Posted on Authorea 5 Apr 2021 | The copyright holder is the author/funder. All rights reserved. No reuse without permission. | https://doi.org/10.22541/au.161766187.74749853/v1 | This a preprint and has not been peer reviewed. Data may be preliminary. pce onaisi h es ide–tsigtehptei fmcoedms na in micro-endemism Wu of Huini hypothesis Nate the S. fungi testing lichen desert – Jorna fog Jesse middle coastal messy of lineage the diverged recently in boundaries phylogeographic our recent Species of complex, models results with delimitation The Niebla, species as delimitations. such The species groups consistent in Ramalinaceae. robust, particularly family infer species, the to delimiting unable histories. in of micro-endemism were problem Bowler for but the & potential groupings, highlight Rundel the specious study Niebla more and support incomplete species fungi, boundaries to lichen-forming species and molecular tend investigate hybridization/introgression of empirical we e.g., lineage even Here However, factors, a limitations. evolutionary in computational boundaries. confounding and by species sorting, biased delimit lineage SNAPP+Bayes be to and can BPP developed as approaches been such delimitation analyses, have boundaries. delimitation species (BFD*), species ambiguous numerous delimitation previously increased, it resolve has factor sequencing, data can genome-scale high-throughput which of of data availability advent genome-scale the the acquire As With to cost-effective results. morphol- increasingly conflicting e.g., become produce approaches, has often phenotype-based characteristics, traditional chemical because or challenging is ecological, ogy, species related closely among delimitation Species Abstract 2021 5, April 4 3 2 1 Spjut Richard Nate Madeleine 5 M´exico, Mexico 4 De Ciudad 04510, 70-367, Postal 3 USA IL, Chicago, 2 1 Jorna Jesse fog coastal of fungi lineage hypothesis lichen diverged the desert recently testing a – in middle micro-endemism messy of the in boundaries Species ot .Ba ieSineMsu,BihmYugUiest,Poo T USA UT, Provo, University, Young Brigham Museum, Science Life USA Bean CA, L. Bakersfield, Monte 81145, Box PO Associates, Botanical World M´exico, Apartado Aut´onoma de Drive, Nacional Biolog´ıa, Shore Universidad Lake de Bot´anica, S. Instituto 1400 de Museum, Departamento Field The Center, Bioinformatics Grainger The Education, USA & UT, Science Provo, University, Young Brigham Biology, of Department ol oaia Associates Botanical Biolog´ıaWorld de M´exico Aut´onoma Instituto de Nacional History Universidad Natural of Museum Field University Young Brigham 1 aai .Saxton A. Natalie , 2 ra Ho Brian , 1,* 1 ako .Linde B. Jackson , ako Linde Jackson , 4 un Wu Huini , 1 aai Saxton Natalie , 2 .Tose Lumbsch Thorsten H. , 1 ei Grewe Felix , 2 ra Ho Brian , 1,* 1 ee Searle Peter , ee .Searle C. Peter , 1 ei Grewe Felix , 2 2 a´ad o nee Herrera-Campos Angeles los Mar´ıa de , tvnLeavitt Steven , 2 tvnD Leavitt D. Steven , 1 bgi Jackson Abigail , 1,* 1 bgi .Jackson C. Abigail , 2 a´ad o nee Herrera-Campos Angeles los Mar´ıa de , 1 n hrtnLumbsch Thorsten and , ,5 * 5, 1, 1 ayEieNielsen Mary-Elise , 1 ayEieNielsen Mary-Elise , 3 ihr .Spjut W. Richard , 2 1 1 Madeleine , , 3 , 4 , Posted on Authorea 5 Apr 2021 | The copyright holder is the author/funder. All rights reserved. No reuse without permission. | https://doi.org/10.22541/au.161766187.74749853/v1 | This a preprint and has not been peer reviewed. Data may be preliminary. cnie ta. 06.Itrrtn ntmcl hmcl n opooia hrceso ihn within lichens of characters 2010; morphological Printzen, and 2011; chemical, fungi Leavitt, anatomical, lichen-forming and Interpreting (Lumbsch in data 2016). diversity sequence al., species-level DNA et interpreting incorporating Schneider by groups, po- revolutionized organismal 2015; in been (Rannala, non-model has differences other species subtle as to of Similar populations also detection may recognizing the data 2017). inappropriately for these Knowles, allows and 2020), and loci populations (Huang, Sukumaran divergence additional over-splitting species-level While to recent 2020). lead resolving (Faircloth al., potentially loci et genomic structure, appropriate Kapli pulation semipermeable data selecting 2012; data remain for genome-scale genome-scale al., boundaries approaches harnessing Furthermore, using divergent et species 2018). and when Leach´e recently al., when challenges 2003; et in even computational al., and especially However, et creates introgression, Hey blurred, 2018). 2014; of to remain Larson, hundreds al., cases can and generating (Harrison boundary zones, et strategies, species hybrid McKain cost-effective the species, 2020; and analyses, diverged time- al., delimitation of species et range empirical (Hale wide and genome- a loci for using allow of technologies generated sequencing thousands a be high-throughput 2005; traditional Particularly, be (Dayrat, to 2005). can framework data using integrative al., data an et scale sequence within Will molecular boundaries 2012; especially of al., boundaries, inclusion species et The species Fujita 2013). robust of challenging al., inferring for et becomes delimitation Wagner tool characteristics, 2010; critical Printzen, species, chemical 2020; or al., diverged et ecological, (Poelstra morphology, recently species e.g., of or approaches, challenges species-level phenotype-based related biggest becomes the (Cars- closely structure of evidence population With one of intra-specific Hence, lines in 2008). boundaries differences independent Mallet, species when among 2015; where divergence. determining Knowles, area signals in grey and conflicting a de is Huang for 2005; often delimitation 2013; Queiroz, potential is al., de there and et 1998; speciation, at ascertain Queiroz, tens of arise (de to process may speciation difficult the properties of in are Different process within early the 2004). used Thus, during Marshall, be 2007). orders re- and can Queiroz, different that (Sites population in evidence or species weighing a of times delimiting than of how lines different for indicative rather to become framework is simply concepts USC, empirical properties isolation the species an these reproductive from Under lineage, away or 2007). independent monophyly, debate Queiroz, an morphology, the presenting (de as shifts delimited such – empirically property, (USC) which be concep- concept are can metapopu- species species evolving boundaries unified how “separately to species the as part, – species in conceptualizing due, lineages” However, task, lation 2011). straightforward Wilkins, or 1997; simple (Mayden, a tualized been never has delimitation Species Introduction delimitation delimiting species of seq, problem unable the were highlight words: study but Key as our groupings, such of specious groups results in more The particularly delimitations. support species, species to consistent and tend robust, boundaries models fungi, infer lichen-forming to species delimitation of investigate species lineage we The a Here in Ramalinaceae. species limitations. micro-endemism molecular computational for hybridization/introgression empirical potential e.g., and even the factors, sorting, However, delimitati- evolutionary lineage confounding boundaries. factor incomplete by data SNAPP+Bayes species biased and genome-scale and delimit be of BPP to can availability approaches as developed delimitation the such been As have analyses, (BFD*), boundaries. delimitation genome-scale on acquire species species With to numerous ambiguous results. cost-effective increased, previously increasingly conflicting has resolve ap- become produce can has phenotype-based often which it traditional sequencing, characteristics, data because high-throughput chemical of challenging or advent is the ecological, species morphology, related e.g., closely proaches, among delimitation Species Abstract equally contributed authors three *first aaClfri,Bysfco eiiain(F*,gnaoia iegneidx( index divergence genealogical (BFD*), delimitation factor Bayes California, Baja Niebla ihcmlx eetpyoegahchistories. phylogeographic recent complex, with , 2 Niebla udl&Bwe ntefamily the in Bowler & Rundel gdi ,RAD- ), Posted on Authorea 5 Apr 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161766187.74749853/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. ta. 08.Wt P’ siae fcaecn oehihs( heights node coalescent of estimates BPP’s ( With 2018). al., et . pcmnsampling lineages. 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Niebla spe- N. Posted on Authorea 5 Apr 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161766187.74749853/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. rmSjte l 22)adainduigtepormMFTv Kthe l 05 ao n Toh multiple and The Katoh values. default 2005, to set al. offset were an et parameters with (Katoh remaining matrix, the v7 those scoring and MAFFT K=2’ with 0.2, / combined program = ‘1PAM were ‘unalignlevel’ the and the study using algorithm 0.1, this alignment species aligned of G-INS-i for value of and the generated process (2020) implemented sequences We by provides the ITS al. ranked ASAP 2008). in et partitions step region. species. 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Leach´e time. analyses et in in BPP equation backwards the traced the is used We genealogy the 2020). of al., estimates sympatric et BPP’s putatively e (Poelstra used with we comparisons and species, our micro-endemism species candidate of coupled pairwise sampled ( pattern we our index proposed in limits, divergence the common species genealogical With species empirical 2018). not heuristic al., Leach´e and et proposed 2017; loci boundaries recently al., Tracer population informative the runs. in with delimit highly of analyzed analyses may convergence were of BPP and sets files values replicates data log ESS Resulting six genome-scale delimitation assess Because datasets. the to species’ loci 2005) between each sampled Drummond, of possibility randomly and four probability the aggregate (Rambaut runs repeated of posterior v.1.7.1 an between replicates subsets The variation for 12 independent for or subsets). three pooled control datasets, loci and to then selected used datasets, was was randomly loci analysis model the informative ‘A00’ for most the each the in file times (Dryad for used iterations as runs burn-in size independent 20,000 sample including of (six same generations, exception 100,000 the the for with were and run 2018), Parameters S52), were al., analyses. chains et BPP MCMC (Flouri ‘A10’ dataset, tutorial our each 4.3.0 for v. above BPP defined in as τ found loci “frog.ctl” of the subsets with different consistent the used we analyses, -2τ/θ ros smnindaoe h ae1 pce hlgntcte ro a nlddi l nlss For analyses. all in included was prior tree phylogenetic species 17 same The above. mentioned as priors, gdi eiiain eecmae oteiiil‘1’aayi efre sdsrbdabove. described as performed analysis ‘A10’ initial the to compared were delimitations , cr eut rmtedffrn apesbesuiganraiainfnto nEcl sarl of rule a As Excel. in function normalization a using subsets sample different the from results score ocluaetepoaiiyta w eune olsebfr ecigseisdvrec ( divergence species reaching before coalesce sequences two that probability the calculate to gdi θ values and τ values. < . ugs igeseisand species single a suggest 0.2 .juncosa N. .cornea N. gdi eueBysfco pce eiiain(F* nlssi NP v.1.3 SNAPP in analyses (BFD*) delimitation species factor Bayes use we , s183F&sl16857BF]; & [sl16863BF var. juncosa gdi s175&s177;and sl16707]; & [sl16705 crsfrec oprsnbtenete igespecies’ single either between comparison each for scores gdi 7 θ , values τ gdi n aalbl sdsrbdaoe Candidate above. described as labels taxa and gdi .dilatata N. crswr acltdidvdal o each for individually calculated were scores > crsfloigJcsne l 21)and (2017) al. et Jackson following scores . ugs w itntseis while species, distinct two suggest 0.7 .juncosa N. s190&sl16946]; & [sl16940 τ and θ var. ocluaethe calculate to gdi τ spinulifera and crsn longer no scores gdi gdi N. θ ako et Jackson ; gdi ausfrom values aff. cr from score gdi gdi gdi τ [sl16706 o each for gdi isidiosa when ) gdi scores scores score. θ and =1- gdi for Posted on Authorea 5 Apr 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161766187.74749853/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. a obndwt nte atto opiigtermidro h pcmn ntesm ld o all for clade same the to in similar specimens morphologically the was of remainder specimen the This comprising analyses. partition subsequent topologies with another ML coincided with and largely combined SVDQuartets+PAUP* ( marker were the was sl16849BF barcode in samples fungal Specimen by data two standard 2). reprsented RADseq the least were for (Fig. from inferred which at spe- inferred clades different of containing partitions monophyletic as 13 partitions ASAP reciprocally identified ASAP, The ASAP specimens using 2). of e.g., 2020) (Fig. specimens, al. majority cies generated polymorphic et The sequences morphologically/chemically Spjut RADseq. (ITS of in dataset for comprised ITS reported selected combined sequences samples the ITS from the + inferred study were partitions this species for 32 barcode of fungal maximum standard A the using delimitation species ASAP 3.2.1. (Fig. reconstruction ML well-supported delimitation the Species generally from 3.2. a strikingly in differ S55). resulted relationships SVDQuartets+PAUP* file backbone Dryad 2020). cases 2; al., some et in (Spjut although topology, location ( a sl-161006 at diffe- saxicolous in recognized and metabolites—obviously ( acid), secondary sl-161004 salazinic their terricolous , by example, more for species clades—include, as ( rent differentiated sl-16775 proximity saxicolous close include in growing that specimens varieties—occurred acid or divaricatic spinulifera are species examples distinct proximity 2); as isidiosa close (Fig. morphologically in clades collected differentiated different specimens chemotype—but in those Additionally, same 2). except the Fig. monophyletic the 1; having as to (Appendix recovered sister Mexico not clade California, were Phenotypical- Quint´ın, acid Baja samples subclades. divaricatic depsidone multiple larger and as by much acid identified represented sekikaic a into species with divided we- circumscribed 2) clade acid ly (Fig. a divaricatic or clades or distinct clade, polyphyletic acid of (nonterpenoid) sekikaic two identi- production depsidone either recovered the in of data with recovered depsides SNP coincided each producing here the re recovered specimens and clades although data Major metabolites, RAD-Seq 2). secondary concatenated (Fig the topologies both well-supported from cal, inferred variable topologies 198K ML including of The Mb, total 3.6 a was reconstructions in loci Phylogenetic resulted RADseq 3.1. dataset concatenated the RADseq of final length Our total loci/sample). sites. 326,580 The (mean=22,373 of loci. average 39,941 143-bp the an to 25,086 to with 2,534 mapped 1,151,143, from reads to ranged of 58,930 75.3% of from number to varied The 7.3 sample 1). with each sample, for in per reads sequences clade Illumina each of across number averaged The was value of likelihood convergence marginal and Results values the 3. ESS and files convergence. assess log times ensure to Resulting four further burn-in. 2005) to run 10% Drummond, order was and path-sampling gamma of and BFD* model generations a a distribution (Rambaut the 1,000,000 Each using a v.1.7.1 using of utilized runs. obtained Tracer have We length set were in chain to generations. were values steps, analyzed set 1,000 theta 48 likelihood were was every with and Marginal Theta Coalescence sampling BEAST (2014). lambda 1,000,000, in 500). al. to both analysis Leach´e G(2, dataset. et set for by of was SNP Priors outlined distribution length 500 methods chain a sample. have The entire to to 250). the set specified G(1, S54). using was and file Lambda BEAUTi Dryad 10.0 (see in at distribution. v2.6.3 set directly BEAST was in calculated analysis rate were separate a rates as Substitution run clade each with clades, major n l178( sl-16778 and ) ,s-60 ( sl-16707 ), .cornea N. 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No reuse without permission. — https://doi.org/10.22541/au.161766187.74749853/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. 5) F*aaye upre h otdvsv pce eiiainmdl ntoo h he ucae – file subclades Dryad three 2; the of (Fig. two in analysis models ASAP delimitation single-locus species the divisive than most the species supported putative analyses more BFD* delimited S54). BFD* cases, all In and SNAPP of estimated in dataset the the implemented small of very BFD* because deviation a 3.2.4 caused standard of the primarily exception is the is so with trend but loci, This variable subsets, for most trend. 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(Fig. subsets remaining the with compared when scores gdi juncosa θ fec niiulcniaeseiswr lohge hnuigtems informative most the using when higher also were species candidate individual each of N. siain.Seisdlmtto sn xdgiete nlss‘1’i BPP in ‘A10’ analysis – tree guide fixed a using delimitation Species estimations. aff. .cornea N. i ld H) and ‘H’), clade (in isidiosa .fimbriata N. rmteCanlIlnswt w pcmn rmBj aiona(in California Baja from specimens two with Islands Channel the from . gdi , scores .palmeri N. .flagelliforma N. gdi 9 Niebla crscluae rmalohraaye suggested analyses other all from calculated scores and , gdi crs erpr average report we scores, θ pcmn Fg E&)adcletdfrom collected and &K) 1E (Fig. specimens .suffnessii N. values. with .rugosa N. gdi i ld ‘M’), clade (in crsspotdcollapsing supported scores i ld B)(i.3). (Fig. ‘B’) clade (in τ auswsobserved was values gdi gdi gdi crsars all across scores ausgenerally values .flagelliforma N. scores τ siaeis estimate < 0.2), Posted on Authorea 5 Apr 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161766187.74749853/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. ubr fsmldlc n addt pce,cmuainllmttosmycnri h tlt fthis limitations of these circumvent utility to the attempted contrain we may increasing study, limitations with this 2014), In computational 2014). al., species, BFD* (Leach´e al., et the S54), candidate (Leach´e models 3). et While approach file and species models. 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Baja al., (Leach´e et diverged recently (2 size population l uprigfie r vial nDyd https://datadryad.org/stash/share/XD Dryad: on available are files supporting All Accessibility: in Data speciation in with define chemical work thorough for to future more used difficult in Nevertheless, in characters 2011). considered notoriously chemical species Leavitt, some and are and clarify morphological (Lumbsch fungi may lineages traditional Lichen-forming investigations natural cases, reflect group. some to in this models fail and species taxonomy in boundaries specific delimitation, taxonomy for work species species evidence Future the of system. of “messy” history. lines resolve terms non-model evolutionary additional all-too-often to add complex a the to order provide their here in on presented of in they hypotheses methods literature understanding the delimitation, delimitation our upon of species build complicate species should body of further empirical growing that terms these the organisms in of in to ambiguous use add somewhat the they are deserts. of Furthermore, fog analysis perspective coastal infer our valuable in to of a only crucial results be occurring will fungi the Domingo, lichen-forming While Santo of Morro genus and important Negra, in Punta processes and diversification Rosalillita Santa Punta between ridges and individuals founder few a by heuristic established The was inferences. pos- population consistent if a loci in where analyzed result cases of can in number loci the biased sampled N maximize be randomly to also of important may number is criterion lower it that loci, higher RADseq but consistent, short sible, largely using in when resulted that loci, suggest RADseq selected these randomly locus lowest contrast, and that the In here in 5). show resulted (Fig. we loci tasets 2018), RADseq on al., variable, Leach´e impact et highly 2017; substantial but al., a et have species (Jackson can of boundaries selection number species from modest structure a lation even heuristic with the models efforts, While those in Despite issue subsets. an data S54). feasible remained file computationally (Dryad runs analyze independent to order among in convergence topology the subdividing by A and θ Α gdi gdi a evr ml.Bcuetecieindpnso h ouaindvrec ierltv to relative time divergence population the on depends criterion the Because small. very be may tmyntb osbet ne ihcndneseisbudre ie h aueo the of nature the given boundaries species confidence with infer to possible be not may it , crswr oecnitn ihte50ms aibeRDe oidtst hs results These dataset. loci RADseq variable 500-most the with consistent more were scores τ gdi / θ Α rtro o pce eiiainpoie erct ud h itnto fpopu- of distinction the guide to metric a provides delimitation species for criterion ,teueof use the ), cige l,22;Shc ta. 02,atog suswt sn hsmre in marker this using with issues although 2012), al., et Schoch 2020; al., et ucking ¨ Niebla gdi Niebla a edt liso pce ttsee fteppltosare populations the if even status species of claims to lead may gdi omnte,icuigMs aah,Ms at Catarina, Santa Mesa Camacho, Mesa including communities, Niebla. crs nteeprclseai of scenario empirical the In scores. nadto,eooia n aeciai etr hudbe should feature paleoclimatic and ecological addition, In . 11 gdi Niebla crs pcfial nte1-ad13mre da- 163-marker and 10- the in specifically scores, Niebla iegs n twudntb unreasonable be not would it and lineages, nerdfo hlgnmcdt (Fig. data phylogenomic from inferred Niebla Niebla eetn limited, selecting , ouain in populations cige al., et ucking ¨ gdi scores; Niebla kUF- gdi Posted on Authorea 5 Apr 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161766187.74749853/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. ietyfo ilei eei akr:BpsigGn re naFl olsetAayi.Molecular Analysis. Coalescent Full a in Trees Gene 1917-1932. Bypassing State 29, Trees Evolution Markers: Arizona Species and Inferring Genetic Unlimited, Biology 2012. Lichens Biallelic A., RoyChoudhury, 2. N.A., from Rosenberg, Vol. Directly J., Felsenstein, Region, R., Bouckaert, Desert D., Sonoran Bryant, Greater 36-80. the pp. of Arizona, Tempe, Flora University, Lichen (Eds.), C., evolutionary F. Zhang, Bayesian 2004. D., for J.E., platform Xie, Marsh, P.A., software Bowler, C.-H., e1006650. advanced Wu, 15, An M.A., Biology 2.5: Suchard, Computational BEAST I., PLOS 2019. Siveroni, analysis. A.J., D., Drummond, Rasmussen, T., A., Stadler, Rambaut, J., A., Heled, Popinga, A., Gavryushkina, L., M., Fourment, 159-171. K Duchˆene, S., 108, G., J., Botany Jones, Barido-Sottani, of T.G., lichen Vaughan, Journal reindeer R., American a Bouckaert, woodlands. of lichen genomics Population American 2021. North J.C., from A., species American endangered, Villarreal S., structure. the Payette, population of F., strong Grewe, Alonso-Garc´ıa, analysis M., and genome-wide 1556-1567. distance First 105, by Botany 2018. isolation of S.E., reveals Journal Alter, linearis R.S., Cetradonia lichen Sleith, endemic S.K., and McKenzie, Sciences J.L., discussions. Field Life Allen, fruitful the of for at Serusiaux College Center Emmanuel the Bioinformatics and Grainger Muscavitch, for References Zachary the support Jensen, and acknowledge Enrique Jacob University and Young also thank acknowledge We Mazo We Brigham gratefully Museum. Punta at We guidance. Natural Foundation. Biology and Reserva Negaunee of in expertise The Department collections by his field funded for facilitating was for Alfaro study Peninsular Terra this from of support portion pending. work # field project The under Archive Read Short NCBI Acknowledgements in deposited are reads RADseq All link) (temporary pRIek8qAngIa3OIEclxC0JePCDmZAyCx1ClyI • • • • • • • ra l S7 file Dryad loci. bootstrap RADseq indicate of nodes subsets at Values S6 file 2). 100%. Dryad the from than Fig. 3’, rankings less in and ‘clade when (summarized scores support in PAUP+SVDquartets BF models; using – inferred ‘clade 13-species 3 tree and Page and S5 2’ 2- using models. file ‘clade the 3-species selected analyses over Dryad for and was SNAPP+BFD* selected 2- models specific model the was clade delimitation 16-species over model the species selected the 5-species was different assessed; the model three species 2’, 7-species the 16 ‘clade – to in 2 3 3’; Page from (BF). ranging factors 1’, Bayes ‘clade for models S4 file Dryad calculate attoso neett aete optbewt P.Frhr tcetsa.x l ihtenames data the S3 RAD-Seq with the file file of .txt Dryad a file interest, file. S2 .phy creates of “.phy” a it file partitions Further, the output Dryad the BPP. from to of with used species names compatible are candidate the them files of make with three to file Those interest “.txt” itself. of a file partitions data, partition RAD-Seq “.txt” the the of and file “.fasta” a using files S1 file Dryad het . eMi,N,Mtcie,M,Mne,FK,M F.K., Mendes, M., Matschiner, N., Maio, De D., uhnert, ¨ gdi crsdrcl rmBP‘0’tp nlssoutput. analysis ‘A00’-type BPP from directly scores eut fteSAPBD nlss ae1–tefiedffrn pce delimitation species different five the – 1 Page analyses. SNAPP+BFD* the of Results . . Srp ’–cso yhnsrp o octntn AT eune noBPP-style into sequences FASTA concatenating for script python custom – 1’ ‘Script . Srp ’–cso yhnsrp oextract to script python custom – 3’ ‘Script . eut rmteBP+ BPP the from Results . Srp ’–cnrlfiefrteA0tp nlssi BPP. in analysis A10-type the for file control – 2’ ‘Script . ieb-iecmaio fteM oooyifre sn Qreadtespecies the and IQtree using inferred topology ML the of comparison Side-by-side . gdi crs&MM usfo h A0 pce eiiainaaye sn different using analyses delimitation species ‘A10’ the from runs MCMC & scores Niebla n ah .. I,Ra,BD,Deeih .. re,C,Bungartz, C., Gries, P.C., Diederich, B.D., Ryan, III, T.H., Nash, In: . d “ gdi 12 i-on pce eiiainapproach. delimitation species tip-down” τ and le,NF,Oive .. uPlessis, du H.A., Ogilvie, N.F., uller, ¨ θ ausfo h P nlssto analyses BPP the from values Posted on Authorea 5 Apr 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161766187.74749853/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. on,DT,Cenmr . o aslr . ih .. ih .. 08 Fot:Ipoigthe Improving UFBoot2: 2018. L.S., Vinh, B.Q., 518-522. Minh, 35, Evol A., confronting Biol Haeseler, Mol and Approximation. von Understanding Bootstrap O., 597. 2003. Ultrafast 18, Chernomor, R.G., Evol. D.T., Harrison, Ecol. Trends Hoang, R.K., conservation. Boundaries. and Butlin, biology Species M.L., in of Arnold, uncertainty species Nature R.S., Waples, the J., and Hey, Introgression, in 795-809. Hybridization, 105, Applications Heredity 2014. plants. of E.L., in Journal per-sample Larson, genomics reducing R.G., population for Harrison, and Strategies phylogenomics 2020. e11337. for M.G., 8, Johnson, sequencing Sciences L., capture Plant Pokorny, target J., in Viruel, costs E.M., Gardner, RAD species H., of fungal Hale, analyses lichen-forming genomic the Population of 2018. H.T., relationship aurantiacoatra Lumbsch, Usnea phylogenetic C., the Printzen, resolves H., Wu, sequences Reports E., Scientific Lagostina, DNA. 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J.D., Satler, N.M., n/a-n/a. ecology, Reid, (Ed.), T.A., I. Pelletier, Pavlinov, B.C., In: InTech. Carstens, Renaissance. Issues. the Ongoing After - Decade A Problem Delimitation: Species Species The 2013. J.W., Sites, A., Camargo, yoes 91-113. MycoKeys, . 13 se antarctica Usnea and Posted on Authorea 5 Apr 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161766187.74749853/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. hka . api .(d.,Rcn dacsi ihnlg.Srne ni,p.11-44. pp. India, Springer Lichenology. in P.K., Divakar, Advances L D.K., Recent Upreti, (Eds.), Progress In: R. Delimitation: Populations. Bajpai, Species Natural of V., in Discipline Shukla, Boundaries Dynamic Species The Recognizing 2015. Effectively H.T., Toward Lumbsch, C.S., Moreau, S.D., evolutionary Leavitt, Resolving 2016. Scientific approaches. 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J.W., Cladonia barcoding in Poelstra, DNA delimitation the Species to 2013. challenge H.T., a Lumbsch, mycota): British A.R., lichens. Burgaz, Mart´ın, of M.P., R., identification Pino-Bodas, the for methods Microchemical stochastic London. 2001. effective Society, F.J., and Lichen 268-274. White, P.W., fast 32, James, a Evol A., IQ-TREE: Biol Orange, 2015. Mol B.Q., phylogenies. Minh, maximum-likelihood A., estimating Haeseler, for von algorithm H.A., Schmidt, L.T., Nguyen, 358-385. 65, Systematics < i > Parmeliaceae < /i > nClmi sn h nenltasrbdsae IS.PatadFungal and Plant (ITS). spacer transcribed internal the using Colombia in ) cig . 00 etn N acdn in barcoding DNA Testing 2020. R., ucking, ¨ ße,D,Bac,MB,Adimeoo,JB,Buhz O., Bouchez, J.B., Andriambeloson, M.B., Blanco, D., ußler, ¨ 15 < i > Usnea < /i > Posted on Authorea 5 Apr 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161766187.74749853/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. nWs-aigsoe fMneCnz,PnaMz.D. Mazo. Punta Ceniza, Monte of slopes dwelling West-facing soil on B. species Mazo. of Punta method doeste, coalescent diverse Bayesian supporting a California, of Evaluation 1 2011. Figure Z., loci. 747-761. Yang, multiple 60, T., Biology from Zhu, Systematic data D.-X., delimitation. sequence Zhang, DNA C., using Zhang, delimitation species Evolution. Unguided and of 2014. Biology Proceedings Molecular B., data. Rannala, sequence Z., multilocus 9264-9269. Yang, using 107, delimitation Sciences of species Academy 61, Bayesian National Zoology 2010. the Current B., delimitation. Rannala, species Z., and Yang, estimation tree species for program populations? BPP 854-865. divergent The Molecular 2015. genetically habitats. Z., karst or Yang, isolated species from 219-231. 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PLoS uncorrected N., conditions. methods tree simulated Nagarajan, Sovi´c, species of I., and R., range Vaser, Concatenation a 2015. under G., accuracy Orti, indistinguishable M., Shcheglovitova, statistically D., of exhibit Stern, Proceedings A., Moore, species. J., 1607. not Tonini, 114, structure, Sciences delimits of coalescent Academy Multispecies National 2017. the L.L., Knowles, J., Sukumaran, Press. 1. 1996. 73, R.W., MycoKeys Ramalinaceae the history. Spjut, Ecology, in evolutionary genera of and fruticose diversity The Review S´erusiaux, 2020. their N., E., Annual Lecanoromycetes): Magain, M., (Ascomycota, species. Guissard, A., delimiting Simon, R., for Spjut, criteria 199-227. Operational 35, Systematics 2004. and J.C., Evolution, Marshall, J.W., Sites, yi osa o eetcmuiyi ut aoNtr eev,na a Quint´ın, Baja San near Reserve, Nature Mazo Punta in community desert fog coastal Typic . Niebla and Niebla Vermilacinia iic . 07 atadacrt env eoeasml rmlong from assembly genome novo de accurate and Fast 2017. Siki´c, M., ˇ omnte.A. communities. ibaarenaria Niebla Rmlnca)fo aionaadBj aiona BRIT California. Baja and California from (Ramalinaceae) 16 erBhı as,SnQuint´ın. C. San Bah´ıa Falsa, near Niebla ibaflagelliforma Niebla omnte nvlai lpso Volc´an Su- on slopes volcanic on communities ( evt 16856BF Leavitt Niebla communities .E. ). Niebla Posted on Authorea 5 Apr 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161766187.74749853/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. .N. ). h A0 pce eiiainmdli h rga P.Terslsaesmaie rmalaaye of species. analyses of number all inferred from the summarized for are aggregate, in results 5 probability, The the Figure BPP. shows Y-axis program the the with in subsets, data model RADseq delimitation species ‘A10’ the varia- 100 2017, 4 of al. Figure subsets et random of Jackson three following subsets loci; with random loci); variable comparisons variable three most RADseq blue), most 163 loci; in subsampled ten (highlighted the variable different the species loci; 1000 using variable and of and most loci; subsets – 500 ble random ‘A’ the three panel loci; variable in loci; 500 RADseq species variable candidate (5649 “tip-down” see the datasets – in comparisons tree collapsed species species tips the didate SVDquartets+PAUP); is in tree model “shadow” subdi- coalescent phylogenetic (Pink BPP+ on inferences multispecies based tree the SVDquartets+PAUP defined under and species inferred ML candidate the represent from boxes, inferred pink vision in highlighted and ‘Q’, through that standard partitions the 3 ASAP – of separate Outgroup Figure representing analyses analyses. indicate tips from subsequent shaded branches all partitions double blue for ASAP with light combined (ITS), to were fungi and correspond for clades); boxes marker with (two barcoding Grey indicated DNA acid acid. clades), are (two sekikaic salazinic clades acid divaricatic producing producing Chemical producing panel. lichens lichens lichens PAUP+SVDquartets. bottom-left indicate indicate branches in in branches black map model topology: dark-blue coalescent in ML sites the multispecies on sampling the branches to under colored correspond inferred tips tree at species Colors the is tree “shadow” 2 Figure Spjut. R. by identified diva- terminal isidiosa rigid aff. Niebla the ( to ‘K’ contrast panel var. in in apex shown F. near segments Guadalpe. downwards reflex curved to branches the ricate by traits morphological isidiosa aff. juncosa ibajuncosa Niebla gdi Ltplg nerdfo 9,0 aibestsdsrbtdars vr2,0 Asqlc.Pink loci. RADseq 25,000 over across distributed sites variable 298,000 from inferred topology ML . itiuinof Distribution . prahaehglgtdwt upebxs .Heuristic B. boxes. purple with highlighted are approach rbblt fdffrn pce eiiainmdl pce A Q,Fg nerdusing inferred – 3 Fig. ‘Q’, – ‘A’ species – models delimitation species different of Probability . addt pce n pce eiiainaaye sn BPP+ using analyses delimitation species and species Candidate . ( evt 16707 Leavitt hsseie a dnie as identified was specimen this ; ibaflabellata Niebla gdi ( ; evt 16853 Leavitt crsbten02ad07aeabgos(ihihe ngrey). in (highlighted ambiguous are 0.7 and 0.2 between scores var. spinulifera .I. ). gdi gdi ( evt 16713 Leavitt crsetmtdfo ieetsbeso Asqlc 13b each). bp (143 loci RADseq of subsets different from estimated scores ibaundulata Niebla crs[]07idct itntseishglgtdi elw n ttsfor status and yellow, in highlighted species distinct indicate 0.7 [?] scores .L. ). ( evt 16719 Leavitt ibamarinii Niebla .G. ). evt 16775 Leavitt N. ( evt 16708 Leavitt aff. Niebla .O. ). 17 isidiaescens ( Vermalicinia evt 16763 Leavitt s.nov” “sp. ibacornea Niebla ,wihcoeyrsmlstetp rmIsla from type the resembles closely which ), .J. ). ibaflagelliforma Niebla . n iesfrom differs and pcmn o shown. not – specimens gdi ( .M. ). evt 16849BF Leavitt ( gdi ausifre rmdffrn can- different from inferred values evt 16863BF Leavitt ibalobulata Niebla crs[]02idct single a indicate 0.2 [?] scores gdi .Cae aee ‘A’ labeled Clades A. . N. .H. ). ( aff. evt 16731 Leavitt ( .Alspecimens All ). evt 16859BF Leavitt isidiosa ibajuncosa Niebla nkey in .K. ). Posted on Authorea 5 Apr 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161766187.74749853/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. 18 Posted on Authorea 5 Apr 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161766187.74749853/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. 100 k m 98 51 0 . 0 0 3 substitutions/sites 19 N. laminaria N. flagelliforma sl161137 N. cornea N. aff.isidiosa N. aff.isidiosa N. cornea sl16856BF N. cornea 50 99 N. cornea N. cornea k16681 79 N. eburnia N. josecuervoi N. lobulata N. laminaria 46 k16576 N. flagelliforma sl16775 sl16853BF k16128 N. juncosa N. juncosa N. juncosavar. N. flagelliforma N. juncosa N. flabellata sl16863BF sl16857BF sl161164 N. brachyura 92 N. flabellata

N. arenaria N. marinii sl161027 N. “sp.nov.” sl16778 N. cf.limicola

N. marinii sl161045 N. lobulata var. spinulifera var. spinulifera N. rugosa var. juncosa sl161019

sl16713 N. dilatata N. suffnesiisl161017 N. dilatata sl16731 sl16763 N. palmeri sl16973 sl16937 sl161005 N. fimbriatasl161098 N. turgida N. undulata sl16907 N. flagelliforma N. rugosa sl16859BF sl16915 sl16849BF N. undulata sl16945 N. podetiaforma sl16705 sl16946 sl16940 sl16707 sl16706 sl16719 sl161004 sl161047 sl161013 sl161006 sl16708 sl161049

sl16704

Posted on Authorea 5 Apr 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161766187.74749853/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. B A 98 51 0 . 0 0 3 substitutions/sites N. laminaria N. flagelliforma sl161137 97 N. corneak16681 N. isidiosa N. corneak16576 20 sl16856BF N. corneak16128 N. corneasl16863BF&sl16857BF N. eburnia sl16775 &sl16853BF N. josecuervoi N. lobulata N. laminaria N. flagelliforma N. juncosavar.spinuliferasl16706&sl16719 N. flagelliforma 99 N. flabellata N. juncosa 99 sl161164 N. brachyura

N. flabellata N. arenaria N. marinii sl161027 N. “sp.nov.” sl16778 N. cf.limicola N. marinii sl161045 N. lobulata var.juncosasl16705&sl16707 N. rugosa sl161019 sl16713 N. dilatata N. suffnesiisl161017 sl16731 sl16763 N. palmeri sl16973 sl16937 sl161005 N. fimbriatasl161098 N. turgidasl161047 N. undulata sl16907 N. flagelliforma N. rugosa sl16859BF sl16915 sl16849BF N. undulata sl16945 N. podetiaforma sl16940 &sl16946 sl161004 sl161013 sl161006 sl16708 sl161049

sl16704

M G Q O C K B D H N A E P F L J I Posted on Authorea 5 Apr 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161766187.74749853/v1 — This a preprint and has not been peer reviewed. Data may be preliminary.

0 1 Frequency2 3 4 probability of species model 0.25 0.05 0.10 0.15 0.20 0.00 1 2 3 0.25 4 Number ofinferredspecies 5 6 7 21 8 GDI 0.50 9 10 11 12 RADseq subsample 0.75 13 100 randomloci 1000 randomloci 163 mostvariableloci 5649 variableloci 500 randomloci 10 mostvariableloci 500 mostvariableloci 14 15 16 1.00 17