Evidence for Multiple Evolutionary Origins in the Moss Flora of Macaronesia

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doi:10.1111/j.1558-5646.2009.00787.x

AND IF ENGLER WAS NOT COMPLETELY WRONG? EVIDENCE FOR MULTIPLE EVOLUTIONARY ORIGINS IN THE MOSS FLORA OF MACARONESIA

Delphine A. Aigoin,1,2,3 Nicolas Devos,1,4 Sanna Huttunen,5,6 Michael S. Ignatov,7,8 Juana M. Gonzalez-Mancebo,9,10 and Alain Vanderpoorten1,11 1Institute of Botany, University of Liege,` 27 Blvd du Rectorat, B22, Sart Tilman, 4000 Liege,` Belgium 2E-mail: [email protected] 3Institute of Evolutionary Sciences, University of Montpellier II, Place Eugene` Bataillon, 34095 Montpellier Cedex 5, France 4E-mail: [email protected] 5Laboratory of Genetics, Department of Biology, University of Turku, 20014 Turku, Finland 6E-mail: shuttu@utu.ﬁ 7Main Botanical Garden of Russian Academy of Sciences, Botanicheskaya 4, 127276 Moscow, Russia 8E-mail: [email protected] 9Department of Botany, University of La Laguna, 38271 La Laguna, Tenerife, Canary Islands, Spain 10E-mail: [email protected] 11E-mail: [email protected]

Received March 27, 2009 Accepted June 23, 2009

The Macaronesian endemic ﬂora has traditionally been interpreted as a relict of a subtropical element that spanned across Europe in the Tertiary. This hypothesis is revisited in the moss subfamily Helicodontioideae based on molecular divergence estimates derived from two independent calibration techniques either employing fossil evidence or using an Monte Carlo Markov Chain (MCMC) to sample absolute rates of nucleotide substitution from a prior distribution encompassing a wide range of rates documented across land plants. Both analyses suggest that the monotypic Madeiran endemic genus Hedenasiastrum diverged of other Helicodontioideae about 40 million years, that is, well before Macaronesian archipelagos actually emerged, in agreement with the relict hypothesis. Hedenasiastrum is characterized by a plesiomorphic morphology, which is suggestive of a complete morphological stasis over 40 million years. Macaronesian endemic Rhynchostegiella species, whose polyphyletic origin involves multiple colonization events, evolved much more recently, and yet accumulated many more morphological novelties than H. percurrens. The Macaronesian moss ﬂora thus appears as a complex mix of ancient relicts and more recently dispersed, fast-evolving taxa.

KEY WORDS: Bryophytes, fossils, macronesian endemism, molecular dating, morphological evolution.

Macaronesia is a string of North Atlantic volcanic islands (the demism (see Juan et al. 2000 for review). Engler (1879), fol- Azores, Madeira, Canaries, and Cape Verde) that emerged 0.4– lowed by many biogeographers (see Vanderpoorten et al. 2007 20 million years ago and are characterized by high rates of en- for review), proposed that Macaronesian endemics are the relics

C 2009 The Author(s). Journal compilation C 2009 The Society for the Study of Evolution. 3248 Evolution 63-12: 3248–3257 BRIEF COMMUNICATION

of biota that were widespread across Europe during the Tertiary phological stasis since the Tertiary period among Macaronesian and decimated on the continent during the glaciations. In con- endemics. trast with the expectations of the refugium concept, however, several lines of evidence from analyses of moss species distributions (Vanderpoorten et al. 2007) and molecular evolution Material and Methods rates in angiosperms (Carine 2005) have recently questioned En- TAXONOMIC AND MORPHOLOGICAL CHARACTER gler’s hypothesis. In mosses, the hypothesis of extinction of a SAMPLING Tertiary flora in all but Macaronesian areas is solely supported The 17 genera of Helicodontioideae as circumscribed by Aigoin by extremely limited fossil evidence in the genus Echinodium et al. (2009) were sampled (Table 1). Each genus was represented (Frahm 2004). This interpretation is, however, weakened by the by one to three species, with a special emphasis on Rhynchoste- polyphyletic origin of the genus (Stech et al. 2008), thereby giella, for which all eight species were sampled. Three other raising doubts about the actual sister relationship between fos- species, namely R. papuensis, R. leptoneura,andR. muriculata, sil and extant Macaronesian Echinodium species. By contrast, clearly do not belong to the genus, and the appropriate taxonomic the nested phylogenetic position of the Azorean endemic liver- changes will be presented elsewhere. Aerobryidium filamentosum, wort Leptoscyphus azoricus within a Neotropical clade (Devos a species of the sister family Meteroriaceae, as well as representa- and Vanderpoorten 2009); the close biogeographic affinities of tive taxa of each of the three subfamilies of Brachytheciaceae was several Macaronesian groups with the North and South Ameri- sampled as outgroups (Table 1). Forty-three morphological char- can continents (see Vanderpoorten et al. [2007] for review); and acters that are variable across the Helicodontioideae, including the polyphyletic origin among Macaronesian populations of the 34 gametophytic and nine sporophytic ones, were scored for each moss Grimmia montana, which are genetically identical or most of the sampled taxa (Appendices S1 and S2) (see Huttunen and closely related to those of different continents (Vanderpoorten Ignatov [2004] for a thorough account on character significance et al. 2008) all point to recurrent migrations between the latter and description). and the Macaronesian archipelagos, potentially followed by in situ MOLECULAR PROTOCOLS AND PHYLOGENETIC speciation. ANALYSES The continental extinction and recent speciation hypothe- Four chloroplast regions (trnL-trnF, atpB-rbcL, psbT-psbH, and ses make different predictions regarding the rates of speciation psbA-trnH) were selected for exhibiting the appropriate level of and morphological evolution. In fact, as opposed to neoendemics, variation at the genus level in the Brachytheciaceae (Huttunen and which originated from local speciation processes and often under- Ignatov 2004). DNA extraction, PCR and sequencing protocols, went spectacular adaptive radiations involving a sudden burst of sequence editing, alignment, indel scoring, and selection of mod- morphological diversification (see Gillepsie and Roderick [2002] els for DNA substitutions and indel evolution follow Aigoin et al. for review), paleoendemics, which survived continental extinc- (2009). Phylogenetic reconstruction was conducted with MrBayes tions on the islands, have most often retained a highly conserved 3.1.2. Four independent Monte Carlo Markov Chains (MCMCs) morphology for millions, or tens of million years, to such an extent of 2,000,000 iterations each were run and trees and model param- that extant taxa appear conspecific with fossil species (Sunding eters were sampled every 10,000 generations. The convergence 1979). of the MCMCs was verified visually from the likelihood values, In this article, we revisit hypotheses on the origin of Mac- and trees of the “burn-in” were discarded. aronesian endemism in the mosses from the Brachytheciaceae, A significant departure of alternative topologies involving a subfamily Helicodontioideae. This group includes several Mac- monophyletic origin of the three Macaronesian endemic Rhyn- aronesian endemics, namely the monotypic Madeiran Hedenasi- chostegiella species, namely R. bourgeana, R. macilenta,andR. astrum percurrens,andthreeRhynchostegiella species: Rhyn- trichophylla, was tested by constrained analyses. The MCMC chostegiella bourgeana and R. trichophylla, which are restricted analysis described above was rerun under the constraint that only to the Canary Islands, and R. macilenta, whose distributions trees fitting with a monophyletic origin of the Macaronesian en- span Madeira and the Canaries. In addition, and unlike most demic Rhynchostegiella species were sampled. We then deter- bryophytes, a fairly well-documented fossil record is available mined whether the constraint induced a significant loss of like- (Miller 1984). We produced a molecular phylogeny of the Heli- lihood by means of the Bayes factors, as assessed by twice the codontioideae to test the hypothesis of a radiation within Rhyn- difference in the log marginal likelihood between the two runs. chostegiella and date the origin of its Macaronesian endemic lineages, contrasting the results derived from a fossil calibration and MOLECULAR DATING an analysis employing absolute rates of molecular evolution. We Times of divergence were calculated to determine the origin then used the phylogeny to test the hypothesis of a long mor- of the most recent common ancestor (hereafter, MRCA) of H.

EVOLUTION DECEMBER 2009 3249 BRIEF COMMUNICATION (MO) (H) (H) (H) Continued. (LG) (S) (S) (H) (H) (H) (H) (NY) (LG) (H) (LG) (LG) (LG) (LG) (S) (S) (S) (BM) (L) (MA) Koponen 32122 Koponen 33007 (MO) ¨ ¨ ¨ as B9050 as B9057 as B42883 Hylander B78518 Hylander B78534 Vanderpoorten V180 Newton 4399 Dirkse 1882 Streimann 49544 Heden Heden Heden Toapanta & Caranqui 1437 Koponen & Huttunen 1324 Allen & Redfearn sn Vanderpoorten 401 Vanderpoorten 421 Vanderpoorten 413 Vanderpoorten 4415 Sotiaux & Sotiaux 14670 Koponen et al. 53920 Koponen et al. 48969 He 36074 Koponen et al. 51775 Bowers 22214 Vanderpoorten 4748 , Koponen et al. 49717 (herb. Sotiaux) H Voucher specimen for sequences /trn A psb H /psb T psb L /rbc B atp F /trn L trn ¨ ull. Hal.) AY184776 AY663296 AY184757 – ¨ ull. Hal. AY306783 – AF417418 FJ262514 Suriname, ´ er.) Ignatov and Huttunen FJ262419 FJ262447 FJ262478 FJ262506 China, ¨ ull. Hal.) A. Jaeger AF397877 EF530978 AF417466 – ¨ ull. Hal.) M. Fleisch. FJ262421 FJ262449 FJ262480 FJ262530 South Africa, ¨ as) Ignatov and Vanderpoorten – FJ262442 FJ262475 FJ262500 Madeira, (Mont.) Ignatov and Vanderpoorten FJ262428 FJ262459 FJ262486 FJ262519 Madeira, (Hedw.) Broth. FJ262423 FJ262451 F417462 FJ262513 U.S.A., ¨ ull. Hal.) M (Hedw.) Ignatov and Huttunen FJ262420 FJ262448 FJ262479 FJ262507 France, Cardot FJ262431 FJ262462 FJ262489 FJ262522 China, (Grout) H.A. Crum and L.E. Anderson AY009848 AF322323 – – (Mitt.) Broth. FJ262426 FJ262457 FJ262484 FJ262517 El Hierro, (Wilson) Ignatov and Vanderpoorten FJ262434 FJ262466 FJ262493 FJ262509 France, (Schleich. ex M (Hedw.) M. Fleisch. AF397797 AF233567 AF417470 – (Hook.) M. Fleisch. AF397789 – AF417347 – (Taylor) Loeske and M. Fleisch. FJ262415 FJ262443 FJ262476 FJ262502 France, (Heden (Hedw.) Dixon AY857573 AY857595 – – (Hoffm. ex F. Weber and AF397857 AY663309 AF417448 AY312896 (Mitt. ex M (Brid.) Limpr. FJ262427 FJ262458 FJ262485 FJ262518 France, (Cardot) Nog. FJ262424 FJ262452 AY184771 FJ262515 China, (Hook. f. and Wilson) M. Fleisch. AY184791 – FJ262483 FJ262516 Australia, (M (Cardot and Th (Hedw.) H. Rob. AF397819 – AF417365 FJ262501 USA, (Schimp. ex Besch.) Broth. AF397859 FJ262454 AF397859 FJ262510 China, (Hedw.) A. Jaeger FJ262422 FJ262450 FJ262481 FJ262512 Venezuela, (De Not.) Limpr. FJ262432 FJ262463 FJ262490 FJ262523 Madeira, (Broth.) Broth. FJ262429 FJ262460 FJ262487 FJ262520 South Africa, (Hornsch.) M. Menzel FJ262414 FJ262441 FJ262474 FJ262499 Ecuador, (Ignatov) Ignatov and Huttunen FJ262416 FJ262444 AF417446 FJ262503 Papua New Guinea, (A. Jaeger) Ignatov and Huttunen AF397862 FJ262455 AF417450 FJ262511 China (Sull. and Lesq.) Grout AF161122 AJ288397 AF417405 – (Hedw.) Grout FJ262417 FJ262445 AF417403 FJ262504 Finland, (Hampe) Sull. FJ262418 FJ262446 FJ262477 FJ262505 USA, (M. Fleisch.) Ignatov and Huttunen AF397854 FJ262456 AF417423 – Papua New Guinea, (Spreng. ex M (Hedw.) Loeske FJ262425 FJ262453 FJ262482 FJ262508 France, Taxon sampling, voucher information (for sequences produced for the present study), and GenBank accession numbers. D. Mohr) Schimp. Ignatov and Huttunen Brachytheciastrum collinum Cirriphyllum crassinervium Cirriphyllum piliferum Platyhypnidium austrinum Pseudorhynchostegiella duriaei Species Aerobryidium filamentosum Aerolindigia capillacea Brachythecium salebrosum Bryhnia novae-angliae Bryoandersonia illecebra Cirriphyllum koponenii Clasmatodon parvulus Donrichardsia patulifolia Eurhynchiella zeyheri Helicodontium capillare Homalothecium fulgescens Microeurhynchium pumilum Oxyrrhynchium hians Oxyrrhynchium vagans Platyhypnidium riparioides Pseudoscleropodium purum Remyella brachypodia Rhynchostegiella bourgeana Rhynchostegiella holstii Rhynchostegiella litorea Eurhynchiastrum pulchellum Homalotheciella subcapillata Meteoridium remotifolium Okamuraea brachydictyon Oxyrrhynchium savatieri Rhynchostegiella leptoneura Donrichardsia macroneuron Hedenasiastrum percurrens Rhynchostegiella curviseta Table 1.

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percurrens and of the three Macaronesian endemics of Rhyn- chostegiella. Examination of rate variation among branches of the

(MW) phylogeny suggested a strong departure of the sequence data from the molecular clock (see below). Divergence times were therefore (S) (L) (LG) estimated using a Bayesian MCMC method under a relaxed-clock (R) (H) (LG) (S) (S) model employing an uncorrelated lognormal model of rate vari-

51803 ation among branches in the tree as implemented by BEAST ¨ as B11895 1.4.8. Nonequivocal Miocene fossils of Cirriphyllum piliferum ¨ ¨ as B9096 as B4503

Dirkse 13843 and Oxyrrhynchium hians, and Pliocene fossils of Clasmatodon Heden ´ ocs et al. 88161 parvulus (Miller 1984) were used for calibration at three internal Streimann 49628 P Heden Heden Vanderpoorten M16 Vanderpoorten 362 Ignatov & Ignatova 05-6143

Koponen et al. nodes. In bryophytes, wherein the probability of fossilization is very low owing to the absence of lignified tissues and much re- duced cuticle, the presence of a species is likely to be recorded in the sediment only well after its actual phylogenetic origin. We therefore used asymmetric distribution priors with a mode at the H Voucher specimen for sequences beginning of the period considered and asymptotically skewed /trn

A toward the end of it (Fig. 1).

psb In a second set of analyses, absolute nucleotide substitution rates documented for a large number of plant lineages (in-

H cluding bryophytes) were used. Accordingly, a normal distribu- −10 /psb tion, with a mean and standard deviation of 5.0 × 10 and T 10−10 substitutions/site/year, respectively, which corresponds to psb the average absolute substitution rate of cpDNA across land plants and largely encompasses their variation range (see Schnabel and L Wendel [1998] for review), was used as a prior on the absolute /rbc

B rates of evolution for the four cpDNA regions combined.

atp Although BEAST allows for topologies, branch lengths, and rates to be coestimated, we chose to perform the dating analyses

F onto topologies that were derived independently from the kind of

/trn calibration used. Therefore, we constrained the topology to match L the 50% majority-rule consensus of the trees sampled from the trn posterior probability distribution generated by MrBayes, but used the MCMC implemented by BEAST to sample branch lengths and substitution rates depending on their posterior probabilities. A Yule prior on branching rates was employed and two independent MCMC analyses were each run for 10,000,000 steps. Param- eter values were sampled every 1000 cycle over the 10,000,000 MCMC steps. Convergence and acceptable mixing of the sampled parameters was checked using Tracer 1.4. After discarding the burn-in steps, the two runs were combined to obtain an es- Dirkse and Bouman FJ262438 FJ262470 FJ262497 FJ262529 Gran Canaria,

Ignatov and Huttunen FJ262439 FJ262471timate FJ262498 of FJ262531 the posterior China, probability distribution of the divergence (Hook. f. and Wilson) Broth. DQ208214 FJ262465 FJ262492 FJ262525 Australia, E.B. Bartram – – AF417439 – (Renauld and Cardot) Cardot FJ262433 FJ262464 FJ262491 FJ262524 Madeira, (Mont.) Dirkse and Bouman #1(Mont.) Dirkse and FJ262430 Bouman #3 FJ262435 FJ262461 FJ262488 FJ262467 FJ262494 FJ262521 Madeira, FJ262526 Switzerland, (Mont.) Dirkse and Bouman #2 FJ262437 FJ262469 FJ262496 FJ262528 France, dates of the ancestral nodes. (Dicks.) Limpr. FJ262436 FJ262468 FJ262495 FJ262527 Russia, (Hornsch.) Broth. FJ262440 FJ262473 AF417393 FJ262532 Tanzania, (Hedw.) Manuel AF397787 – AF417362 – (Brid.) M. Fleisch. and Loeske AF397834 FJ262472 AF417410 – France,

ANCESTRAL CHARACTER STATE RECONSTRUCTIONS Ancestral character state reconstructions were performed to contrast the hypotheses that Macaronesian endemics are characterized by plesiomorphic or derived character states. All reconstructions Continued. were performed after pruning the outgroups from the trees. The probabilities of a change in a branch were calculated by esti- Rhynchostegiella teneriffae Rhynchostegiella teneriffae Rhynchostegium psilopodium Species Rhynchostegiella macilenta Rhynchostegiella muriculata Rhynchostegiella tenella Rhynchostegiella teneriffae Rhynchostegiella trichophylla Scorpiurium circinatum Zelometeorium patulum Squamidium brasiliense Rhynchostegiella papuensis

Table 1. mating the instantaneous forward and backward rates among the

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Figure 1. Fifty percent majority-rule consensus tree from the Bayesian analysis of four chloroplastic genes in Helicodontiodeae, with branch lengths averaged over the 284 trees sampled from the posterior probability distribution. Posterior probabilities are indicated under each branch. Fossil calibration points are labeled by stars and the range of the prior distribution encompassing the geological era during which the fossil was found is illustrated in boxes. The dates indicated at ﬁve nodes (black circles) correspond to the median (and 95% conﬁdence interval) of the age of selected nodes, as inferred from the molecular dating analysis when the fossil calibration (F) or absolute substitution rates of chloroplastic DNA (R) were used. The four Macaronesian endemic species of Helicodontioideae appear in bold. two states by implementing the Markov model of “Multistate” in absence of information on rates, uniform distributions ranging BayesTraits 1.0. between 0 and 100 were used as priors. The likelihood of the To contrast alternative hypotheses regarding the morphol- new combination of a rate and a tree was calculated and this new ogy of the MRCA of the Macaronesian endemics, we used the state of the chain was accepted or rejected following evaluation “local” approach, wherein the significance of the reconstruction by the Metropolis–Hastings term. The rate, at which parame- is explicitly tested at each node of interest (Pagel 1999). For ters were changed (“ratedev”), was set at the beginning of each that purpose, we fixed each of the MRCAs of H. percurrens and run so that the acceptance rate of the proposed change globally Rhynchostegiella at one of the two states it can take. Then, an ranges between 20% and 40%. The chain was run for 10,000,000 MCMC was used to visit the sample of trees generated by the generations and was sampled for rate parameters every 10,000 MrBayes analysis and the space of rate parameter values. In the generations. A second, independent chain was run to sample rate

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parameters and derive overall likelihoods of the reconstructions whereas R. macilenta is estimated to have diverged more recently, when the node of interest was fixed at its alternative state. Bayes 1 [0;4] million year according to the fossil calibration, and 1 [0;4] factors were then used to determine the support for alternative when absolute rates were used (Fig. 1). state at each node of interest. ANCESTRAL CHARACTER STATE RECONSTRUCTIONS The marginal log-likelihoods of the reconstruction of ancestral Results morphological character states at the MRCA of the Macaronesian PHYLOGENETIC RELATIONSHIPS endemics are presented in Table 2. When the MRCA of the Heli- The chloroplast matrix contains 19% variable sites. The 50% codontioideae was constrained to be morphologically identical to majority-rule consensus of the 284 trees sampled from the poste- the Madeiran endemic H. percurrens, the marginal log-likelihoods rior probability distribution is presented in Figure 1. Macaronesian of the reconstruction were significantly higher for 20 of the 32 endemics within the Helicondontioideae appear at the two extrem- investigated gametophytic characters. For the remaining 12 char- ities of the phylogeny. The Madeiran endemic Hedenasiastrum acters, the marginal log-likelihood obtained after fixing the root percurrens is sister to all other genera of the Helicodontioideae, of the Helicodontioideae at one or the alternative state were not and this relationship is supported with a posterior probability significantly different (i.e., the Bayes factors were <2), so that the (hereafter, p.p.) of 1.00. By contrast, Rhynchostegiella is one of state at the root was ambiguous for those characters. Altogether, the most recently diverging groups of species of the Helicodon- the two Canarian endemics R. bourgeana and R. trichophylla dif- tioideae. Within Rhynchostegiella, the sub-Saharan African R. fer from the MRCA of the genus by six character states (#12, 13, holstii is part of a polytomy with two other clades. The first clade 15, 20, 23, 26). Finally, R. macilenta, which is the phylogeneti- includes the Canarian endemics R. bourgeana and R. trichophylla cally most recently diverging Macaronesian endemic within the with a p.p. of 0.98. Within the second clade, the Macaronesian Helicodontioideae, differs from the MRCA of Rhynchostegiella endemic R. macilenta is resolved as sister to a Madeiran accession by nine character states (#5, 10, 11, 12, 13, 18, 20, 26, and 35). of R. teneriffae with a p.p. of 0.96. The hypothesis of a monophyletic origin of the three Macaronesian endemic species can be significantly rejected. In fact, constraining all three Macaronesian Discussion endemic species to monophyly resulted in a significantly lower The two calibration techniques used in the present study, that log marginal likelihood by comparison with the unconstrained is the use of fossils and the sampling of absolute substitution analysis (log marginal likelihood of −6884.07 and −6861.42 for rates from a prior distribution encompassing a wide range of the constrained and unconstrained analyses, respectively). rates documented across land plants, rely on completely different assumptions. Yet, they returned highly congruent results, which MOLECULAR DIVERGENCE DATING strongly reinforces the confidence in the molecular dating in- The coefficient of variation of rates among branches of the phy- ferences. The MRCA of Rhynchostegiella dates back to about logeny was 0.63 (95% highest posterior density 0.44–0.83) for the 10 million years. This timing is compatible with both a paleo- first analysis (fossil calibration) and 0.60 (95% highest posterior and a neo-endemic origin of its Macaronesian endemic species. density 0.43–0.77) for the second one (rate sampling from a prior The polyphyletic origin of the latter, however, does not lend sup- distribution). This suggests a strong departure of the data from a port to Engler’ refugium hypothesis. Like in some angiosperms molecular clock, a condition in which the coefficient of variation (see Carine et al. [2004] for review), the colonization of Mac- equals zero. The two kinds of calibration used for molecular dat- aronesia by Rhynchostegiella species seems to have involved at ing by a relaxed-clock model provided congruent results regard- least two dispersal events. Rhynchostegiella was one of the few ing both the medians of the posterior probability distributions and bryophyte genera, wherein several Macaronesian endemics had their 95% confidence intervals (Fig. 1). Hedenasiastrum percur- been described, and therefore was one of the few candidates for rens emerged 38 [20;74] million years with the fossil calibration adaptive radiations. The polyphyletic origin of its Macaronesian and 42 [25;65] when absolute substitution rates were employed. endemics invalidates that hypothesis, although the sister relation- The divergence and diversification of Rhynchostegiella, respec- ship between R. bourgeana and R. trichophylla suggests that local tively, date back to 20 [9;35] and 11 [4;20] million years with speciation may, to a minimal extent, have happened. Furthermore, the fossil calibration and 22 [13;36] and 12 [6;20] with absolute and although this needs to be confirmed by additional sampling, rates of molecular evolution. The MRCA of the Macaronesian the Macaronesian endemic R. macilenta is resolved as a sister to endemics R. bourgeana and R. trichophylla was dated back to 7 a sympatric accession of R. teneriffae, rendering the latter species [2;14] million years with the fossil calibration and 8 [3;15] when paraphyletic. Such a situation has been recently increasingly de- a distribution of absolute substitution rates was used as a prior, scribed in other bryophyte species and has been interpreted in

EVOLUTION DECEMBER 2009 3253 BRIEF COMMUNICATION ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗ ∗∗ ∗ ∗ ∗ ∗∗∗ ∗∗∗ ∗∗ ∗∗∗ ∗ ∗∗∗ ∗∗∗ ∗ ∗ ∗∗∗ ∗∗∗ ∗ ∗∗ ∗∗∗ ∗∗∗ 8 6 0 2 2 8 7 9 4 3 3 2 5 4 3 9 6 5 7 8 1 2 8 4 4 5 4 5 5 8 ...... Continued. 61 14 539582 14 15 16 7907 5 6 172514 10 9 15 1303 10 12 7548 13 14 0192 12 10 42 3 28 18 39 12 27 0 40 1 59 4 02 2 5634 2 4 7179 3 10 1950 4 6 33 1 34 0 ...... ndix S1 for character 19 19 20 21 19 24 22 16 24 21 21 21 20 18 22 23 18 20 23 24 22 24 21 15 22 10 23 14 21 20 − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − he MRCA is successively / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / ). The state returning the 97 21 24 47 73 19 68 82 31 42 98 88 13 23 79 78 39 14 55 90 16 13 39 02 38 75 92 50 06 74 ...... ∗∗∗ HM BF 9 12 13 14 11 22 18 10 19 13 15 14 14 11 15 18 20 14 21 22 21 22 23 21 24 13 13 22 20 22 − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − /1 /1 /1 /1 /1 /1 /1 /1 /1 /1 /1 /1 /1 /1 /1 /1 /1 /1 /1 /1 /1 10 very strong evidence ( MRCA Constrained > ), and ∗∗ ), 5–10 strong evidence ( ∗ 00 00 00 00 00 10 00 00 00 00 00 00 00 10 01 00/1 01 00 00 00 00 00 01 10 00 00 00 00 –0 00 00 00 00 00 R. R. R. bourgeana trichophylla macilenta Rhynchostegiella ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗ ∗ ∗ ∗∗∗ ∗ ∗∗ ∗ ∗∗ ∗∗ ∗∗ ∗ ∗ 9 41 – – 0/1 11 1 05– 0/1 070 020 011 0/1 1 1 1 0 050 120 0/1 0 0/1 19– 190 1 0 0 1 0/1 81 091 0 0 0 – 0 0 – 0/1 0/1 3 8 3 9 4 1 9 8 1 5 9 3 5 7 8 3 5– 0 0 0 ...... 65 9 33 10 65 0 20 8154 1 71 1 0 8249 1 0 2301 1 0 7152 1 1 51 8 750741 6 6 7 61 2 8441 4 3 4693 4 6 8945 3 2576 6 6 3996 4 4 91 0 ...... 21 1 . 14 15 21 23 22 22 16 23 20 22 23 22 10 15 16 18 15 23 20 20 21 16 19 17 17 14 18 20 14 14 − − − − − − − − − − − − − − − − − − − − − − − − − − − − − / / / / / / / / / / / / / / / / / / / / / / / / / / / / / − / 71 95 61 97 94 92 66 05 39 27 55 61 49 5 35 34 93 46 42 79 46 42 66 95 77 02 39 98 83 65 ...... 9 9 21 22 21 21 16 23 20 21 22 23 11 13 11 13 14 11 22 18 18 19 13 17 14 14 11 15 18 14 HM BF − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − /1 /1 /1 /1 /1 /1 /1 /1 /1 /1 /1 /1 /1 /1 /1 /1 /1 Constrained MRCA Character state of Character states of H. percurrens Ancestral character state reconstructions at the most recent common ancestors of the Macaronesian endemics within the Helicodontioideae. See Appe Character 1Character 2 0 0Character 4Character 5 0Character 6 0 0 Character 7 0 0/1 Character 8 0 0 0 Character 10 0 Character 0 11 0 Character 0 12 0 Character 0 13 0/1 Character 0 14Character 1 0 15Character 0 0/1 16Character 0 0 17Character 0 0/1 18Character 0 0/1 19Character 1 0 20Character 0 0 21Character 0 0 22Character 0 0/1 23Character 0 0/1 24Character 0 0 25Character 0 0 26Character 1 0/1 27Character 1 0/1 28 0 0 Character 0 30 0 0/1 0/1 0/1 0 Character 9 0 0 Character 29 0 0 Character 3 1 0/1 highest log marginal likelihood is underlined. Table 2. identiﬁcation and description. Bayes factors (BF)set measure twice at the its difference two between the possible log states, of with the a harmonic means difference (HM) of returned 2–5 by considered the model as when positive t evidence (

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terms of recent speciation and incomplete lineage sorting (see ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗ ∗∗∗ ∗∗ ∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗∗∗ 6 5 2 2 1 5 2 3 2 3 3 8 8 Sotiaux et al. [2009] for review), thereby reinforcing the idea of ...... a neoendemic origin of R. macilenta. Although the polyphyletic origin and recent evolution of the Macaronesian endemic Rhynchostegiella species is thus sugges- 1737 10 17 848917 11 15 15 41 4 93 10 73 7 78 2 76 7 44 7 63 8 9 13 ...... tive of multiple colonization events followed by rather recent di- 22 19 17 17 20 17 18 16 16 16 15 16 17 − − − − − − − − − − − − − vergence, the Madeiran endemic moss H. percurrens diverged / / / / / / / / / / / / / about 40 million years, that is well before Madeira actually 87 63 19 22 08 73 79 89 97 20 62 85 71 ...... HM BF 16 10 12 10 18 11 14 15 12 13 11 11 10 emerged, 5.2 million years. This time frame, together with the − − − − − − − − − − − − − fairly long branch leading to this unique species, is suggestive of numerous extinctions and a relictual origin. Hedenasiastrum percurrens actually becomes the only clear-cut documented case of relict in the Macaronesian bryophyte flora. In the context of a “dispersalist counter-revolution” fueled by the outcome of recent molecular phylogenies and molecular dating studies (de Queiroz /1 /1 /1 /1 /1 /1 /1 /1 /1 /1 /1 /1 /1 MRCA Constrained 2005), and of a reassessment of the recent origin of emblematic island endemics (e.g., Goldberg et al. 2008; Grandcolas et al. 2008), including Macaronesia (e.g., Jordal and Hewitt 2004; Emerson and Orom´ı 2005; Dimitrov et al. 2008), the present study argues for a pluralistic view of Macaronesian biogeography. Hedenasias- trum percurrens thus falls within the increasingly limited category of confirmed Tertiary relicts. The acceptance of a relictual origin of H. percurrens, together with its morphological identity with the MRCA of the Helicodontioideae, dated at 40 million years, lends support to the traditional view of bryophytes as “unmoving sphinxes of the past” (see Devos and Vanderpoorten [2009] for review). In fact, the morphological similarity between extant and 00 00 00 00 R.bourgeana trichophylla macilenta R. Rhynchostegiella R. 00 00 00 00 00 10 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 fossil species suggests that some groups of mosses have persisted with little morphological change for at least 80 million years ∗ ∗∗ 8 0 . . (see Devos and Vanderpoorten [2009] for review). The question of whether this similarity is indeed due to the direct sister relationship or homoplasy remains, however, open. For example, the 0269 3 9 . . oldest liverwort fossil, the Devonian Pallaviciinites devonicus, ex- 19 14

− − hibits a striking resemblance with the unrelated Pallavicinia and / / 11 21

. . Symphyogyna (Heinrichs et al. 2007), suggesting that the similar- –– –– –– –– –– –– –– –– –– –– –– 17 10 HM BF ity between fossil and extant taxa likely results from homoplasy. − − Thus, although evidence for cryptic speciation in bryophytes is mounting (see Devos and Vanderpoorten [2009] for review), morphological stasis over periods of time of a range of tens of million years actually appears as an extremely rare phenomenon. /1 /1 Constrained MRCA In contrast, fast rates of morphological evolution or diversification have been repeatedly reported in bryophytes (Shaw et al. 2003; Wall 2005; Devos and Vanderpoorten 2009; Sotiaux et al. 2009). In the case of H. percurrens, however, although character evolu- Character state of Character states of percurrens H. tion might be constrained, so that all the space of morphologies cannot be necessarily explored (Beldade et al. 2002), convergence or parallelism occurring to develop what is the same taxon two Continued. or more times is, arguably, less likely than plesiomorphy (Zander 2008). The retention of so many plesiomorphic traits during a 40- Character 31Character 32 0Character 33 0Character 34 –Character 35 –Character 36 – 0 Character 37 – 0 Character 38 – 0/1 Character 39 – 0/1 Character 40 – 0/1 Character 41 – 0/1 Character 42 – 0/1 Character 43 – 0/1 – 0/1 0/1 0/1 0/1 0/1

Table 2. million-year period is remarkable and can be compared to other

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spectacular examples of living fossils (see Lee et al. [2006] for Grandcolas, P., J. Murienne, T. Robillard, L. Desutter-Grandcolas, H. review). Surprisingly, and despite their much more recent origin, Jourdan, E. Guilbert, and L. Deharveng. 2008. New Caledonia: a all three Macaronesian endemic Rhynchostegiella species accu- very old Darwinian island? Philos. Trans. R. Soc. Lond B. 363:3309– 3317. mulated many more morphological novelties than H. percurrens. Heinrichs, J., J. Hentschel, R. Wilson, K. Feldberg, and H. Schneider. Although the remarkable stasis of H. percurrens might originate 2007. Evolution of leafy liverworts (Jungermanniidae, Marchantio- from stabilizing selection, the reasons for such striking differences phyta): estimating divergence times from chloroplast DNA sequences in evolutionary rates within the same group of mosses remain using penalized likelihood with integrated fossil evidence. Taxon 56:31– 44. completely unexplained. Huttunen, S., and M. S. Ignatov. 2004. Phylogeny of the Brachytheciaceae (Bryophyta) based on morphology and sequence level data. Cladistics ACKNOWLEDGMENTS 20:151–183. DA, ND, and AV acknowledge financial support from the Belgian Funds Jordal, B. H., and G. M. Hewitt. 2004. The origin and radiation of Macarone- for Scientific Research (F.R.S.–FNRS) and the Leopold´ III Funds, and sian beetle breeding in Euphorbia: the relative importance of multiple MI the RFBR 07-04-00013. Many thanks are due to M. Carine, P.-H. data partitions and population sampling. Syst. Biol. 53:711–734. Fabre, R. G. Gillepsie, F. Kjellberg, and three anonymous reviewers for Juan, C., B. C. Emerson, P. Orom´ı, and G. M. Hewitt. 2000. Colonization their constructive comments on an earlier draft of this article. and diversification: towards a phylogeographic synthesis for the Canary Islands. Trends Ecol. Evol. 15:104–109. Lee, J., J. Alrubaian, and R. M. Dores. 2006. Are lungfish living fossils? 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Supporting Information The following supporting information is available for this article:

Appendix S1. List of 43 morphological binary characters scored on the sample of taxa from the Helicodontioideae. Appendix S2. Matrix of 43 morphological characters (see Appendix S1) scored for taxa of the Helicodontioideae used in the present study.

Supporting Information may be found in the online version of this article. (This link will take you to the article abstract).

Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

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