Posted on Authorea 26 Jun 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159318488.82442436 — This a preprint and has not been peer reviewed. 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All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159318488.82442436 — This a preprint and has not been peer reviewed. Data may be preliminary. l,20;Rente l,20;Rent&Ewrs 01.Hr,w aegnrtdanwdatgenome draft new a approach integrative generated an have et employing we Bazin while Here, individual 2004; 2011). white-faced Whitlock, Edwards, a & & on (Ballard based Rheindt which Wren-babbler sweeps 2018b), 2009; Limestone al., selective al., L´opez-Baucells the et frequent 2014; et of largely and al., have Rheindt et introgression speciation 2006; Wang in genetic al., 2008; traits to al., behavioural et susceptible and (Rheindt morphological is DNA of mitochondrial importance vocal on relative and relied coloration the most of of the importance tests be relative Previous to the thought process. test Hpa- are the differentiation to at vocalizations to the model least which individuals in ideal at in brown-faced parameters an divergence, complex provides cross-river and songbird character plumage-based west colored defining conspicuous the cryptically important a otherwise to such an of individuals in discovery white-faced individuals The An, with brown-faced river. and Salween, the (16 white-faced commonly of the of Wimpong east of be distribution as side exclusive such can either mutually it, individuals a on along of established white-faced directly has inspections or River, that museum River Salween revealed Salween Subsequent description Myanmar the face. (16 original of across Moulmein brown the west entire on range a localities the Based at wider has of 1963). found specimen specimen a (Deignan, variously type type from border River The has Salween Thai the specimens the 2018). but the of (1855), al., east to 1a), mountain Blyth et close a (Figure Taung, by River) (Collar Mulayit Myanmar from Thanlwin “morph” in 1855 Burmese: white in distribution collected (in or individual the form Myanmar of a leucistic is west complex a far of as population the regarded white-faced species, from been conspicuously distinct historically a a known complexities, evaluation as been taxonomic recent taxon these a biological rufous-bellied However, to of the 2019). lack Adding al., split general et specimens a Cai far museum and 2002; these morphology of Allen, conserved calcicola of differences & members’ identity (Robson plumage its birds taxonomic on to of the based due group to this species points of single obs.) understanding a pers. as (PDR, treated been border as Lao birds the Thai northern abutting Province nominate Nan to grey – in known a then either nominate population and Thai taxa: genus, three the 1a) comprises to characterized (Figure complex common are species throat complex The Thailand streaked species belly. western distinctly this rufous a of dark plumage, Members or brown striking Asia. inconspicuous associated a Southeast closely an provides across complex by species equally formations songbird karst which conserved limestone morphologically but complex non-dispersive, with reproduction, highly species a for a of important requires example Wren-babbler, an ideally be Limestone one to The traits, known contrast. bioacoustic morphological are as and species vocalizations such morphological understood. which bioacoustic traits, between poorly biological in time, interplay of remain species same interplay this thus cryptic and the test process differentiated importance To At genetically speciation relative in the 2019b). 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Deignan, 1922; (Baker, 38’E) etrdi ita n as and Laos, and Vietnam in centered ge rai iue1) h ietn rnbblrhstraditionally has Wren-babbler Limestone The 1a). Figure in area (grey .crispifrons N. crispifrons 2 h oercn icvr fa daetpopulation adjacent an of discovery recent more the , aohr crispifrons Napothera Cla ta. 2018). al., et (Collar pe,21) nfc,agoignme of number growing a fact, In 2018). ¨ opfer, calcicola crispifrons rmlwrnrhatThailand northeast lower from seatysc oe.I is It model. a such exactly is , m&Rnt 03 Philippe 2003; Ranft, & ¨ om N. rmMamrand Myanmar from ° [ 9N 97 49’N, c. ] crispifrons .crispifrons N. ° 8E and 28’E) has N. Posted on Authorea 26 Jun 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159318488.82442436 — This a preprint and has not been peer reviewed. Data may be preliminary. h irre eesqecduigteIlmn ie 00pafr ih10b ardedruns. paired-end bp barcodes. 150 and with adapters platform including 4000 assembly Hiseq bp genome size Illumina 370 Draft fragment the approximately and using concentration was sequenced DNA unique size library were check a Each DNA libraries fragment to with UK). The input Analyzer prepared peak Biolabs, Fragment was average an England AATI sample (New an The with Each Illumina and respectively. (New cycles. samples for All Qubit PCR Kit Oligos except with eight Prep protocol. 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Sma ta. 05,wihsoe httegnm a 14 opeewt the genome the with draft against complete the genome 81.4% draft of al., was completeness et genome with (Smit the the synteny 4.0.9 assessed that conserved RepeatMasker We showed scaffolds with which 36,669. of regions short 2015), to total removing gallus repeat al., scaffolds (Sim˜ao by a filtering et 3.1 of output bp, and BUSCO number scaffold 48,862 script using = the the custom N50 processed a of reducing with overlap further with thus We minimum genome bp 2015), Assembler a Gb. a 3000 Celera requires 1.08 produced than it and of assembly less as bp size initial of scaffolds The a billion and stringent. and 5 contigs more scaffolds of of thus 82,195 assembly size is the jellyfish and for a bp chosen with 40 was 2000) run al., was et pipeline (Myers MaSuRCA The assemblies. read h oat pini NS .2 Kreise ta. 04.TejckieRsrp nAGD0.923 ANGSD with in obtained script was jackknife.R alignment The consensus 2014). a al., and et of above (Korneliussen assembly as 0.923 genome ANGSD pipeline whole in same the option the for 2 using doFasta sequenced the processed for reads were raw applied the reads as Babbler raw obtained parameters Rufous-capped We with same and ruficeps the population. bp, C. with (H4) 50,000 2014) outgroup of ABBA- size an al., computed We block as et a 2012). 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No reuse without permission. — https://doi.org/10.22541/au.159318488.82442436 — This a preprint and has not been peer reviewed. Data may be preliminary. n hiad eeietfid hs ekrgoswr lge gis h er ic eoe(assembly genome Finch Zebra the against aligned were regions peak These bTaeGut identified. were Thailand) d and net the brown in two found the between regions highly-conserved are d but net individuals high brown a 99.9 expect the We above baseline. d windows the Outlier using populations d size. net window size computed step we per Additionally, bp identified. present 10,000 were window, be comparison sliding must pair bp sites 50,000 population three each parameters: following least the at with and pair population each for values Spotn nomto al 3.Dsiealrepnlyipsdo h ariecmaiosinvolving comparisons pairwise the on imposed between penalty detected large diagnostic we a as criterion, Despite emerged Isler S3). the Table parameters information Using vocal (Supporting between 1b). the parameters (Figure of vocal investigated none diagnostic parameters two bioacoustic to the one of basis the that on revealed measurements vocal annamensis of results PCA taxa and three (ML) all of with likelihood comparison aligned Bioacoustic maximum and (NJ), 2016) al.’s joining al., each. neighbour et et bootstraps a mitogenomic Lim Results (Kumar 10,000 construct of following MEGA7 with coverage to tree to sequence (MP) low used exported ND2 parsimony were a also a the maximum sequences had with was retrieve ND2 which reads MEGA7 to the All samples ClustalW. separately All blood format. sequencing FASTA For protocol. 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[ . ouain from populations ausars the across values c c , .] .] calcicola crispifrons individuals crispifrons and . Posted on Authorea 26 Jun 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159318488.82442436 — This a preprint and has not been peer reviewed. Data may be preliminary. ht ae r ieyarcn curnedie ymttosi e oaie regions localized few a population in Lao mutations by the driven with occurrence recent continuum a a likely form (PDR, are may gap faces geographic populations White a Thai fills to northern border, belong that Lao ultimately suggests the may from abutting and and Province, Citing populations Nan obs.) herein. assigned in included pers. population 1963) not adjacent (1945, populations an to of of Deignan Province discovery affinity Phrae details, the in limited on Thailand light providing northernmost further but a shed on differences, to able relying morphological be in taxonomy. the will integrative summary, limitation sampling of In the Future for importance highlights 2009). the proclivity (Hall, patterns advocates their parameters bioacoustic and temporal to vocal and approach for some attributed single plumage difficulties of in be between important. measurements resulting may in detected less wren-babblers, variation of divergence differentiation incongruence taxa large vocal bioacoustic two a these for perceived and in need usual of analysis is the lack which render partners, the may between hand, duetting colors other belly the different their On grey-bellied 1b): the integrity hand, (Figure species one study rufous-bellied maintaining the the in On important from limited. especially guishable are be may cues traits visual bioacoustic when that suggests species distinct plumage grey-bellied namensis similar a sharing Despite the congruent? demonstrates traits ultimately morphological but and 2019), bioacoustic alone. Are al., data extinct et mitochondrial of Zhang on haplotypes mito-nuclear 2011; mitochondrial relying This Edwards, of of introgression’ & S2). shortcomings ‘ghost Figure (Rheindt undi- as information remaining populations such (Supporting and unsampled causes, analysis S1) of or Structure variety Figure differentiation, in a information have limited K Supporting may exhibited of 1, discordance levels populations (Figure higher two clusters at these Kerr PCA even genomically 2003; tight vided Yet al., same et 2016). di- the (Hebert al., mitochondrial in groups et of occurring songbird depth other Campbell in this 2009; species delimitation, al., sister species et distinct of between found purposes sometimes for is borderline vergence While S4). Table information of populations Thai mensis western and Myanmar the Unexpectedly, relying of primarily separation of dangers color-based the crispifrons (2018) (Hebert to N. phylogenetics al.’s attest bird et results in Collar that Our pairs indicates overhauls: 2016). species taxonomic al., sister et modern Table typical calcicola Campbell in information between 2009; characters (Supporting those al., plumage yardstick as et convenient on Kerr deep a 2003; as provide al., twice incorporating 6.4% et over Table analyses – 1; are in (Figure ˜5 flow and clusters gene of S4), secondary well-separated divergences or intermediacy in Mitochondrial of level: emerged signs species 1). any three without the markers, All genome-wide at of lineages thousands 1). diverged (Figure deeply Thailand three north-eastern for support crispifrons strong N. revealed approach genome-wide complexOur the in lineages diverged qualitative 2018). of deeply al., assessment Three et rough plumage a (Collar a than taxa as more River among no Salween differences included the and vocal of variation, west geographic birds than of rather phenotype polymorphism species, information. white-faced distinct bioacoustic the two interpreted existing to erroneously into to due treatment Wren-babbler attention species Limestone of single the lack a split previous and as classification a treated taxonomic and been recent plumage long A its has of Wren-babbler conservatism Limestone overall The the diversity. taxon generating in .calcicola N. and u not but , Fgr b.Ti togvcldvrec ewe w opooial iia u genomically but similar morphologically two between divergence vocal strong This 1b). (Figure calcicola Fgr 1). (Figure .annamensis N. rmMamradwsenThailand, western and Myanmar from ae nterge n uosblis epciey(olre l,22) oee,this However, 2020). al., et (Collar respectively bellies, rufous and grey their on based , .annamensis N. ipae eaieyhg iohnra iegneo prxmtl .%(Supporting 2.7% approximately of divergence mitochondrial high relatively a displayed ) .annamensis N. hrsacoe ffiiywith affinity closer a shares .calcicola N. samntpcseisi o upre ytegnmcrsls which results, genomic the by supported not is species monotypic a as , .crispifrons N. ge rai iue1a). 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Data may be preliminary. ti qal ocial htti ouainmyb vltoaiyehmrl n ucm oclimatic allows. connectivity to as succumb soon and the as range of ephemeral, main beginning evolutionarily the very be from the populations may Conclusion divergence, at non-leucistic genomic population disappear population by of population probably overrun a this absence peripheral be captured can that the or innovation a we in conceivable vagaries plumage that evident in equally conceivable already a found is is is such Being differentiation it it range, phenotypic While the when isolated. process, of otherwise arises. 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Long-term the only preventing capability the barrier, to perhaps dispersal towards secondary river leading and poor formidable the recently, the genes a across by relatively be these innovation amplified therefore occurred the in effect may have Given founder mutations River to a Salween 2), 2018). by likely al., driven (Figure genomic are substantive and et peaks faces of (Yu individuals divergence lack white bone virtual few and of the those muscle evolution and skin, of population chicken Fukuda, white-faced outside of the & differentiation pigmentation Ohbayashi of the divergence 2001; mitochondrial in al., shallow role et skin important Matesic lightened as an 2000; such play al., coat, gene et the the in (Wilson to pigmentation et humans white-faced of mutations Chabrillat in loss 2003; Therefore, hair a al., silvery to et 2012). lead and Chakraborty can 2000; mice genes al., in these et shown colour to (Araki been mutations have transport and genes proteins melanosome 2005), Rab in two Various al., role including identified form. pathway, significant GTP-bound a we pigmentation active play the their Specifically, to into in proteins involved Rab albinism. GDP-bound be our partial inactive may 2013), this that al., with 18 SLC16A3 of et and associated populations 1A Switonski be 2016). brown chromosomes 2012; may al., from and al., et which white-faced Bourgeois et regions the 2006; pigmentation Cibois outlier of Toews al., of et genomes 2009; expression 2012; Cheviron the the al., al., 2003; with 2001; Comparing et et al., associated al., ( et indirectly Uy Cibois (MacDougall-Shackleton et be receptor 2007; 2009; rudimentary may (Theron melanocortin-1 that still al., al., species loci the is et et candidate animal of Baiao other Uy various involvement pigmentation of 2004; across 2004; the understanding in documented al., While al., differences well et et plumage 2019). extremely Mundy Mundy genomic stark al., is 2004; limited for et polymorphisms that al., Knief responsible melanic shown et 2016; has be Kerje that al., can research 2001; et loci of al., single body et sizeable even confirmed (Theron a work sometimes genomic supporting and of our 1), brown-plumaged morph of regions (Figure the leucistic results Thailand with a The cohesive and as genomically recognition. mar regarded is subspecies population been of white-faced always deserving the has ar- race that it Myanmar geographic yet from a plumage, population of distinct white-faced instead most the Wren-babbler, the Limestone has of guably populations different the Among h gene The . .crispifrons N. RAB3IP niiul.Tegene The individuals. noe o a for encodes RAB3IP RAB3A SLC16A3 a nietyaetteepeso fpgetto in pigmentation of expression the affect indirectly may 8 neatn rti,wihi epnil o converting for responsible is which protein, interacting lokonas known also , .crispifrons N. MCT4 .crispifrons N. eietfidseveral identified we , a ensonto shown been has , MC1R .crispifrons N. RAB3IP rmMyan- from eein gene ) and Posted on Authorea 26 Jun 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159318488.82442436 — This a preprint and has not been peer reviewed. Data may be preliminary. rwig .L,&Y,Z 20) iutnosgntp aln n altp hsn mrvsgenotype studies. improves association phasing genome-wide haplotype and for calling associations C. genotype false-positive Thebaud, Simultaneous reduces & (2009). and B., Z. 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Environmental Tien us collection and providing Duc and for Resources Bui research UK, Natural Tring, Mr. at of thank Museum (R- also Ministry History grant We Development Technology Natural 2 H.L.M). and the Tier Science to thank Education for 106-NN.05-2015.34 Foundation Department of National Singapore Ministry (no. Vietnam of Singapore grant the University the and National F.E.R), F.E.R), from to to 154-000-A59-112 fund (R-154-000-060-001 undergraduate Sciences an Biological by of supported was research This Salween the distinct phenotypically as time. a such Acknowledgements of of barrier establishment period the geographical short facilitated secondary have the a a may in within regions of pigmentation localized of population presence few loss the a the In in with mutations associated population. River, apparently elusive this genes the of candidate feathering distinct, localized facial few morphologically a Although identified par- We individually. species, tion. diverged results genomically of vocal three population and of white-faced plumage consists by complex Wren-babbler supported Limestone tially the reveals study Our ultno h rts rihlgss lb 123 Club, Ornithologists’ British the of Bulletin ora fHrdt,107 Heredity, of Journal cec,312 Science, 4,729-744. (4), 5,332-336. (5), 354-364. , uasula Sula 57) 570-572. (5773), .crispifrons N. :amlncri- eetr(CR analysis. (MC1R) receptor melanocortin-1 a ): 4,3735 doi:10.1093/jhered/esw017 327-335. 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(1), (10), Posted on Authorea 26 Jun 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159318488.82442436 — This a preprint and has not been peer reviewed. Data may be preliminary. hw eieec oei h orsodn eunefrteN2te,wiebosrpspotgenerated support bootstrap while tree. tree, SNP Boot- ND2 the (right). the for for Limestone SNPs shown sequence the is genome-wide are corresponding of likelihood 306,874 methods the maximum Phylogeny and likelihood in from c) maximum (left) node and each gene (a). parsimony beside maximum panel ND2 shown neighbour-joining, to the are 95% from indicating correspond of generated results ellipses support dots pairs STRUCTURE with strap of measurements base bioacoustic taxon. Colors map. 887 of respective with using the PCA group. 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Data may be preliminary. hoooeGn Description delta 1 4 kinase only Casein domain LIM 6 protein binding Syntaxin 2 (ENSTGUG00000003904) AT-hook 16 CSNK1D group metallopeptidase mobility NA Matrix High B member (ENSTGUG00000006316) 110 LMO4 similarity sequence H3 with (ENSTGUG00000011795) 2100001-2250000 Family STXBP6 18: 15490001-15640000 8: (putative) (ENSTGUG00000011786) 10A MMP16 33910001-34060000 transporting 5: phospholipid ATPase (ENSTGUG00000011117) FAM110B 131400001-131550000 2: non-selective channel, 119560001-119710000 leak 2: Sodium ENSTGUG00000023478 (ENSTGUG00000010310) 37720001-37870000 ATP10A H2 1A: 35730001-35880000 1A: Gene (ENSTGUG00000010876) 89750001-89900000 NALCN 1: region Intronic genes 82930001-83080000 and 1: shown, are 38950001-39100000 regions 1: outlier the of each Chromosome 7468 bold. within in found highlighted Genes are pathways 13562 pigmentation 6736 Myanmar. to or 7229 associated and/ 13582 Thailand 15579 7770 from 15725 2 Table 15367 H1 15647 0.874 15803 Blocks 0.16 16016 0.168 BABA 0.596 -1.38 0.001 ABBA 0.006 0.53 -0.007 p-value 2.75 0.002 populations. 0.012 (H3) donor Z and (H2) recipient D-stat between sharing allele of excess referring an latter suggests the 3 above arrows, red or 1 black Table by genes. indicated pigment-related regions containing d Outlier regions to high chromosomes. of all d across Net proportion respectively, dii) lower cii, a d chromosomes. had high across d of lines) di) proportion purple chromosomes. higher across and a blue had green, d lines) by yellow (depicted and orange comparisons red, d the by of (depicted proportion d Cumulative each b) in isons. line horizontal red The (chr). of population (VN) 2 Figure XY between ule eoi ein dnie ewe ht-ae n brown-faced and white-faced between identified regions genomic Outlier -ttsiso eetdppltost sespeec fscnaygn o.Aciia au (Z) value critical A flow. gene secondary of presence assess to populations selected of D-statistics bouencetd iegne(d divergence nucleotide Absolute .crispifrons N. N. [ c. ] XY AN(NTU000079 at cdsynthase acid Fatty 3 member 16 family carrier Solute (ENSTGUG00000003719) FASN (ENSTGUG00000003892) SLC16A3 protein interacting RAB3A 1 like beta ENSTGUG00000007004 subunit Integrin ENSTGUG00000006982 (ENSTGUG00000010867) ITGBL1 annamensis ouain ihwiefcs(yna,M)adbonfcs(hiad TH) (Thailand, faces brown and MM) (Myanmar, faces white with populations ewe Myanmar between XY XY setx)o h ariecmaio orsodn o(i n (di), and (ci) to corresponding comparison pairwise the of text) (see n hi(H ouainof population (TH) Thai and XY ltrflcste99.9 the reflects plot XY auso eetdpiso ouain.Itrtxncomparisons Inter-taxon populations. of pairs selected of values fslce ar fppltos )d a) populations. of pairs selected of ) .crispifrons N. 16 naessclioacrispifrons crispifrons crispifrons calcicola calcicola calcicola crispifrons annamensis annamensis annamensis th ouain ihwiefcsadbonfaces brown and faces white with populations ecnietrsodars ariecompar- pairwise across threshold percentile (Myanmar N. [ c. ] crispifrons brown) XY .crispifrons N. aus hl intra-taxon while values, XY ewe h Vietnam the between crispifrons coschromosomes across XY populations aus ci) values. (white) crispifrons (Thailand) (white) (Myanmar (Thailand) brown)