Journal of Systematics and Evolution 51 (2): 142–153 (2013) doi: 10.1111/j.1759-6831.2012.00226.x Research Article Exploring the utility of three nuclear regions to reconstruct reticulate evolution in the fern genus Asplenium 1,2 Harald SCHNEIDER ∗ 2 Adriana NAVARRO-GOMEZ 2 Stephen J. RUSSELL 2 Stephen ANSELL 2 Michal GRUNDMANN 2 Johannes VOGEL 1 (State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China) 2 (Department of Life Sciences, Natural History Museum, London SW7 5BD, UK) Abstract Three nuclear regions nuclear ribosomal internal transcribed spacer (nrITS) and intron regions of two nuclear low-copy genes—gapCp, and pgiC, in combination with one chloroplast genome region were employed to explore patterns of reticulate evolution in the fern genus Asplenium. This is the one of the first studies using DNA sequences of multiple nuclear markers in ferns. All three nuclear markers amplified well with PCR and several copies were recovered by cloning PCR products. All three nuclear regions showed congruent results by recovering the neo-allotetraploid Asplenium adulterinum as the hybrid of diploid A. trichomanes and diploid A. viride. Conflicting results were obtained for several nodes. First, gapCp did not discriminate between A. aethiopicum and A. praegracile whereas the other markers recovered these two taxa as distinct. Conflicts among gene-trees were found in respect to A. monanthes, chloroplast and pgiC suggested a sister relationship of A. monanthes and A. trichomanes but gapCp and nrITS nested A. monanthes within A. normale. Our results confirm: (i) the usefulness of several nuclear regions, in particular gapCp and pgiC, to unravel reticulate evolution in ferns and species differentiation and (ii) highlights the need to employ more than one nuclear region to obtain reliable hypotheses on reticulate events versus incomplete lineage sorting. Especially, if one assumes that the reticulation event might have occurred in the more distant past. Considering the expected high frequency of reticulate evolution in ferns, the establishment of robust and informative nuclear genomic markers is critical to achieve further progress in our efforts to elucidate fern evolution. Key words ferns, gapCp, GAPDH, hybridization, introgression, nrITS, nuclear genes, pgiC, reticulate evolution. Reticulate evolution is a common mechanism in plant evolution (Baack & Rieseberg, 2007; Wood et al., 2009) and in particular in fern evolution (Lo- vis, 1977; Haufler, 2002). Polyploid speciation involv- ing hybridization of diploid parents is well documented for several fern genera including Asplenium (Wagner, 1954; Lovis, 1973, 1977; Brownsey, 1975; Werth et al., 1985; Vogel et al., 1996, 1999), which is in turn the most species-rich fern genus, comprising at least 700 species (Smith et al., 2006). Currently, we cannot fully evaluate the contribution of reticulate evolution to this astonishing fern diversity because only a small num- ber of species have been studied experimentally. The detection of reticulate evolution and its reconstruction using phylogenetic networks may be inferred using ge- nomic marker combinations capable of determining his- toric differences among partitions of genomes, con- Received: 11 June 2012 Accepted: 20 August 2012 ∗ Author for correspondence. E-mail: [email protected]. Tel.: 44-20- 7942-6058. Fax: 44-20-7942-5529. tributing to genomes originated by hybridization (John- son & Soltis, 1998; Wendel & Doyle, 1998; Linder & Rieseberg, 2004; Brysting et al., 2007). While nu- clear markers are commonly used with other vascular plants, studies on ferns including spleenworts mostly rely on sequences from plastid regions (e.g. Pryer et al., 2004; Schneider et al., 2004a, 2004b, 2005; Perrie & Brownsey, 2005b; Smith et al., 2006). This “plastid only approach”—studying only se- quences of the chloroplast genome—has advantages and disadvantages. Chloroplast DNA phylogenies re- construct only the uniparental [usually maternal], inher- ited plastid genome in ferns (Gastony & Yatskievych, 1992; Vogel et al., 1998). As a consequence, chloroplast- based phylogenetic hypothesis are not suited to detect reticulate events, for example, hybridization and intro- gression. Most phylogenetic studies on ferns were able to ignore this shortcoming because the pertinent re- search questions focused on the relationships of deeper nodes in the fern phylogeny, where reticulate evolution is assumed to be less important. However, the “plastid C 2012 Institute of Botany, Chinese Academy of Sciences
Transcript
Journal of Systematics and Evolution 51 (2): 142–153 (2013) doi: 10.1111/j.1759-6831.2012.00226.x
Research Article
Exploring the utility of three nuclear regions to reconstruct reticulateevolution in the fern genus Asplenium
1(State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China)2(Department of Life Sciences, Natural History Museum, London SW7 5BD, UK)
Abstract Three nuclear regions nuclear ribosomal internal transcribed spacer (nrITS) and intron regions of twonuclear low-copy genes—gapCp, and pgiC, in combination with one chloroplast genome region were employedto explore patterns of reticulate evolution in the fern genus Asplenium. This is the one of the first studies usingDNA sequences of multiple nuclear markers in ferns. All three nuclear markers amplified well with PCR and severalcopies were recovered by cloning PCR products. All three nuclear regions showed congruent results by recovering theneo-allotetraploid Asplenium adulterinum as the hybrid of diploid A. trichomanes and diploid A. viride. Conflictingresults were obtained for several nodes. First, gapCp did not discriminate between A. aethiopicum and A. praegracilewhereas the other markers recovered these two taxa as distinct. Conflicts among gene-trees were found in respect toA. monanthes, chloroplast and pgiC suggested a sister relationship of A. monanthes and A. trichomanes but gapCpand nrITS nested A. monanthes within A. normale. Our results confirm: (i) the usefulness of several nuclear regions,in particular gapCp and pgiC, to unravel reticulate evolution in ferns and species differentiation and (ii) highlightsthe need to employ more than one nuclear region to obtain reliable hypotheses on reticulate events versus incompletelineage sorting. Especially, if one assumes that the reticulation event might have occurred in the more distant past.Considering the expected high frequency of reticulate evolution in ferns, the establishment of robust and informativenuclear genomic markers is critical to achieve further progress in our efforts to elucidate fern evolution.Key words ferns, gapCp, GAPDH, hybridization, introgression, nrITS, nuclear genes, pgiC, reticulate evolution.
Reticulate evolution is a common mechanismin plant evolution (Baack & Rieseberg, 2007; Woodet al., 2009) and in particular in fern evolution (Lo-vis, 1977; Haufler, 2002). Polyploid speciation involv-ing hybridization of diploid parents is well documentedfor several fern genera including Asplenium (Wagner,1954; Lovis, 1973, 1977; Brownsey, 1975; Werth et al.,1985; Vogel et al., 1996, 1999), which is in turn themost species-rich fern genus, comprising at least 700species (Smith et al., 2006). Currently, we cannot fullyevaluate the contribution of reticulate evolution to thisastonishing fern diversity because only a small num-ber of species have been studied experimentally. Thedetection of reticulate evolution and its reconstructionusing phylogenetic networks may be inferred using ge-nomic marker combinations capable of determining his-toric differences among partitions of genomes, con-
Received: 11 June 2012 Accepted: 20 August 2012∗ Author for correspondence. E-mail: [email protected]. Tel.: 44-20-
7942-6058. Fax: 44-20-7942-5529.
tributing to genomes originated by hybridization (John-son & Soltis, 1998; Wendel & Doyle, 1998; Linder& Rieseberg, 2004; Brysting et al., 2007). While nu-clear markers are commonly used with other vascularplants, studies on ferns including spleenworts mostlyrely on sequences from plastid regions (e.g. Pryer et al.,2004; Schneider et al., 2004a, 2004b, 2005; Perrie &Brownsey, 2005b; Smith et al., 2006).
This “plastid only approach”—studying only se-quences of the chloroplast genome—has advantagesand disadvantages. Chloroplast DNA phylogenies re-construct only the uniparental [usually maternal], inher-ited plastid genome in ferns (Gastony & Yatskievych,1992; Vogel et al., 1998). As a consequence, chloroplast-based phylogenetic hypothesis are not suited to detectreticulate events, for example, hybridization and intro-gression. Most phylogenetic studies on ferns were ableto ignore this shortcoming because the pertinent re-search questions focused on the relationships of deepernodes in the fern phylogeny, where reticulate evolutionis assumed to be less important. However, the “plastid
SCHNEIDER et al.: Nuclear genes and Asplenium phylogeny 143
only approach” is likely to generate misleading resultswhen applied to the most shallow nodes of the fernphylogeny for the separation of species.
The “plastid only approach” has been also thedominant approach in recent phylogenetic studies ofAspleniaceae (Murakami et al., 1999; Schulze et al.,2001; Pinter et al., 2002; Trewick et al., 2002; Schneideret al., 2004a, 2005; Perrie & Brownsey, 2005b; Shep-herd et al., 2007, 2008a; Bellefroid et al., 2010) becausethe majority of these studies focused on the deeper phy-logeny of this lineage. Studies, which focus on recentdiverging species, will require the unraveling and iden-tification of reticulate networks. Most current studieson reticulate evolution in ferns rely on fingerprintingmethods such as allozymes (Vogel et al., 1999; Yatabeet al., 2009), amplified fragment length polymorphism(Perrie & Brownsey, 2005a), and diversity array tech-nology (James et al., 2008), which were sometimes usedwithout in conjunction with plastid DNA sequence data.Such studies will be advanced by the integration of DNAsequences derived from nuclear genomic regions.
Very few studies have so far utilized DNA se-quences of nuclear markers to study the evolution ofclosely related fern species. The markers which havebeen used include the nuclear ribosomal internal tran-scribed spacer (nrITS) region (Wolf, 1996; Gastony &Rollo, 1998; Maggini et al., 1998; van den heede et al.,2003; Reid et al., 2006), the introns of the transcrip-tion factor LEAFY (LFY ) (e.g. Shepherd et al., 2008b),and introns of two low-copy protein encoding regionsthe plastidic glyceraldehydes-3-phosphate dehydroge-nase (gapCp) (e.g. Ebihara et al., 2005; Schuettpelzet al., 2008; Nitta et al., 2011), and the cytosolic phos-phoglucose isomerase [pgiC] (e.g. Ishikawa et al., 2002;James et al., 2008; Chang et al., 2009). The rare appli-cation of nuclear gene regions in fern phylogeneticsis curious given the frequency of nuclear markers em-ployed in phylogenetic studies of other plant groups.For example, nrITS is one of the most commonly usedDNA markers in plant phylogenetics (Hershkovitz et al.,1999).
DNA sequences of nuclear markers have many ad-vantages in the study of the evolution of species andspecies complexes in ferns. Studies based on low-copygenes such as gapCp and LFY have demonstrated thevalue and advantages of such markers (Ebihara et al.,2005; Schuettpelz et al., 2008; Shepherd et al., 2008b;Nitta et al., 2011), because they elucidate most recentevents of fern evolution in great detail. When applyingDNA sequences of nuclear genome markers, an assess-ment of orthology issues needs to be carried out in part tothe high frequency of polyploidy in some fern lineages(Lovis, 1977). Much can be learned from taking advan-
tage of the numerous studies conducted with nucleargenomic markers in angiosperms such as issues con-cerning orthology, concerted evolution, and PCR bias(e.g. Doyle & Doyle, 1999; Linder & Rieseberg, 2004).Considering the expected high frequency of reticulateevolution in ferns, the establishment of a robust and in-formative set of nuclear genomic markers is critical toachieve further progress in our efforts to elucidate theirevolution.
Previous fern studies using nuclear markers havefocused on the use of a single region, for example,gapCp and LFY . In this study, we explore the utilityof three nuclear markers to reconstruct reticulate evo-lution within the genus Asplenium. We generated se-quence data for three nuclear genomic markers (gapCp,nrITS, pgiC). We also generated sequences of the widelyused chloroplast markers, the trnL-trnF region, whichincludes the intron of the trnL and the trnL-trnF in-tergenic spacer. This study has three main objectives:(i) to explore the reproducibility of PCR and DNA se-quencing of the three nuclear regions; (ii) to exploreincongruence among gene-trees obtained from all fourmarkers; and (iii) to infer the potential of any markerof marker combination to unravel reticulate evolution inAsplenium and other ferns.
1 Material and methods
We assembled a dataset including 11 species thatrepresent four of the eight major clades of asplenioidferns (Schneider et al., 2004a, 2005). Two clades wererepresented by a single species, Asplenium adiantum-nigrum and A. (Phyllitis) scolopendrium. The othertwo clades were represented with three and six species,respectively. Asplenium aethiopicum, A. cuneatiforme,and A. praegracile belong to the Tarachia clade (Schnei-der et al., 2004a, 2005). The black stem rock spleenwortswere represented by A. adulterinum, A. dielerecta, A.monanthes, A. normale, A. trichomanes, and A. viride.Only one specimen was collected for each species, ex-cept for A. normale, which was represented by twospecimens. The sampling was designed to enable anassessment of potential conflicts among different phy-logenetic hypotheses obtained by different markers ormarker combinations. The dense sampling of the blackstem rock spleenworts was justified by the need to bothclarify the relationships among the subclades withinthis lineage and the apparently frequent occurrence ofhybridization within this lineage. We included the neo-allotetraploid A. adulterinum, which is originated bythe hybridization of the diploid A. trichomanes anddiploid A. viride (Lovis, 1968; Vogel et al., 1996, 1999;
144 Journal of Systematics and Evolution Vol. 51 No. 2 2013
Table 1 Taxa, area of origin, collector plus collector number and herbarium code, ploidy level, and GenBank accession numbers. Chromosomenumbers were obtained from Index to Plant Chromosome Numbers at http://mobot.org/W3T/search/ipcn and given here as ploidy levels calculated bydividing the diploid chromosome number by 36
Taxon Area Voucher Ploidy trnL-trnF pgiC gapCp nrITS
JX475196 JX475230 JX475167∗Apomictic species.†This species is better known as Diellia erecta Brack. but Diellia is nested within Asplenium (Schneider et al., 2005).‡Asplenium adiantum-nigrum is most likely an autopolyploid of A. onopteris (Vogel et al., 1996).§Asplenium adulteriunum is a neo-allotetraploid between A. trichomanes and A. viride.�Unknown, the species includes diploids and tetraploids.
James et al., 2008). Table 1 comprises all information onvouchers, ploidy levels, and GenBank accession num-bers of specimens used in this study.
Genomic DNA was extracted using a modifiedCTAB approach as described by Trewick et al. (2002)and we also used the previously published primersand protocols to generate sequences of the chloro-plast marker region trnL-trnF region including the in-tron within trnL and the trnL-trnF intergenic spacer(Trewick et al., 2002). The region from exon 14 to exon16 of the single-copy pgiC nuclear gene was amplifiedand sequenced using primers and protocols designedfor Dryopteris (Ishikawa et al., 2002). DNA amplifi-cation of this region resulted in a single well-definedband. The gapCp region was amplified and sequencedusing primers and protocols developed by Schuettpelz
et al. (2008). The amplification product covers the re-gion between exon 8 and exon 11. DNA amplificationresulted in one main product detectable as a brightband in agarose gels, but the PCR product needed tobe cleaned using GFXPCR DNA and Gel Band Purifi-cation kit (Amersham plc, Buckinghamshire, UK). ThenrITS region was amplified using primers and protocolspublished by White et al. (1990).
SCHNEIDER et al.: Nuclear genes and Asplenium phylogeny 145
each of the PCR products. DNA extraction, PCR am-plification, and cloning were reproduced for selectedspecimens to minimize the possibility that potentiallyconflicting phylogenetic signals as a result of contami-nation.
All sequences were generated using an ABI 3730capillary DNA Analyser (Applied Biosystems Inc., Fos-ter City, CA, USA) using Big Dye version 3.1 sequenc-ing mix (Applied Biosystems Inc.) and the PCR primers.The sequences were assembled and edited using theLaserGene v8.0 software (DNASTAR, Madison, WI,USA). All sequences were aligned and visually checkedusing the software MacClade 4.0 (Maddison & Maddi-son, 2005). Single point mutations were ignored as longas they occur only in one of several copies and are notcomplemented by additional single point mutations inthe same sequences. The ignored sequence readings arelikely the result of reading errors generated as a result ofsingle PCR copy amplification via cloning. All copieswere also checked for hybrid sequences. Two hybridsequences were found among the 10 sequences gener-ated for A. adulterinum. They were excluded from theanalyses because they are likely the product of artifactsgenerated during the amplification process.
Each genome region was studied in independentphylogenetic analyses employing maximum parsimony(MP) analyses with PAUP∗ 4.0 (Swofford, 2002), max-imum likelihood (ML) analyses with GARLI 0.96(Zwickl, 2006), and Neighbour-Net analyses (Bryant& Moulton, 2002) with SPLITSTREE 4.6 (Huson &Bryant, 2006). MP analyses were pursued using heuris-tic mode with 100 random starting replicates and TreeBisection/Reconection (TBR) branch swapping. Strictconsensus trees were estimated if more than one maxi-mum parsimonious tree was found. Bootstrap MP anal-yses were carried out using 1000 bootstrap replicatesusing heuristic search mode, 10 random starting repli-cates, and TBR. ML analyses were run using the defaultconditions in GARLI. We were running two ML anal-
yses for each nuclear dataset: (1) with the GTR modelplus gamma and invariant with all parameter estimatesduring the analyses or (2) with the model and parametersestimated using MODELTEST 3.7 (Posada & Crandall,1998). ML bootstrap values were estimated using 100bootstrap replicates and the default procedure of GARLIfor both model specifications. Neighbour-Net analyseswere run using LogDet distances and maximum of fourdimensions. Each accepted copy of the nuclear gene washandled as a terminal taxon.
Visual inspection was used to determine topolog-ical incongruence among the optimal gene-trees (MPand ML) and bootstrap consensus trees of each genewith the following bootstrap values considered: 100%,≥95%, ≥75% (see Mason-Gamer & Kellog, 1996). Be-sides, we carried out partition homogeneity tests us-ing the Incongruence Length Difference Test (ILD) asimplemented in PAUP (Farris et al., 1995) despite thewell-known shortcomings of this test (Barker & Lut-zoni, 2002). Furthermore, we estimated the number ofvariable sites and parsimonious informative sites foreach dataset in PAUP.
A combined dataset was assembled by using asingle sequence of each region for each species withthe exception of A. adulterinum. This species is rep-resented by two artificial terminal taxa reflecting theheritage of the genomic components: A. adulterinum-trichomanes comprise sequence copies inherited fromA. trichomanes, whereas A. adulterinum-viride com-prise sequence copies inherited from A. viride. Missingdata are used to represent copies that were lacking ei-ther for A. trichomanes or A. viride. Variation amongcopies was handled by generating consensus sequences.The combined dataset was analyzed despite the fact thatsome topological heterogeneity was present in the inde-pendent analyses of each region. The combined datasetwas analyzed using MP, ML, and Neighbour-Net as de-scribed above. To take into account the conflicts amonggene-trees, we performed independent MP analyses forall four genome regions using the taxon sample of thecombined dataset and constructed a Z-closure super-network from partial trees (Huson et al., 2004) as im-plemented in SPLITSTREE 4.6.
2 Results
Sequences were generated successfully for all fourstudy regions and for all samples with the exception ofpgiC for Asplenium scolopendrium. Two or more copieswere recovered for all nuclear markers with the excep-tion of nrITS in some specimens. The number of copiescorresponded to the expected ploidy levels of the taxa
SL, sequence length variation; AL, alignment length; CC, percentageof constant character states; PI, percentage of parsimonious informativesides per sequence; PS, pairwise sequence similarity; GC, GC content.
(Table 1). Two copies were recovered in diploid speci-mens, whereas two or more copies were present in poly-ploidy specimens.
Sequence similarity searches using nucleotidemegablast confirmed the identity of the region se-quenced and excluded contamination by nonfern DNA:pgiC sequences had high-scoring segment pairs (HSP)with pgiC of A. viride (EF645631–EF645642) and A.trichomanes (EF645644) but a low score for pgiC ofseveral species of Dryopteris (EU797707–EU797725)and the angiosperm Shorea argentifolia (AB189592);gapCp sequences produced HSP with gapC se-quences of Hymenasplenium unilaterale (EU551420–EU551424) and the nrITS sequence produced HSP withnrITS sequences of Alsophila firma (EU090937), Azollaspp. (DQ066470–DQ066495), Ceratopteris thalictropi-des (AF218261), Cyathea costaricensis (EU0900938),C. lepifera (AF448790), C. myosuroides (EU090939),Lygodium japonicum (AF448793), and Marsileaquadrifolia (AF448792), but not with nrITS sequencesof Asplenium spp. (AY165440–AY165486) accessibleat GenBank. Direct sequence comparison between ournrITS and the Asplenium nrITS sequences in Genbank(AY165440–AY165486) did not recover significationsimilarities between sequence pairs by sequence pairblasts or by visual inspection.
The gapCp region showed the highest amount ofvariable sites followed by pgiC and trnL-trnF (Table 2).The nrITS region showed the lowest level of variationand the highest GC content of all studied regions (Ta-ble 2). Comparing base pair frequency did not provideevidence of significant differences in base pair compo-sition among sequences of the same region. Sequencesof the nrITS2 region were often incomplete as a resultof frequent long homo-polymeric regions. Comparisonof the results of maximum parsimonious trees (Table 3)recovered slightly conflicting evidence concerning themost informative regions: trn-trnF region recovered thelowest number of most parsimonious trees whereas thetwo nuclear regions pgiC and gapCp showed the betterCI and RC values (Table 2).
Independent phylogenetic analyses of all four re-gions recovered similar relationships for most of the
Table 3 Results of maximum parsimony analyses for the four regions
NT, number of most parsimonious trees; TL, length of most parsimonioustree(s); CI, consistency index excluding uninformative characters; RI,retention index; RC, rescaled consistency index.
taxa with some notable exceptions (Fig. 1; Tables 4 and5). All regions provided evidence for the Tarachia cladewith A. cuneatiforme being sister to the clade includingA. aethiopicum and A. praegracile. These two specieswere distinguishable within three of the four regions.Only gapCp did not separate between these two species.The black-stemmed rock spleenworts were recovered asmonophyletic in trnL-trnF, gapCp, and pgiC, but not innrITS. Asplenium scolopendrium was nested within theblack-stemmed rock spleenwort clade in nrITS but notin any other gene-tree and A. scolopendrium was sisterto black-stemmed rock spleenworts with trnL-trnF orsister to the Tarachia clade with gapCp. Conflicting re-lationships were also found within the black-stemmedrock spleenwort clade. These conflicting relationshipsare partly due to both the hybrid origin of A. adulter-inum and the ambiguous relationships that exist amongthe remaining species. The visually observed topologi-cal heterogeneity was consistent with the results of theILD test that recovered evidence for incompatibility.
The uniparentally inherited chloroplast markertrnL-trnF recovered A. adulterinum to be closely re-lated to A. viride, whereas two of the three bi-parentally inherited nuclear markers (gapCp, pgiC)found close relationships to A. viride for some copies butclose relationships to A. trichomanes for other copies(Fig. 1). These results were expected because thisspecies is known to be the allopolyploid derivative ofthese two species. Only copies of the A. viride ancestrywere found for nrITS (Table 1).
Conflicting results were also found among gene-trees concerning the relationships of A. monanthes(Fig. 1). The chloroplast marker trnL-trnF and pgiCsuggested sister relationships between A. monanthesand A. trichomanes, whereas nrITS and gapCp sug-gested close relationship of A. monanthes and A. nor-male. These relationships were recovered with highbootstrap values (≥95%) in gapCp and pgiC.
Most terminal branches had high bootstrap values(≥95%), whereas the majority of deeper nodes lackedbootstrap values of more than 50%. The Tarachia cladehad a bootstrap value ≥ 95% in trnL-trnF, pgiC, andgapCp. The black-stemmed rock spleenworts clade had
SCHNEIDER et al.: Nuclear genes and Asplenium phylogeny 147
Fig. 1. Phylograms of the independent maximum likelihood (ML) analyses of the four generated data sets—clockwise: trnL-trnF, nrITS, pgiC, andgapCp. Maximum parsimony analyses of the same datasets resulted in one of several most parsimonious trees (see Table 3) with identical relationshipsas far as clades were concerned. Thick branches indicate ML bootstrap values ≥ 95%. All accepted copies were included. B, black-stemmed rockspleenworts; T, Tarachia clade. Some clades included alleles from two taxa. They were marked as: adu, Asplenium adulterinum; aet, A. aethiopicum;mon, A. monanthes; nor, A. normale; pra, A. praegracile; tri, A. trichomanes; vir, A. viride.
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Table 4 Congruence of maximum parsimony topologies
trnL-F pgiC gapCp nrITS
Tarachia clade 69 100 93 <50%Black-stemmed rock spleenwords 71 95 100 NMAsplenium adiantum-nigrum NA 100 100 NAA. adulterinum NA NM NM NMA. aethiopicum NA 100 NM 86A. cuneatiforme NA 100 100 100A. dielerecta NA 100 100 100A. monanthes NA 100 NM 100A. normale NA 99 NM 59A. praegracile NA 98 NM NAA. scolopendrium NA NA 100 100A. trichomanes NA NM NM 100A. viride NA NM NM NMA. aethiopicum + A. praegracile 98 100 100 100A. adulterinum + A. trichomanes NA 100 100 NAA. adulterinum + A. viride 100 100 100 59
Bootstrap values are given for recovered nodes, not recovered nodes areindicated by NA. NA, not applicable; NM, non-monophyletic taxon.
Table 5 Congruence of maximum likelihood topologies. See legend ofTable 4 for explanations
trnL-F pgiC gapCp nrITS
Tarachia clade 95 100 100 58Black-stemmed rock spleenwords 77 94 100 NMAsplenium adiantum-nigrum NA 100 100 NAA. adulterinum NA NM NM NMA. aethiopicum NA 100 NM 100A. cuneatiforme NA 100 100 100A. dielerecta NA 100 NA 100A. monanthes NA 100 NM 100A. normale NA 100 NM NMA. praegracile NA 100 NM NAA. scolopendrium NA NA NA 99A. trichomanes NA NM NM 100A. viride NA NM NM NMA. aethiopicum + A. praegracile 99 100 100 100A. adulterinum + A. trichomanes NA 100 100 NAA. adulterinum + A. viride 100 100 100 98
a bootstrap value ≥ 95% only in gapCp. The conflictingrelationship of A. monanthes was the only conflictingtopology with a bootstrap support ≥ 95% (gapCp vs.pgiC). Another conflict among the three nuclear genesis the inability to separate A. aethiopicum and A. prae-gracile with gapCp, whereas pgiC recovered the twospecies as belonging to two separate lineages with abootstrap value of 100%.
Analyses of the combined dataset recovered atopology in which A. scolopendrium is sister to a mono-phyletic Tarachia clade but this sister relationship had abootstrap value of ≤ 80% (Fig. 2). The black-stemmedrock spleenworts were recovered as monophyletic witha bootstrap value of 100% (in MP and ML). Aspleniummonanthes and A. normale were sister taxa with a boot-strap value of 100% (in MP and ML). A super-networkwas reconstructed to integrate the information pro-vided by the four genomic regions studied (Fig. 3). TheTarachia clade is well separated and the relationships
Fig. 2. Phylogram obtained by the maximum likelihood (ML) anal-ysis of the combined dataset. Maximum parsimony (MP) analysis ofthe same dataset results in a single most parsimonious tree of the sametopology. Asplenium adulterinum was separated in two terminal taxa: A.adulterinum-viride copies includes all copies shared with A. viride andA. adulterinum-trichomanes copies consists of all copies shared with A.trichomanes. Exclusion of A. adulterinum and/or its nrITS did not alterthe topology. Thick branches indicate clades with a bootstrap support of≥ 95% in ML and MP bootstrap analyses. Exclusion of nrITS did notalter the bootstrap values substantially and none of the thin branches ob-tained a bootstrap value above 80%. B, black-stemmed rock spleenworts;T, Tarachia clade.
among the members of this clade were fully resolved.The relationships among the four lineages included inthis study were unresolved, as indicated by the rhom-bus connecting A. adiantum-nigrum, A. scolopendrium,the Tarachia clade, and the black-stemmed rock spleen-worts. The relationships within the black-stemmed rockspleenworts are unresolved (Fig. 3).
3 Discussion
3.1 Incongruence amongst the four markersThe topologies obtained in independent phyloge-
netic analyses of the four regions were only partly con-gruent with each other (Fig. 1). Incongruent trees canbe caused by stochastic errors generated by insufficientdata or by evolutionary processes such as interspecificrecombination, gene duplication or deletion, and in-complete gene-lineage sorting (Wendel & Doyle, 1998;Doyle & Doyle, 1999). Observed incongruence in the
SCHNEIDER et al.: Nuclear genes and Asplenium phylogeny 149
Fig. 3. A Z-Super-network based on the most parsimonious trees obtained by independent maximum parsimony analyses of the four gene partitions(trnL-F, nrITS, gapCp, pgiC) of the combined dataset. Connections within the network reflect the relationships found in each of the gene-trees. Onemost parsimonious tree was found for the all partitions with the exception of nrITS with two most parsimonious trees. Asplenium adulterinum wasrepresented with two terminal taxa as in Fig. 2. B, black-stemmed rock spleenworts; T, Tarachia clade.
deeper nodes are to be expected as the result of insuf-ficient data. Many molecular phylogenetic studies havedemonstrated the impact of denser taxonomic samplingon the phylogenetic accuracy (Hillis, 1998) and densersampling results in more robust phylogenetic hypothe-ses, as demonstrated for Aspleniaceae (Schneider et al.,2004a, 2005). Future studies will achieve a denser taxo-nomic sampling as required to fully explore the putativeincongruence among genetic markers and the underly-ing mechanisms.
Asplenium adulterinum is included as a well-studied taxon of known origin, that is, it being a hybridbetween A. trichomanes and A. viride (Lovis, 1968; Vo-gel et al., 1996, 1999). The chloroplast DNA identifiedA. viride as the maternal parent, which is consistent withprevious studies (Vogel et al., 1996, 1999). Sequencesof gapCp and pgiC obtained from A. adulterinum clus-tered in two lineages of distinct origin. One set of copieswas similar to sequences obtained from A. trichomanes,whereas the other set of copies was similar to sequences
of A. viride. The results of our study are congruent withprevious studies demonstrating the hybrid origin of thistaxon (Vogel et al., 1996, 1999; James et al., 2008).While it was expected to recover copies of both par-ents in this allopolyploid taxon, this was not the case fornrITS. This may either be the result of PCR primer pref-erence to one copy or caused by biological processes,such as a deletion of duplicated copies or concerted evo-lution. However, this taxon is interpreted as a F1 hybridand no recombination has been observed. The nrITSresults highlight the challenge of data interpretation ifonly one nuclear marker is employed to study polyploidspeciation patterns and processes.
Asplenium aethiopicum and A. praegracile are twotaxa of a species complex known to be taxonomi-cally challenging (Braithwaite, 1986; Chaerle & Viane,2004). The group is well known for the occurrenceof sexual taxa with high ploidy levels and polyploidapomicts (Braithwaite, 1964, 1986). Our results indi-cate that the use of several nuclear regions aid the
150 Journal of Systematics and Evolution Vol. 51 No. 2 2013
reconstruction of the complex evolution of this lineage,which has its diversity center in eastern and southernAfrica (Braithwaite, 1986). The conflicting results be-tween gapCp and pgiC may be explained by hybridiza-tion, by shared ancient polymorphisms through incom-plete lineage sorting, or by a mixture of both (Linder &Rieseberg, 2004). The duplication/multiplication of thewhole genome in these species introduces further chal-lenges on attempts to reveal the true species phylogeny(Brysting et al., 2007).
The third notable incongruence concerns the re-lationships of A. monanthes. This species is a triploidapomict occurring in the Americas and the Afromada-gascan region (Smith & Mickel, 1977; Mickel & Smith,2004). A previous study using a denser sampling andthe four chloroplast regions, rbcL, rps4, rps4-trnS IGS,and the trnL-trnF region, supported a clade of NewWorld species of A. monanthes to be sister to the A.trichomanes clade (Schneider et al., 2005). From thenew data, only one of three nuclear genomic markersrecovered evidence for the sister relationship betweenA. monanthes and A. trichomanes, whereas the other tworecovered A. monanthes either as nested within a gradeof A. normale sequences (nrITS) or nested within an un-resolved clade including both A. monanthes and A. nor-male sequences (gapCp). Further interpretation of ourresults is limited by the insufficient taxonomic coverageof samples in this study and conclusions will need tobe drawn on future studies including sampling of otherspecies in the A. monanthes complex. The sequences ofthe apomictic A. monanthes were carefully checked forsignatures of gene silencing or other changes such asa higher mutation rate. From our data, we did not findany convincing evidence for any such bias. Incompletelineage sorting and the fate of duplicated gene copieswill need to be addressed in future studies of this speciescomplex. A more detailed phylogenetic study on the A.monanthes complex that samples the putative parentsof this apomict is currently underway. Asplenium nor-male and A. trichomanes are distant relatives but notconsidered to be the parents of A. monanthes.
3.2 The problem with the use of nrITS in fernsThe loci nrITS1 and nrITS2 are among the most
commonly used regions in plant phylogenetics (Her-shkovitz et al., 1999) but less than 100 sequences offern nrITS are currently available in GenBank (April2009). This surprisingly low number may reflect eitherthe preferences of researchers studying these plants ormajor challenges to generate reliable nrITS sequenceswith existing primer sets. In discussions with colleaguesdoubts have been expressed about the identity of fernnrITS sequences generated using standard plant nrITS
primers as exemplified by White et al. (1990). We cannottotally exclude the possibility that even our nrITS datais not from ferns, but possibly from endophytic fungi.However, the remarkable similarities between topolo-gies obtained by all the independent phylogenetic anal-yses of the nrITS data and the other regions makesthis unlikely, if we exclude, as one reasonably can, thehypothesis of a close coevolution of endophytic fungiand their fern hosts. The results of the blast similarityanalyses are the most problematic issue concerning theidentity of the nrITS data. As mentioned in the resultssection of this paper, similarities were found with nrITSdata accessible in GenBank obtained from ferns such astree ferns (Cyathea) and heterosporous ferns (Azolla).Sequence similarities are restricted to the 18S and 5.8Sregions, whereas the nrITS1 and nrITS2 region lackedsimilarities to Cyathea and Azolla sequences. Remark-ably, blast searches did not find similarities between ournrITS sequences and nrITS sequences deposited pre-viously for the A. ceterach and A. cordatum speciescomplexes (van den heede et al., 2003). We further ex-plored these findings by performing blast searchers ofsequence pairs (http://blast.ncbi.nim.nih.gov) with ournrITS sequences versus the ones deposited by van denheede et al. (2003). Our findings did not show any signif-icant similarities among various sequence pairs. Subse-quently, we blasted the sequences deposited by van denheede. These sequences showed a significant similaritywith various groups of angiosperms including mono-cots, asterids 1 and asterids 2. These similarities werepresent across the whole sequence with as much as 98%of the sequence was overlapping and not as one mightexpect only the 18S and 5.8S region. The high similar-ity of nrITS1 and nrITS2 may be explained by varioushypotheses including high conservation of the nrITS1and nrITS2 in land plants, convergent sequence evolu-tion, frequent horizontal gene transfer, or contamina-tion. None of the first three hypotheses are supported byevidence. The possibility of sequencing contaminationsis therefore the most likely explanation. Future studieswill need to carefully scrutinize sequences of nuclearribosomal intergenic spacer regions in ferns. In conclu-sion, all evidence suggests the need for an exhaustivestudy of the ribosomal nuclear gene region in ferns toenable the unambiguous detection of nrITS identity inferns.
3.3 Homology assessment of gapCp and pgiCThe gene identity of gapCp and pgiC sequences
needs careful consideration because of the lack of awhole genome of a fern. All available plant genomes,for example, the moss Physcomitrella patens, thespikemoss Selaginella mollendorfii, the angiosperms
SCHNEIDER et al.: Nuclear genes and Asplenium phylogeny 151
Arabidopsis thaliana and Oryza sativa, are not relatedclosely enough to derived ferns to allow a comprehen-sive identification of gene copy number and identity.
The locus gapCp was amplified with primer sets de-veloped by Eric Schuettpelz (Schuettpelz et al., 2008).Only one copy of gapCp is found in Aspleniaceae.A gapCp sequence of H. unilaterale generated bySchuettpelz (Schuettpelz et al., 2008) is remarkablysimilar and fitted well into our alignment, not surpris-ing given that Hymenasplenium is the sister genus ofAsplenium (Schneider et al., 2004a, 2005). The gapCpsequence of Asplenium is about 300 bp longer than thegapCp sequence of Hymenasplenium, due to a large in-sertion in the intron between exon 9 and exon 10. Asthe primers were designed specifically for gapCp (seeSchuettpelz et al., 2008), we do not anticipate that wehave accidentally sequenced another copy of GAPDHsuch as gapC (Petersen et al., 2003) and the identity ofthe exons among the generated Asplenium sequence andthe Hymenasplenium sequence concurred.
Sequences of the pgiC region were generated witha primer set designed for Dryopteris (Ishikawa et al.,2002). The PCR amplifications generated a single well-defined band and there was no evidence of accidentallysequencing of other members of the pgiC gene family.
4 Conclusion
The three nuclear regions studied amplified consis-tently well with respect to PCR success and sequencingdata production, with the exception of the failure tosequence pgiC for Asplenium scolopendrium. Severalcopies were typically recovered, the number of copiesroughly corresponding to the known or expected ploidylevels. Both loci, gapCp and pgiC, recovered the ances-try of the neo-allopolyploid A. adulterinum. The gene-trees generated by gapCp, pgiC, or nrITS showed sev-eral incongruent topologies suggesting different histo-ries. Our results support the applicability of all threenuclear regions to discover and reconstruct reticulateevolution in the fern genus Asplenium. The discoveryof incongruent gene-trees supports the need to combinethe study of more than one nuclear gene to more accu-rately recover a robust and informative phylogeny in thepresence of reticulate evolution.
Acknowledgements We acknowledge the finan-cial support by the Botany Innovation Fund, theNatural History Museum Research Fund to JCV,and by the Deutsche Forschungsgemeinschaft GrantHSCHN758/4-1 as part of the Radiation Schwerpunktto HS. HS also acknowledges the Senior Visiting Pro-
fessorship granted by the Chinese Academy of Sci-ences. We thank Julia LLEWELLYN-HUGHES and herteam for support with DNA sequencing. We thank PaulWOLF and two reviewers for their helpful comments onan earlier draft of the study.
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