trnT-trnF, and nrITS.

The incongruence length difference test (Farris et al. 1994) in PAUP* ver. 4.0b10 (Swofford 2002) was employed to test for phylogenetic congruence among the four ptDNA regions using a partition homogeneity test with 1,000 replicates. This test did not reveal significant heterogeneity among genes (P value >0.05), and all subsequent analyses were therefore performed with a combined data set of ptDNA.

Phylogenetic inference was performed using maximum parsimony (MP) in PAUP* (Swofford 2002) and Bayesian inference (BI; Yang and Rannala 1997). In the MP analysis, a heuristic search was performed with 100 random addition replicates involving tree-bisection-reconnection (TBR) branch swapping, with the MulTrees option in effect. The MaxTrees option was set at 100,000. Bootstrap analyses (Felsenstein 1985) were performed using 1,000 replicates with TBR branch swapping and simple addition of sequences. The MaxTrees option was set to 1,000. Gaps were treated as binary characters.

BI analyses were conducted with MrBayes ver. 3.2.2 (Ronquist and Huelsenbeck 2003; Ronquist et al. 2012) run on the CIPRES portal (Miller et al. 2010) after the best models had been determined in MrModeltest ver. 3.7 (Nylander 2002); these models were GTR + I + Γ and GTR + Γ for ptDNA and nrITS, respectively. For gap characters, the “datatype = standard” option of MrBayes was used and default prior settings were applied, i.e., “ratepr = variable”. Analyses were run for 670,000 and 415,000 generations for ptDNA and nrITS, respectively, until the average standard deviation of split frequencies dropped below 0.01, sampling every 1,000 generations and discarding the first 25% as burn-in. The convergence and effective sampling sizes (ESS) of all parameters were checked in Tracer ver. 1.6 (Rambaut et al. 2014). All trees were visualized using FigTree ver. 1.3.1 (Rambaut 2009). Nodes are recognized as strongly, moderately, or weakly supported with: ≥95% bootstrap support (BS), ≥0.99 Bayesian posterior probabilities (PP); ≥70% BS, ≥ 0.95 PP; or <70% BS; <0.95 PP, respectively. The data matrices and the MP and BI trees are available at Treebase (TB2:S19401).

Species trees considering all samples were reconstructed for biogeographic analysis. Limosella macrantha was excluded due to incongruent positions between ptDNA and nrITS trees (see “Results”). A multispecies coalescent method (Heled and Drummond 2010) implemented in BEAST ver. 1.7.2 (Drummond et al. 2006; Drummond and Rambaut 2007) was performed. We ran *BEAST using the two data sets (ptDNA and nrITS) from 21 samples from seven ingroup taxa and assigning them to six terminal species, namely L. aquatica, L. australis, L. curdieana, L. grandiflora, L. major, plus Limosella sp.

We performed two independent runs of ten million generations of the MCMC chains, sampling every 1,000 generations. Convergence of the stationary distribution was checked by visual inspection of plotted posterior estimates using Tracer ver. 1.5 (Rambaut and Drummond 2007). After discarding the first 1,000 trees as burn-in, the samples were summarized in the maximum clade credibility tree using TreeAnnotator ver. 1.6.1 (Drummond and Rambaut 2007) with a posterior probability limit of 0.5 and summarizing mean node heights. The results were visualized using FigTree ver. 1.3.1 (Rambaut 2009).

Biogeographic analysis

Reconstruction of historical biogeography of Limosella was performed using RASP ver. 3.2 (Reconstruct Ancestral State in Phylogenies) (Yu et al. 2015). The Bayesian Binary Method (BBM; Ronquist and Huelsenbeck 2003) was selected because it: (1) accepts polytomies; (2) tends to suggest single distribution areas for ancestral nodes more often than others (Müller et al. 2015); and (3) is capable of providing unambiguous and informative results (Ito et al. 2016). BBM was conducted using the post burn-in species trees that resulted from the *BEAST analysis. The following seven biogeographic areas were defined: (a) Europe; (b) temperate Asia; (c) North America; (d) tropical Africa; (e) southern Africa; (f) Australasia; (g) South America (including Falklands). For the distribution of the species we used the area including the locality of accessions from the molecular phylogenetic analysis of the present study. Multiple ancestral states were allowed. The number of generations was set to 10 million and the first 10% of the samples were discarded as burn-in. All other parameters were kept at default settings.

Results

Molecular phylogeny

Sequences of the concatenated four ptDNA regions of Limosella consisting of 22 ingroup and two outgroup samples resulted in an alignment with a total length of 5,067 bp. In total 428 characters including 29 binary-coded indels were polymorphic, of which 214 were parsimony-informative. Percentage of missing characters and gaps were: 15.20 and 0.00% (rbcL); 22.37 and 9.21% (rps16); 19.64 and 12.74% (trnL); 46.58 and 0.30% (ndhF) (Table 2). Analysis of this data set yielded the imposed limit of 100,000 MP trees (tree length = 466 steps; consistency index = 0.96; retention index = 0.96). The strict-consensus MP tree and MrBayes BI 50% consensus trees showed no incongruent phylogenetic relationships. Therefore, the better resolved MrBayes tree is presented (Fig. 4a).
Table 2

Percentage of missing characters and gaps by DNA regions

 

Missing characters

Total charactersa

Percentage of missing characters (%)

Gap length

Total alignment length

Percentage of gaps (%)

rbcL

4,858

31,968

15.20

0

1,332

0.00

rps16

3,705

16,560

22.37

70

760

9.21

trnL

4,035

20,544

19.64

125

981

12.74

ndhF

21,833

46,872

46.58

12

3,937

0.30

nrITS

917

16,296

5.63

12

687

1.75

aGaps are excluded

Fig. 4

MrBayes trees of Limosella based on a plastid DNA and b nuclear ITS. Branch lengths are proportional to molecular divergence among accessions. Numbers above or below the branches indicate bootstrap support (BS) calculated in maximum parsimony and Bayesian posterior probabilities (PP). BS < 50% and PP < 0.9 are indicated by hyphens while those of ≥95% and ≥0.99 are asterisks. Moderately- to strongly-supported groups are surrounded by round rectangles in background in gray and numbered. It should be noted, however, that group III is paraphyletic in ptDNA due to the inconsistent position of L. macrantha (see “Discussion”)

Limosella was divided into two strongly-supported groups: a clade of four accessions of L. grandiflora (group I; 100% BS; 1.0 PP) and the rest of the genus (100% BS; 1.0 PP). In the latter clade, Limosella sp. was placed as sister to the remaining accessions (89% BS; 1.0 PP). The other supported groups were (1) L. australis and L. subulata (group II; 88% BS; 1.0 PP) and (2) L. aquatica, L. curdieana, L. macrantha, and L. major (group III plus L. macrantha; <50% BS; 0.98 PP). Three accessions of L. africana were resolved in a polytomy with L. australis-L. subulata and L. aquatica-L. curdieana-L. macrantha-L. major.

The nrITS alignment consisting of 22 ingroup and two outgroup sequences had a total length of 691 bp. In total 121 characters including four binary-coded indels were polymorphic, of which 63 were parsimony-informative. Percentage of missing characters and gaps were: 5.63 and 1.75%, respectively (Table 2). Analysis of this data set yielded 22 MP trees (tree length = 166 steps; consistency index = 0.86; retention index = 0.89). The strict-consensus MP tree and MrBayes BI 50% consensus trees showed no incongruent phylogenetic relationships. Therefore, the better resolved MrBayes tree is presented (Fig. 4b).

A topology similar to that of ptDNA was recovered, with two strongly-supported clades: one comprising four accessions of Limosella grandiflora (group I; 87% BS; 1.0 PP) and another consisting of the remaining taxa (99% BS; 1.0 PP). In the clade of 17 accessions, L. africana UPS:BOT:V-120091 was placed as sister to the remaining accessions (68% BS; 0.99 PP). A clade of L. macrantha and L. africana β (87% BS; 0.98 PP) positioned sister to the remaining accessions (<50% BS; 1.0 PP). Group II, consisting of L. australis and L. subulata (88% BS; 0.99 PP) and group III (L. aquatica, L. curdieana, and L. major; 83% BS; 1.0 PP) formed a clade in the BI analysis (<50% BS; 0.97 PP), which was placed as sister to L. africana N.H. Helme 1541 (NBG).

The *BEAST species tree analysis retrieved two lineages: (a) BCDGHMPRSTZ (Barthlottia; Chenopodiopsis; Cromidon; Dischisma; Glumicalyx; Hebenstretia; Manulea; Melanospermum; Microdon; Phyllopodium; Polycarena; Pseudoselago; Reyemia; Selago; Sutera; Tetraselago; Trieenea; Zaluzianskya); (b) Limosella and Lyperia (Fig. 5).
Fig. 5

Bayesian *BEAST species tree for Limosella based on plastid DNA and nuclear ITS data. The outgroup clade (BCDGHMPRSTZ: Barthlottia; Chenopodiopsis; Cromidon; Dischisma; Glumicalyx; Hebenstretia; Manulea; Melanospermum; Microdon; Phyllopodium; Polycarena; Pseudoselago; Reyemia; Selago; Sutera; Tetraselago; Trieenea; Zaluzianskya) has been collapsed. Values above or below the branches represent the Bayesian posterior probabilities (PP). PP ≥ 0.95 are indicated by asterisks. Clades without significant support (PP < 0.75) are collapsed. Ancestral areas inferred from Bayesian Binary MCMC (BBM) are shown on each node. Multiple posterior results are shown in a box. The area codes follow Fig. 1 (A Europe; B temperate Asia; C North America; D tropical Africa; E southern Africa; F Australasia; G Central and South America)

Biogeographic reconstruction

The ancestral area of Limosella was inferred in southern Africa (1.0 PP); ambiguous results were obtained for the ancestral area of the most recent common ancestor (MRCA) of L. aquatica, L. curdieana, and L. major with highest probability for Australia (70.9 PP) (Fig. 5; Online resource 2).

Discussion

Phylogeny of Limosella and the position of L. curdieana

The present study reconstructed the most detailed molecular phylogeny of the genus Limosella to date with a primary aim to test two competing hypotheses for ancestral relationships of the Australasian species, L. curdieana, i.e., whether the species is phylogenetically closely related to (1) the African species, L. grandiflora, or (2) a group composed of the two African species, L. africana and L. major, and the Northern circumpolar species, L. aquatica. The topologies recovered based on ptDNA and nrITS, respectively, are mostly congruent, except for a single accession of L. macrantha (see below). Both topologies resolve L. grandiflora as sister to the remaining species, including L. africana, L. aquatica, L. curdieana, and L. major (Fig. 4). The molecular evidence thus clearly rejected the hypothesis proposed by Glück (1934) and instead supported a relationship of L. curdieana with L. africana, L. major and L. aquatica.

Topological incongruence between ptDNA and nrITS

We detected a topological incongruence caused by our single accession of Limosella macrantha. In the ptDNA phylogeny this tropical African species (Ghazanfar et al. 2008; Glück 1934) is resolved in a clade including multiple accessions of the likewise tropical African species, L. major, whereas in the ITS phylogeny it is sister to southern African L. africana. Although morphological evidence suggests a close relationship between L. macrantha and L. australis (including specimens previously segregated as L. subulata) (Cook 2004; Glück 1934), the single accession of L. macrantha obtained from previous phylogenetic studies (Kornhall and Bremer 2004; Oxelman et al. 2005) was resolved as distantly related to L. australis (Fig. 4). Further taxon and data sampling will reveal whether this single specimen is part of an introgressive hybrid swarm or represents a hybrid. Alternatively, incomplete lineage sorting cannot be excluded as cause of the topological conflicts since this is common in closely related lineages such as Limosella.

Biogeography of Limosella and implications for dispersal

The present study supports a basal diversification of Limosella in southern Africa with moderate to strong support (Fig. 5), confirming a southern African origin of the genus as indicated by species richness of the ingroup and the distribution of sister genera. Thus, southern Africa seems to be a cradle for not only a number of drought-adapted taxa such as Crassulaceae (Mort et al. 2001), Scrophulariaceae (Oxelman et al. 2005), Thesium L. (Moore et al. 2010), Amaryllidoideae (Rønsted et al. 2012), and Asparagus L. (Norup et al. 2015) but also aquatics such as Limosella.

Bell et al. (2010) provided an estimated origin of Scrophulariaceae earlier than the mid-Paleogene, since tribes Myoporeae and Scrophularieae diversified ca. 51–53 mya. As Limosella is derived much later in the evolution of the family (Oxelman et al. 2005), vicariance due to plate tectonics is not an option to explain the discontinuous distribution worldwide and instead seed dispersal is more likely as is mentioned by Cook (2004): “the disseminules are small seeds, dispersed in mud and perhaps otherwise.” The current distribution of L. australis (including L. subulata) in e.g., Ecuador, New Zealand, and the Falklands could be a case of “wind highways” existing in the Southern Hemisphere (Muńoz et al. 2004).

The biogeographic origin of Limosella curdieana remains uncertain, but a close relationship with L. aquatica from Europe, temperate Asia, and North America, and L. major from tropical Africa (Ethiopia) is moderately supported (Fig. 4). The biogeographic analysis suggests Australasia as the ancestral area for the MRCA of the three species and thus implies a dispersal route from southern Africa to Australasia, and then to temperate Asia and tropical Africa (Fig. 5). Alternatively, considering the facts that (1) migratory bird routes are well documented between Asia and Australasia (Boere and Stroud 2006), (2) case studies exist that reveal disjunct distributions between Asia and Australasia (Lobelia L.: Kokubugata et al. 2012; Solenogyne Cass.: Nakamura et al. 2012), and (3) young plant groups in Australia have predominantly migrated from Asia (Crisp and Cook 2013), dispersal to Australasia via temperate Asia may also explain the geographic isolation of L. curdieana in Australia.

Implications for taxonomy of Limosella

Our molecular phylogenetic results provide additional insights into the taxonomy of Limosella. The three specimens of L. africana show close relationships, especially in ptDNA analysis, but do not form a clade in either of the analyses. No significant morphological differences are observed among the specimens except leaf shape, i.e., linear-lanceolate leaves for L. africana α and ovate leaves for L. africana β and L. africana γ. Still, all three specimens could belong to L. africana var. africana Glück, even in the strict sense, because none of these have much-clustered, short-pedicellate flowers or fruits (Cook 2004; Glück 1934). The sterile Limosella sp. shows affinities to L. africana var. africana in vegetative morphology, i.e., ovate leaves. Additional taxon sampling in flower and fruit from the locality and/or nearby habitats will provide further insight into the affinities of this accession, e.g., whether it belongs to either of the varieties, or represents an undescribed taxon. Since non-monophyletic taxa may be the result of various processes such as incomplete lineage sorting, a proper investigation of the species status of L. africana would require sampling from multiple loci in a species tree context.

The single accession of L. subulata from Ecuador forms a clade with three accessions of L. australis from New Zealand and the Falklands. This result agrees with the taxonomic treatment by Cook (2004) who synonymized L. subulata with the widespread L. australis. Further studies based on increased taxon and data sampling focusing on the taxonomic status of these taxa are needed.

Conclusions

Within a phylogenetic framework we provided new insights into the evolutionary origin of L. curdieana, the only Australasian endemic species in the genus. Our molecular phylogenetic analyses based on ptDNA and nrITS data sets revealed a basal diversification in southern Africa and close relationships between L. curdieana and northern circumpolar as well as a species from tropical Africa. The biogeographic analysis points to an out-of-southern Africa into the Northern Hemisphere and subsequent dispersal back into the Southern Hemisphere. Past dispersal from southern Africa to Australasia is suggested, however, we cannot exclude the existence of a route via tropical Africa and Asia.

Notes

Acknowledgements

The authors thank R. Kaul (NEB) for providing Limosella materials, T. Trinder-Smith (BOL), J. Palmer (CANB), C. Gallagher, P. Milne (MEL), C. Cupido (NBG), and E. van Wyk (PRE) for arranging loans from their institutions and/or for hospitality during our research visits; M. Hjertson (UPS) for sending scanned images of voucher specimens of Limosella at UPS; C. Ishii (Tsukuba) for help with DNA sequencing; J. Guerin (South Australian Seed Conservation Center, Australia) for providing L. curdieana photos. We would also like to thank P. B. Pelser (CANU), J. Murata, H. Ikeda, and T. Ohi-Toma (TI), and J. Li (Kunming) for their continuous encouragements and supports. YI and AMM received plant collecting permit in Western Cape, South Africa from Cape-Nature. This research was supported by FY 2012 Researcher Exchange Program between Japan society for the promotion of science (JSPS) and Royal Society of New Zealand (RSNZ) to YI, JSPS KAKENHI Grant Number 25440224 to NT, and the South African National Research Foundation (NRF) to AMM.

Supplementary material

10265_2016_872_MOESM1_ESM.docx (18 kb)
Supplementary material 1 (DOCX 17 kb)
10265_2016_872_MOESM2_ESM.docx (251 kb)
Supplementary material 2 (DOCX 251 kb)

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Copyright information

© The Botanical Society of Japan and Springer Japan 2016

Authors and Affiliations

  • Yu Ito
    • 1
    • 2
  • Norio Tanaka
    • 3
  • Dirk C. Albach
    • 4
  • Anders S. Barfod
    • 5
  • Bengt Oxelman
    • 6
  • A. Muthama Muasya
    • 7
  1. 1.Biological SciencesUniversity of CanterburyChristchurchNew Zealand
  2. 2.Xishuangbanna Tropical Botanical GardenThe Chinese Academy of SciencesKunmingPeople’s Republic of China
  3. 3.Tsukuba Botanical GardenNational Museum of Nature and ScienceTokyoJapan
  4. 4.Institute of Biology and Environmental Sciences (IBU)Carl von Ossietzky-University OldenburgOldenburgGermany
  5. 5.Department of BioscienceAarhus UniversityAarhus CDenmark
  6. 6.Department of Biological and Environmental SciencesUniversity of GothenburgGothenburgSweden
  7. 7.Department of Biological SciencesUniversity of Cape TownCape TownSouth Africa

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